HK1139589A - Compositions and methods for treating pathologic angiogenesis and vascular permeability - Google Patents

Description

Compositions and methods for treating pathological angiogenesis and vascular permeability

Statement regarding federally sponsored research

The invention was made with government support sponsored by the National Institutes of Health under lot number 1R01 HL 77671-01. The government has certain rights in this invention.

Background

Although the formation of the vasculature of any vertebrate organ system is a complex process, orchestrated by a pool of growth factors and targeting signals (Jain et al, 2003), recent studies have greatly increased our understanding of the signaling cascades that govern angiogenesis. For example, it is becoming increasingly clear that molecular programs that direct axonal pathways and the formation of neural networks play an important role in generating a highly stereographic pattern of mature vascular networks (Carmeliet et al, 2005; Urnesss et al, 2004; and Jones et al, 2007).

In the initial stage of vascular development, known as angiogenesis (vasculogenesis) in mammals, endothelial cells differentiate, migrate and coalesce to form a central axial tube, the dorsal aorta and the main veins. The second stage is known as angiogenesis (angiogenesis), which is characterized by the sprouting of new blood vessels from the primary plexus to form a mature circulatory system. VEGF (or VPF) is critical for both of these first two phases: endothelial cell differentiation and survival during angiogenesis, and proliferation and permeability during angiogenesis. Following this angiogenic remodeling, the endothelium secretes Platelet Derived Growth Factor (PDGF), inducing recruitment and differentiation of vascular smooth muscle cells. Vascular smooth muscle cells then secrete angiogenin, which ensures proper interaction between endothelial cells and vascular smooth muscle cells. Finally, vascular smooth muscle cells deposit matrix proteins, such as elastin, which inhibit proliferation and differentiation of vascular smooth muscle cells, thereby stabilizing mature blood vessels. Thus, in order to establish and maintain a mature vascular network, the endothelium and smooth muscle compartment of the blood vessels must interact through autocrine and paracrine signaling. The gaps (cell junctions) between endothelial cells forming the vascular endothelium are tightly regulated depending on the type of tissue and physiological state. For example, in mature vascular beds, endothelial cells behave not independently of one another; instead, they form a monolayer that prevents proteins, fluids (flu) and cells from migrating from the endothelial lumen into the surrounding tissue.

Even after development, the vascular system is continuously exposed to events, conditions or pathogens that cause injury, ischemia, and inflammation, which often result in the release of cytokines and angiogenic factors, such as Vascular Endothelial Growth Factor (VEGF). VEGF was originally described, purified and cloned as a Vascular Permeability Factor (VPF) based on its ability to induce vascular leakage. VEGF causes destabilization of endothelial cell-cell junctions, produces endothelial permeability, stimulates endothelial proliferation and migration, and promotes sprouting and edema of blood vessels. These functions serve to deconstruct the stable vascular network, creating leaky new blood vessels. In many cases, it is desirable to release cytokines and angiogenic factors in response to injury, ischemia, and inflammation, as such responses result in the initiation of a healing or healing process. However, excessive angiogenesis and vascular leakage (e.g., endothelial hyperpermeability) potentiate the pathology of several diseases and pathological conditions.

For example, pathological angiogenesis and endothelial hyperpermeability in the retinal or choroidal vascular beds are the most common causes of catastrophic vision loss in developed countries. New and dysfunctional vascular leakage, hemorrhage or irritation fibrosis, which in turn can contribute to edema, hemorrhage, or vision-threatening retinal detachment. The major diseases that share this pathogenesis include proliferative Diabetic Retinopathy (DR), non-proliferative Diabetic Macular Edema (DME), and age-related macular degeneration (AMD) (Dorrell et al, 2007; Afzal et al, 2007). About fifteen million americans over 65 years of age suffer from AMD, and 10% of these patients will experience vision loss due to choroidal neovascularization. In addition, over one thousand six million americans suffer from diabetes, and over 400,000 new patients suffer from retinal edema or neovascularization. Given that the current number of 2 billion diabetic patients worldwide may double in the next 20 years, more than 8% of these patients will suffer from microvascular complications, and there is an unfortunate trend toward a rapid increase in the number of patients who will experience vision loss due to diabetic eye disease. Although less prevalent than DR, DME and AMD, retinopathy of prematurity (ROP) and Ischemic Retinal Vein Occlusion (IRVO) are also associated with pathological angiogenesis and endothelial hyperpermeability in the retina or choroidal vascular bed, and lack an effective treatment.

In addition to ocular diseases, pathological angiogenesis is also associated with tumor formation and growth. Tumor angiogenesis is the proliferation of a network of blood vessels that penetrate into the growth of the tumor, supply nutrients and oxygen, and remove waste products. Tumor growth continues with angiogenesis, without which tumor growth ceases. Angiogenesis in tumors actually begins as malignant cells release molecules that transmit signals to surrounding normal host tissues. This signaling activates certain genes in the host tissue, which in turn make proteins to promote the growth of new blood vessels. Angiogenesis is regulated by activator and inhibitor molecules. Under normal conditions, the inhibitor dominates, blocking growth. However, during tumor formation and growth, tumor cells release angiogenic activators, resulting in an increase in the number/concentration of these activators. This increase in angiogenesis activators causes the growth and division of vascular endothelial cells, ultimately forming new blood vessels.

More than ten different proteins, as well as several smaller molecules, have been identified as "angiogenic". Of these molecules, two proteins appear to be most important for the sustained growth of tumors: vascular Endothelial Growth Factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF and bFGF are produced by a wide variety of cancer cells and certain types of normal cells. VEGF and bFGF are first synthesized inside tumor cells and then secreted into the surrounding tissues. When they encounter endothelial cells, they bind to specific proteins called receptors located on the outer surface of the cell. Binding of VEGF or bFGF to its appropriate receptor activates a series of relay proteins (relay proteins) that transmit signals into the nucleus of endothelial cells. Nuclear signaling ultimately drives a set of genes to produce new products required for endothelial cell growth. Activation of endothelial cells by VEGF or bFGF initiates a series of steps toward the generation of new blood vessels. First, activated endothelial cells produce Matrix Metalloproteinases (MMPs), a special class of degrading enzymes. These enzymes are then released from the endothelial cells into the surrounding tissue. MMPs break down extracellular matrix-supporting substances formed of proteins and polysaccharides that fill the spaces between cells. The breakdown of the matrix allows endothelial cells to migrate. As they migrate into the surrounding tissue, the activated endothelial cells begin to divide and organize into hollow tubes, gradually evolving into a mature vascular network.

Other diseases and conditions characterized by detrimental vascular permeability include, for example, edema associated with brain tumors, ascites associated with malignancies, Meigs' syndrome, pneumonia, nephrotic syndrome, pericardial effusion, pleural effusion, acute lung injury, inflammatory bowel disease, ischemia/reperfusion injury in stroke, myocardial infarction, and infectious and non-infectious diseases that cause cytokine storms (cytokine storms). Although the cytokine storm is a systemic manifestation of the healthy and powerful immune system, it is an excessively increased immune response by rapidly proliferating and highly activated T cells or Natural Killer (NK) cells, resulting in the release of over 150 inflammatory mediators (cytokines, oxygen radicals and coagulation factors). Both proinflammatory cytokines (e.g., tumor necrosis factor-alpha, interleukin-1 and interleukin-6) and anti-inflammatory cytokines (e.g., interleukin-10 and interleukin-1 receptor antagonist) are elevated in serum, and the violent and often lethal interaction of these cytokines is called a "cytokine storm".

Cytokine storms can occur in a number of infectious and non-infectious diseases, including, for example, Graft Versus Host Disease (GVHD), Adult Respiratory Distress Syndrome (ARDS), septicemia, avian influenza, smallpox, and Systemic Inflammatory Response Syndrome (SIRS). Without timely intervention, cytokine storms can lead to permanent lung injury and in many cases death. Many patients will develop ARDS characterized by pulmonary edema, which is not associated with volume overload (voumeover), or left ventricular hypofunction. End-stage symptoms of diseases that contribute to cytokine storm may include one or more of the following: hypertension, tachycardia, dyspnea, fever, ischemia or tissue hypoperfusion, uncontrolled bleeding, severe metabolic disorders, and multiple system organ failure. Death from infection contributing to a cytokine storm is generally attributable to symptoms caused by the cytokine storm and, therefore, is not directly caused by the pathogen of interest. For example, death in severe influenza infections such as avian influenza or "bird flu" is typically the result of ARDS, which is caused by a cytokine storm by viral infection.

Much attention has been focused on Vascular Endothelial Growth Factor (VEGF) due to its association with angiogenesis and vascular permeability. Products capable of reducing VEGF-mediated angiogenesis and vascular edema are currently marketed and available to patients. For example, the anti-VEGF antibody Ranibizumab (Lucentis), an antibody fragment of Bevacizumab (Avastin), which is itself a VEGF antibody (Rosenfeld et al, 2006; Brown et al, 2006), is commercially available for the treatment of AMD. The development and success of such products has stimulated great commercial interest in alternative strategies for treating diseases and conditions associated with pathological angiogenesis or endothelial high permeability. Other methods for inhibiting VEGF signaling include, for example, anti-VEGF aptamers, soluble VEGF receptor extracellular domains, receptor tyrosine kinase inhibitors, and sirnas directed to VEGF or its receptor. Such strategies have shown promise for AMD. However, there remains great interest in similar approaches to the treatment of other diseases associated with pathological angiogenesis and vascular leakage. Furthermore, because VEGF is only one of many angiogenic, permeability, and inflammatory factors that contribute to angiogenesis and vascular permeability, identification pathways and development methods that affect VEGF functionality, as well as the functionality of other angiogenic, permeability, and inflammatory factors, continue to be of value.

Summary of The Invention

Generally, described herein are compounds, compositions and methods for inhibiting vascular permeability and pathologic angiogenesis. Also described herein are methods for producing and screening compounds and compositions capable of inhibiting vascular permeability and pathologic angiogenesis. Pharmaceutical compositions are included in the compositions described herein.

The compositions described herein are useful, for example, in methods of inhibiting vascular permeability and pathologic angiogenesis, including methods of inhibiting vascular permeability and pathologic angiogenesis induced by specific angiogenic, permeability, and inflammatory factors such as VEGF, bFGF, and thrombin. Also provided herein are methods for treating specific diseases and disorders.

Other aspects of the description provided herein will become apparent by reference to the detailed description, including the examples and materials and methods, the claims, and the accompanying drawings, which include a brief description of the drawings.

Brief Description of Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions. The term "mimetic" as used herein refers to a pseudo-preparation that does not contain active Slit proteins.

FIG. 1 shows the cytoplasmic tail of the receptor required for Robo 4-mediated vascular targeting. Confocal microscopy results of 48hpf TG (fli: egfp) y1 embryos are shown, (A) not injected, (B) robo4 morpholino injected, (C) robo4 morpholino and wild type murine robo4 RNA, and (D) robo4 morpholino and robo4 delta tail RNA. The quantification is shown in figure 7. FIG. 1E shows a model of a vascular guide defect in a robo4 morphant embryo. The 5X and 20X images are shown in the left and right panel sets, respectively. DLAV is a dorsal longitudinal anastomotic vessel. PAV is a paraspinal blood vessel. DA ═ the dorsal aorta. PCV-posterior major vein.

FIG. 2 shows that Robo 4-dependent thigmotaxis requires the amino-terminal half of the cytoplasmic tail. FIG. 2A shows a schematic representation of a cDNA construct used in a thigmotaxis assay. TM represents a transmembrane domain. CC0 and CC2 are conserved cytoplasmic signaling motifs found in members of the Robo family. HA is a hemagglutinin epitope. FIGS. 2B and 2C show that HEK 293 cells were co-transfected with GFP and the indicated constructs and performed chemotactic migration after 36 hours on membranes coated with 5. mu.g/ml fibronectin and mock preparation or Slit 2. Expression of the Robo4 construct was confirmed by Western blotting (inset). Results are expressed as mean ± SE.

FIG. 3 shows the interaction of Robo4 with Hic-5 and paxillin in HEK 293 cells. FIG. 3A shows that HEK 293 cells were co-transfected with Robo4 cytoplasmic tail-HA and Hic-5-V5, or empty vector (pcDNA3) and Hic-5-V5. Robo4 was immunoprecipitated with HA antibody and Hic-5 was detected by western blotting using V5 antibody. FIG. 3B shows the probing of whole cell lysates from Cho-K1, HEK 293 and NIH 3T3 cells with antibodies against Hic-5 and paxillin. FIG. 3C shows that HEK 293 cells were co-transfected with paxillin-V5 and Robo4 cytoplasmic tail-HA or empty vector (pcDNA 3). Robo4 was immunoprecipitated with HA antibody from cell lysates and paxillin was detected by western blotting using V5 antibody. FIG. 3D shows HEK 293 cells transfected with full-length Robo4-HA and paxillin-V5 and stimulated with Slit2 for 5 min. Robo4 was immunoprecipitated with HA antibody from cell lysates and paxillin was detected by western blotting using V5 antibody.

FIG. 4 shows that paxillin interacts with a novel motif required for Robo4 inhibition by Robo 4-dependent thigmotaxis. FIG. 4A shows a schematic of GST-Robo4 fusion protein used in the pull down assay shown in Panel B. Fig. 4B shows the purification of GST-Robo4 fusion protein from e.coli (e. Paxillin was detected by western blotting using paxillin-specific monoclonal antibodies. FIG. 4C shows a schematic of the GST-Robo4 fusion protein used in the pull down assay described in FIG. D. FIG. 4D shows the purification of GST-Robo4 fusion protein from E.coli and incubation with recombinant purified paxillin. Paxillin was detected by western blotting using paxillin-specific monoclonal antibodies. FIG. 4E shows GST-Robo4 wild type or GST-Robo 4. delta. PIM was purified from E.coli and incubated with recombinant purified paxillin or in vitro transcribed/translated Mena-V5. Paxillin and Mena were detected using paxillin-specific monoclonal and V5 antibodies, respectively. FIG. 4F shows HEK 293 cells transfected with GFP and the indicated constructs and performing chemotactic migration after 36 hours on membranes coated with 5. mu.g/ml fibronectin and mock preparations or Slit 2. Expression of the Robo4 construct was confirmed by Western blotting (inset). Results are expressed as mean ± SE.

FIG. 5 shows that Robo4 inhibits cell spreading by inactivating Rac. Fig. 5A, 5D and 5G show HEK293 cells transfected with GFP and the indicated constructs and cell spreading analysis performed after 36 hours on coverslips coated with 5 μ G/ml fibronectin and mock preparation or Slit 2. Results are expressed as mean ± SE. FIGS. 5B and 5E show HEK293 cells transfected with the indicated constructs and plated after 36 hours on dishes coated with 5. mu.g/ml fibronectin and mock preparation or Slit 2. After 5 min incubation, cells were lysed and GTP-Rac was precipitated with GST-PBD. Rac was detected by western blotting using Rac-specific monoclonal antibodies. FIG. 5H shows incubation of HUVEC with Slit2 for 60 min, stimulation with 25ng/ml VEGF for 5 min, lysis, and precipitation of GTP-Rac with GST-PBD. Rac was detected by western blotting using Rac-specific monoclonal antibodies. (C) And (F) Slit 2-dependent inhibition of adhesion-induced and (I) VEGF-induced Rac activation, quantified by densitometry. Results are expressed as mean ± SE.

Figure 6 shows that the paxillin Δ Lim4 mutant does not interact with Robo4 or does not support Slit2-Robo4 mediated inhibition of cell spreading. Figure 6A shows a schematic of the paxillin construct used in blocks B, C and D. FIG. 6B shows that HEK293 cells were co-transfected with Robo4 cytoplasmic tail-HA and paxilin-V5, or empty vector (pcDNA3) and paxilin-V5. Robo4 was immunoprecipitated from cell lysates using HA antibody and paxillin was detected by western blotting using V5 antibody. FIG. 6C shows that HEK293 cells were co-transfected with Robo4 cytoplasmic tail-HA with wild-type paxilin-V5 or paxilin Δ Lim 4-V5. Robo4 was immunoprecipitated with HA antibody and paxillin was detected by western blotting using V5 antibody. Figure 6D shows endogenous pilin knockdown using siRNA in HEK293 cells and reconstitution with wild-type pilin or pilin Δ Lim 4. Knock-out and reconstitution were visualized by western blot using pilin antibody and quantified by densitometry. Expression of paxillin was measured at 35% of wild type levels. Fig. 6E shows knockout/reconstituted HEK293 cells, spread analysis was performed on coverslips coated with 5 μ g/ml fibronectin and mock preparation or Slit 2. Results are expressed as mean ± SE.

Figure 7 shows that the paxillin interaction motif is required for repulsive vessel guidance. Fig. 7A shows the quantification of defects in vascular pattern formation in TG (fli: egfp) yl embryos injected with no (n ═ 66), robo4 morpholino (n ═ 56), robo4 morpholino, and wild type murine robo4 RNA (n ═ 60), robo4 morpholino, and robo4 Δ tail RNA (n ═ 17), and robo4 morpholino, and robo4 Δ PIM RNA (n ═ 45). A representative image is shown in fig. 1. FIG. 7B shows a model of the Slit2-Robo4 signaling axis that inhibits cell migration, spreading, and Rac activation.

FIG. 8 shows that splicing-blocking morpholinos inhibited the expression of robo4 in zebrafish embryos. FIG. 8A shows a schematic of Robo4 locus and encoded Robo4 protein in zebrafish (Danio rerio). The splicing blocking morpholino targeted exon is indicated, as is the position of the primer used to amplify robo4 cDNA. FIG. 8B shows RNA isolated from uninjected embryos and embryos injected with robo4 splicing blocking morpholinos and used for reverse transcription of cDNA. The cDNA was then used to amplify robo4, and the resulting fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide.

FIG. 9 shows that Hic-5 is a Robo4 interacting protein. FIG. 9A shows a schematic of full-length Hic-5 and cDNA clones recovered from the yeast two-hybrid screen. FIG. 9B shows s.cerevisiae strain PJ694-A transformed with the indicated plasmids and plated on synthetic medium without leucine and tryptophan, or without leucine, tryptophan, histidine and alanine. Colonies that were able to grow on the auxotrophic medium were spotted on the same medium, replica plated and photographed or used for β -galactosidase analysis.

FIG. 10 shows that the paxillin interaction motif is located between CC0 and CC2 in the Robo4 cytoplasmic tail. Schematic representation of murine Robo4 protein and identification of amino acids comprising paxillin interaction motifs.

Figure 11 shows that the Robo4 cytoplasmic tail does not inhibit Cdc42 activation, nor does it interact with srGAP 1. FIG. 11A shows the plating of HEK 293 cells expressing Robo4 on bacterial dishes coated with 5. mu.g/ml fibronectin and mock preparations or Slit 2. After 5 min incubation, cells were lysed and GTP-Cdc42 was precipitated with GST-PBD. Cdc42 was detected by western blotting using a monoclonal antibody specific for Cdc 42. FIG. 11B shows HEK 293 cells transformed with the indicated plasmids and then immunoprecipitated with HA antibody on Robo1/Robo 4. srGAP1 was detected by western blotting using Flag M2 antibody.

Fig. 12 shows that slit reduces retinopathy of prematurity, a FDA criterion for factors affecting diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. Figure 12A shows the percent of neovascularisation of the retina in wild-type mice receiving mock preparations compared to mice receiving Slit protein. In mice treated with Slit, there was a 63% reduction in new blood vessels compared to wild-type mice. N is 6, P is less than 0.003. Figure 12B shows the percent neovascularization of the retina for wild type mice receiving the mock preparation compared to mice receiving the saline control. N is 5, P is less than 0.85. Fig. 12C shows the percent neovascularization of the retina in knockout mice compared to Slit treated mice. N is 1.

Fig. 13 shows that slit and homing factor (netrin) are able to decrease VEGF-induced skin permeability.

FIG. 14 shows that slit is able to decrease VEGF-mediated retinal permeability.

Figure 15 shows that semaphorin-like VEGF increases skin permeability.

FIG. 16 shows that Robo4 blocks Rac-dependent prominent activity by inhibiting Arf 6. CHO-K1 cells stably expressing the cytoplasmic tail of α IIb or α IIb-Robo4 were plated on fibronectin or fibronectin and fibrinogen coated dishes, lysed, and GST-GGA3 was used to precipitate GTP-Arf 6. Arf6 was detected by western blotting using an Arf6 specific monoclonal antibody (see fig. 16A). CHO-K1 cells stably expressing either the α IIb or α IIb-Robo4 cytoplasmic tail were co-transfected with GFP with empty vector or GIT1-PBS and spread analyzed on fibronectin or fibronectin and fibrinogen coated slides. Areas of GFP positive cells were determined using ImageJ and error bars represent SEM (see fig. 16B). HEK 293 cells were co-transfected with GFP and the indicated constructs and spreading analysis was performed 36 hours later on fibronectin and mock preparation or Slit2 protein (see figure 16C). Error bars represent mean ± SE in all blocks. Expression of Robo4 and ARNO was confirmed by western blotting (data not shown). HEK 293 cells were co-transfected with GFP and the indicated constructs and plated 36 hours later on dishes coated with fibronectin and mock preparation or Slit2 protein. GTP-Rac was precipitated with GST-PBD and Rac was detected with a monoclonal antibody specific for Rac1 (see FIG. 16D).

Fig. 17 shows the results of the immunoprecipitation reaction, confirming the binding of the Robo4 receptor to the Slit ligand. FIG. 17A shows the results of immunoprecipitation of cell lysates from untransfected human embryonic kidney cells (HEK), HEK cells transfected with Slit bearing a myc epitope tag (Slit-myc), HEK cells transfected with Robo4 bearing an HA epitope tag (Robo4-HA), and HEK cells transfected with a control vector (control-HEK). Western blot analysis of lysates of Slit-myc cells was used as a control, demonstrating a mass of Slit protein of approximately 210kD, as in previous reports (lane 1). Slit-myc protein was also detected by Western blot analysis using anti-myc antibodies after mixing the Slit-myc and Robo4-HA cell lysates and immunoprecipitation with anti-HA antibodies (lane 6). The specificity of this interaction was confirmed by the inability to detect Slit proteins using all other combinations of lysates. The same amount of lysate was used in each experiment. The lower band in lanes 2-6 corresponds to the immunoglobulin heavy chain. FIG. 17B shows the results of immunoprecipitation from conditioned media of untransfected HEK cells (HEK CM), HEK cells transfected with Slit bearing a myc epitope tag (Slit-myc CM), HEK cells transfected with the N-terminal soluble extracellular domain of Robo4 bearing an HA epitope tag (NRobo4-HA CM), and HEK cells transfected with a control vector (control-HEK CM). The full-length Slit-myc protein (210kD) and its C-terminal proteolytic fragment (70kD) were detected in Slit-myc CM by an anti-myc antibody (lane 1). As in FIG. 17A, after mixing Slit-myc and Robo4-HA conditioned media and immunoprecipitation with anti-HA antibodies, Slit-myc protein was also detected by Western blotting (lane 6). The specificity of this interaction was confirmed by the absence of Slit protein when all other combinations of conditioned media were used. As shown in fig. 17C-17F, Slit proteins bound to the plasma membrane of cells expressing Robo 4. Binding of Slit-myc protein was detected using anti-myc antibodies and anti-mouse antibodies to which Alexa 594 bound. Binding was detected on the surface of Robo4-HEK cells (FIG. 17F) but not on the surface of control-HEK cells (FIG. 17D).

FIG. 18 shows that Robo4 expression is endothelial cell specific and stem cell centered. FIG. 18A shows a diagram from P5 Robo4^+/APRetinal accessory discs (flatmounts) were prepared from mice and stained for endothelial mucin (endothelial cells), NG2 (pericytes) and alkaline phosphatase (AP; Robo 4). The top most arrow pointing to the right in the upper left panel indicates the top cells, the remaining arrows indicate the pericytes (NG2 positive). "T" also means apical cells. FIG. 18B shows Robo4 from adult^+/APRetinal accessory discs were prepared from mice and stained for NG2 (pericytes) and AP (Robo4), with the arrows contained in fig. 18B indicating pericytes (NG2 positive). FIG. 18C shows the results of quantitative RT-PCR (qPCR) performed on indicated samples using primers specific for PECAM, Robo1, and Robo 4. When used in fig. 18C: "HAEC" means human aortic endothelial cells; "HMVEC" refers to human microvascular endothelial cells; "HASMC" refers to human aortic smooth muscle cells. FIG. 18D shows the results of probing whole cell lysates from HMVEC and HASMC using antibodies against Robo4, VE-cadherin, smooth muscle actin and ERK 1/2.

FIG. 19 shows that Robo4 signaling inhibits VEGF-A induced migration, tube formation, permeability, and Src Family Kinase (SFK) activation. From Robo4 ^+/+And Robo4^AP/APMouse isolated lung Endothelial Cells (ECs) were used for endothelial cell migration (fig. 19A), tube formation (fig. 19B), in vitro permeability (fig. 19C), Miles assay (fig. 19D), and retinal permeability assay (fig. 19E). Human microvasculatureEndothelial cells were stimulated with VEGF-a for 5 min in the presence of mock preparation or Slit2 protein, lysed, and subjected to western blotting using phospho-VEGFR 2 antibody (fig. 19F), western blotting using phospho-Src antibody (fig. 19G), and Rac activation analysis (fig. 19H). In all figures, p < 0.05, p < 0.005, p < 0.0005, NS "not significant" and SEM error bars.

Fig. 20 shows that Robo4 signaling inhibits pathological angiogenesis in an animal model of oxygen-induced retinopathy ("OIR") and in an animal model of choroidal neovascularization ("CNV"). For newborn Robo4^+/+And Robo4^AP/APMice were subjected to oxygen-induced retinopathy and perfused with Fluorescein Isothiocyanate (FITC) -dextran (green). Retinal accessory discs were prepared for each case and analyzed by fluorescence microscopy. Arrows indicate areas of pathological angiogenesis (fig. 20A to 20D). Quantification of pathological angiogenesis observed in fig. 20A through 20D is provided in fig. 20E. In the CNV model, for Robo4 at 2-3 months of age ^+/+And Robo4^AP/APMice underwent laser-induced choroidal neovascularization. Choroidal accessory disks were prepared, stained with isolectin, and analyzed by confocal microscopy (fig. 20F to fig. 20I). Quantification of pathological angiogenesis observed in fig. 20F through 20I is provided in fig. 20J. In all figures, p < 0.05, p < 0.005, p < 0.0005, NS "not significant" and SEM error bars.

FIG. 21 shows that Robo4 signaling inhibits bFGF-induced angiogenesis and thrombin-stimulated endothelial hyperpermeability. In performing experiments that provided the results shown in fig. 21A, tube formation analysis was performed on murine lung endothelial cells on matrigel in the presence of bFGF and mock preparation or Slit2 protein. In performing experiments that provided the results shown in fig. 21B, thrombin-induced permeability assays were performed on fibronectin-coated Transwells.

Figure 22 shows that Robo4 signaling reduces injury and inflammation in an acute lung injury model. Mice were exposed to intratracheal LPS and treated with Slit protein or mock preparations. The concentration of inflammatory cells and proteins in bronchoalveolar lavage (BAL) is significantly reduced by Slit protein treatment.

FIG. 23 shows different constructs of Slit proteins and shows that recombinant Slit peptides as small as Slit2-D1(40kD) are also active. In fig. 23A, different constructs of Slit proteins are depicted. Four leucine rich domains (LRRs), epidermal growth factor homology regions (EGF) and C-terminal tag (MYC/HIS) are indicated. The inhibition of VEGF-mediated endothelial cell migration by different Slit constructs (2nM) is shown in fig. 23B.

Fig. 24 shows the effect of administering Slit protein on survival of mice infected with avian influenza virus, following a mouse model of avian influenza.

Figure 25 shows the genomic traits of the knockout mice described in example 14.

Detailed description of the invention

Materials, compositions, and ingredients useful for, with, or products of the methods and compositions of the present disclosure are disclosed. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific meanings of each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a polypeptide is disclosed and discussed, and a number of modifications that can be made to a number of molecules comprising the polypeptide are discussed, each and every possible combination and permutation of the polypeptide and modifications are specifically contemplated unless specifically indicated to be so. Thus, if a class of molecules A, B and C and a class of molecules D, E and F are disclosed, and an example of a combined molecule A-D is disclosed, then each is considered individually and collectively even if not individually recited. Thus, in this example, each combination A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F is specifically contemplated and should be considered to be from A, B and C; D. e and F; and example combinations a-D are disclosed in the disclosure. Likewise, any subset or combination thereof is also specifically contemplated and disclosed. Thus, for example, the subgroups of A-E, B-F and C-E are specifically contemplated and should be considered to be from A, B and C; D. e and F; and example combinations a-D are disclosed in the disclosure. This concept is applied to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the present disclosure. Thus, if there are a variety of other steps that can be performed it is understood that each of these other steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods and that each such combination is specifically contemplated and should be considered disclosed.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

It is to be understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the context of this specification.

It must be noted that, as used herein and in the appended claims, the term "a" or "an" without an adjective includes the plural form of the term, unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a plurality of such polypeptides, reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth.

"optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or exist, and that the description includes instances where the event, circumstance, or material occurs or exists and instances where it does not occur or does not exist.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Likewise, when values are expressed as approximate values by the use of "about" in the foregoing, it should be understood that the particular values form another embodiment. It will be further understood that the endpoints of each of the ranges are obviously both related to the other endpoint, and independent of the other endpoint. It will also be understood that many values are disclosed herein, and that each value herein is also disclosed as "about" that particular value, in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values are also disclosed, as would be understood by one of ordinary skill in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout this application, data is provided in a number of different formats, with the data representing end and start points, and any range of combinations of data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15, and between 10 and 15, are considered disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13 and 14 are also disclosed.

The term "subject" as used herein refers to any target of administration. The subject may be a vertebrate, such as a mammal. Thus, the subject may be a human. The term does not specify a particular age or gender. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject suffering from a disease or disorder. The term "patient" includes both human and veterinary subjects.

"inhibit" refers to a decrease in activity, response, disorder, disease, or other biological parameter. This may include, but is not limited to, complete elimination of the activity, response, condition or disease. This may also include, for example, a 10% reduction in activity, response, condition, or disease as compared to native or control levels. Thus, the reduction may be any reduction between 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or the specifically stated percentage as compared to the native or control level.

By "promoting" is meant an increase in activity, response, disorder, disease, or other biological parameter. This may include, but is not limited to, the initiation of an activity, response, condition, or disease. This may also include, for example, a 10% increase in activity, response, condition, or disease as compared to native or control levels. Thus, an increase in activity, response, condition, disease, or other biological parameter can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or more, including any increase between the specifically recited percentages, as compared to native or control levels.

The term "therapeutically effective" means that the amount of the composition used is sufficient to alleviate one or more causes or symptoms of a disease or disorder. Such mitigation is only required to be reduced or altered, and not necessarily eliminated.

The term "carrier" refers to a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates in the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of such compound or composition for its intended use or target. For example, the carrier may be selected to minimize any degradation of the active ingredient in the subject and to minimize any adverse side effects in the subject.

The term "regulatory sequences" refers to sequences that affect gene expression, typically within 100-1000 kilobases (kb) of the coding region of a locus, but they may also be further from the coding region. Such regulation of expression includes transcription of the gene, as well as translation, splicing and stability of messenger RNA.

The term "operably linked" refers to adjacent positions in which the described components are in a relationship that allows them to function in the intended manner. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. The term "operably linked" can refer to a functional association between a nucleic acid expression control sequence (e.g., a promoter, enhancer, or arrangement of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the corresponding nucleic acid of the second sequence.

"isolated," when used to describe a biomolecule disclosed herein, refers to, for example, a peptide, protein, or nucleic acid that has been identified and isolated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would normally interfere with diagnostic or therapeutic applications of the isolated molecules and may include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. Methods of isolating and purifying the biomolecules described herein are known and available in the art, and one of ordinary skill in the art can determine the appropriate method of isolation and purification based on the substance to be isolated or purified. Although an isolated biomolecule will typically be prepared using at least one purification step, "isolated" as used herein may also refer to, for example, a peptide, protein, antibody or nucleic acid species in situ in a recombinant cell, even if expressed in a homologous cell type.

Furthermore, when the terms "isolated," "substantially pure," and "substantially homogeneous" are used to describe a monomeric protein, they are used interchangeably herein. Monomeric proteins are substantially pure when at least about 60% to 75% of the samples exhibit a single polypeptide sequence. Generally, a substantially pure protein may comprise about 60-90% W/W of a protein sample, and a substantially pure protein may be greater than about 90%, about 95%, or about 99% pure, if desired. The purity or homogeneity of a protein can be demonstrated by a variety of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band after gel staining. For some purposes, HPLC or other methods for purification well known in the art may be used to provide higher resolution.

Throughout the description and claims of this specification, the word "comprise" means "including but not limited to", and is not intended to exclude, for example, other additives, components, integers or steps.

As used herein, "vascular permeability" refers to the ability of small molecules (e.g., ions, water, nutrients), macromolecules (e.g., proteins and nucleic acids), or even whole cells (lymphocytes on their way to the site of inflammation) to pass through the walls of blood vessels.

The term "pathogenic" or "pathogenic state" refers to any deviation from a healthy, normal, or effective state, which may be the result of a disease, condition, event, or injury.

Proteins and peptides

The terms "protein" and "peptide" as used herein, simply refer to polypeptide molecules in a broad sense and are not intended to refer to polypeptide molecules of any particular size, length or molecular weight. Protein variants and derivatives are well known to those skilled in the art and may include amino acid sequence modifications. For example, modifications of amino acid sequences typically fall into one or more of three types: substitution, insertion or deletion variants. Insertions include amino-and/or carboxy-terminal fusions as well as insertions within the sequence of single or multiple amino acid residues. Insertions are generally smaller insertions than amino-or carboxy-terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing polypeptides large enough to confer immunogenicity to a target sequence by in vitro cross-linking, or by transforming recombinant cell cultures with DNA encoding the fusion protein. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about 2 to 6 residues are deleted at any one site within the protein molecule. Such variants are typically prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby generating DNA encoding the variant, which DNA will then be expressed in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically single residues, but may occur at many different positions at once; insertions are typically around about 1 to 10 amino acid residues; deletions are in the range of about 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., two residues are deleted or two residues are inserted. Substitutions, deletions, insertions, or any combination thereof may be combined to obtain the final construct. Mutations can in no way place the sequence out of reading frame and preferably do not produce complementary regions that can give rise to secondary mRNA structure. Substitution variants are those in which at least one residue is removed and a different residue is inserted in its place. Such substitutions are generally made according to table 1 below and are referred to as conservative substitutions.

Table 1: the substitution of an amino acid(s),

conservative substitutions of original residue examples

Others are known in the art.

Significant changes in functional or immunological characteristics are produced by selecting substitutions that are less conservative than those in table 1, i.e., selecting residues that differ more significantly in their effect in maintaining the following properties: (a) the structure of the polypeptide backbone in the substitution region, e.g., a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. In general, the substitutions expected to produce the greatest change in the properties of the protein will be those in which: (a) a hydrophilic residue, such as a serine or threonine residue, substituted for (or by) a hydrophobic residue, such as a leucine, isoleucine, phenylalanine, valine, or alanine residue; (b) cysteine or proline in place of (or substituted for) any other residue; (c) a residue having a positively charged side chain, such as a lysine, arginine or histidine residue, substituted for (or by) a residue having a negative charge, such as a glutamic acid or aspartic acid residue; or (d) a residue with a bulky side chain, such as phenylalanine, is substituted for (or by) a residue without a side chain, such as glycine, in which case (e) by increasing the number of sites for sulfurization and/or glycosylation.

For example, the replacement of an amino acid residue with another biologically and/or chemically similar residue is referred to by those skilled in the art as a conservative substitution. For example, a conservative substitution would be the replacement of one hydrophobic residue for another, or one polar residue for another. Substitutions include combinations, for example Gly, Ala; val, Ile, Leu; asp, Glu; asn, Gln; ser, Thr; lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each of the specifically disclosed sequences are encompassed by the chimeric polypeptides provided herein.

Substitution or deletion mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). It may also be desirable to delete cysteine or other possible residues. Deletion or substitution of a possible proteolytic site, e.g. Arg, can be achieved, for example, by deleting one of the basic residues or replacing one with a glutamine or histidine residue.

Certain post-translational derivatizations are the result of recombinant host cells acting on the expressed polypeptide. Glutamine and asparagine residues are often post-translationally deamidated to form the corresponding glutamic and aspartic acid residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the o-amino groups of lysine, arginine and histidine side chains (T.E.Creighton, Proteins: Structure and Molecular Properties), W.H.Freeman & Co., San Francisco pp 79-86[1983]), acetylation of N-terminal amines, and, in some cases, amidation of the C-terminal carboxyl group.

It will be appreciated that one way to determine variants and derivatives of the proteins and peptides disclosed herein is by determining variants and derivatives based on homology/identity to particular known sequences. Specifically disclosed are variants of these and other proteins disclosed herein that have at least 70% or 75% or 80% or 85% or 90% or 95% homology to the described sequences. One skilled in the art would readily understand how to determine the homology of two proteins. For example, homology can be calculated after aligning the two sequences so that homology is at the highest level.

Another method of calculating homology can be performed by published algorithms. Optimal sequence alignments for comparison can be performed by the local homology algorithm of Smith and Waterman (adv.Appl.Math.2: 482(1981)), the homology alignment algorithm of Needleman and Wunsch (J.mol.biol.48: 443(1970)), the similarity search method of Pearson and Lipman (Proc.Natl.Acad.Sci.U.S.A.85: 2444(1988)), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575Science Dr., Madison, Wis), or by visual inspection.

For nucleic acids, the same type of homology can be obtained, for example, by homology as described in Zuker, m.science 244: 48-52, 1989; jaeger et al, proc.natl.acad.sci.usa 86: 7706 7710, 1989; jaeger et al, Methods Enzymol.183: 281, 306, 1989, at least the materials relating to nucleic acid alignment are incorporated herein by reference.

It is understood that the descriptions of conservative mutations and homology may be combined together in any combination, such as embodiments having at least 70% homology to a particular sequence, where the variant is a conservative mutation.

Because this specification discusses a variety of different proteins and protein sequences, it is understood that nucleic acids capable of encoding those protein sequences are also disclosed. This is intended to include all degenerate sequences which relate to a particular protein sequence, i.e.all nucleic acids whose sequence encodes a particular protein sequence, as well as all nucleic acids which encode disclosed variants and derivatives of a protein sequence, including degenerate nucleic acids. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that in fact each and every sequence is disclosed and described herein by way of the disclosed protein sequences.

It is understood that there are numerous analogs of amino acids and peptides that can be incorporated into the disclosed compositions. For example, there are numerous D-amino acids or amino acids with different functional substituents than those shown in Table 1. Opposite stereoisomers of naturally occurring peptides are disclosed, as well as stereoisomers of peptide analogs. These amino acids can be readily incorporated into the polypeptide chain by loading the tRNA molecule with selected amino acids and engineering a Genetic construct using, for example, an amber codon to insert similar amino acids into the peptide chain in a site-specific manner (Thorson et al, Methods in molecular. biol.77: 43-73 (1991); Zoller, Current opinion in Biotechnology, 3: 348-.

Molecules can be produced that resemble peptides, but cannot be linked by natural peptide bonds. For example, a bond for an amino acid or amino acid analog can include CH ₂NH--、--CH₂S--、--CH₂--CH₂-, - -CH- - - - (cis and trans) - -, - -COCH₂--、--CH(OH)CH₂- - -and- -CHH₂SO- - (these and other bonds can be found in Spatola, A.F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, chemical and biochemical Biochemistry of Amino Acids, Peptides and Proteins) B.Weinstein, Main edition, Marcel Dekker, New York, p.267 (1983); Spatola, A.F., Vega Data (March 1983), Vol.1, Issue 3, Peptide Back bone Modifications (for Peptide Backbone modification); Morley, Trends Pharm Sci (1980) pp.463-468; Hudson, D.et al, Int J Pept Probe 14: 177-₂NH--、CH₂CH₂- - - -; spatola et al, Life Sci 38: 1243-₂-S); (1982) (-CH- -CH- -, cis and trans) Hann J.chem.Soc Perkin Trans.I 307-314; almquist et al, j.med.chem.23: 1392-1398(1980) (- -COCH)₂- - - -; ) (ii) a Jennings-White et al, Tetrahedron Lett 23: 2533(1982) (- -COCH)₂- - - -; szelke et al, European Appln, EP 45665CA (1982): 97: 39405(1982) (- -CH (OH) CH₂- - - -; holladay et al tetrahedron.lett 24: 4401-4404(1983) (- -C (OH) CH₂- - - -; and Hruby Life Sci 31: 189- - ₂- - -S- -); each of which is incorporated herein by reference. A particularly preferred non-peptide bond is- -CH₂NH- -. It is understood that peptide analogs may have more than one atom between the bonding atoms, such as beta-alanine, gamma-aminobutyric acid, and the like.

Amino acid analogs and peptide analogs typically have enhanced or desired properties, such as more economical production, greater chemical stability, enhanced pharmaceutical properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, and the like.

D-amino acids can be used to produce more stable peptides because D-amino acids are not themselves recognized by peptidases. Systematic substitution of one or more amino acids of the conserved sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to produce more stable peptides. Cysteine residues may be used for cyclization or to link two or more peptides together. This is beneficial for confining the peptide to a specific conformation. (Rizo and Gierasch Ann. Rev. biochem.61: 387(1992), incorporated herein by reference).

Nucleic acids

Disclosed herein are a variety of nucleic acid-based molecules. The disclosed nucleic acids are composed of, for example, nucleotides, nucleotide analogs, or nucleotide substitutions. Non-limiting examples of these and other molecules are discussed herein. It will be understood that, for example, when the vector is expressed in a cell, the expressed mRNA will typically consist of A, C, G and U. It will also be appreciated that it would be advantageous, for example, if an antisense molecule were introduced into a cell or cellular environment by, for example, exogenous delivery, the antisense molecule being comprised of nucleotide analogs that reduce degradation of the antisense molecule in the cellular environment.

Nucleotides are molecules that contain a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together by creating internucleoside linkages through their phosphate and sugar moieties. The base portion of the nucleotide may be adenin-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U) and thymine-1-yl (T). The sugar portion of the nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is a pentavalent phosphate. Non-limiting examples of nucleotides are 3 '-AMP (3' -adenosine monophosphate) or 5 '-GMP (5' -guanosine monophosphate).

Nucleotide analogs are nucleotides that contain some type of modification to the base, sugar, or phosphate moiety. Modifications to the base moiety include natural and synthetic modifications to A, C, G and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl (. psi.), hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo is in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other base modifications can be found in, for example, U.S. Pat. Nos. 3,687,808; englisch et al, Angewandte Chemie (applied chemistry), International Edition, 1991, 30, 613 and Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-. Certain nucleotide analogs, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine, can increase the stability of duplex formation. Many times, modifications of bases can be combined with modifications of, for example, sugars, such as 2' -O-methoxyethyl, to achieve unique properties, such as increased duplex stability. There are numerous U.S. patents such as 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617 and 5,681,941 which describe in detail the scope of base modifications. Each of these patents is incorporated herein by reference.

Nucleotide analogs may also include modifications of the sugar moiety. Modifications of sugar moietiesDecorations include natural modifications to ribose and deoxyribose as well as synthetic modifications. Modifications of the sugar include, but are not limited to, the following at the 2' position: OH, F, O-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁To C₁₀Alkyl or C₂To C₁₀Alkenyl and alkynyl groups. 2' sugar modifications also include, but are not limited to, -O [ (CH)₂)_nO]_mCH₃、-O(CH₂)_nOCH₃、-O(CH₂)_nNH₂、-O(CH₂)_nCH₃、-O(CH₂)_n-ONH₂and-O (CH)₂)_nON[(CH₂)_nCH₃)]₂Wherein n and m are from 1 to about 10.

Other modifications at the 2' position include, but are not limited to: c₁To C₁₀Lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH₃、OCN、Cl、Br、CN、CF₃、OCF₃、SOCH₃、SO₂CH₃、ONO₂、NO₂、N₃、NH₂Heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties of the oligonucleotide, or groups that improve the pharmacodynamic properties of the oligonucleotide, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly at the 3 'terminal nucleotide or at the 3' position of the sugar and at the 5 'position of the 5' terminal nucleotide in 2 '-5' linked oligonucleotides. Modified sugars also include those containing a modification on the oxygen of the bridging ring, such as CH ₂And a sugar of S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are a number of U.S. patents that teach the preparation of these modified sugar structures, such as 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 519,134, 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, each of which is incorporated herein by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, modified phosphate groups that can be modified such that the bond between two nucleotides contains: thiophosphates, chiral thiophosphates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates include 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3' -amino phosphoramidates and aminoalkyl phosphoramidates, thiocarbonyl alkylphosphonates, thiocarbonyl alkylphosphotriesters, and borane phosphates. It is understood that the linkages of these phosphates or modified phosphates between two nucleotides may be via a 3 '-5' linkage or a 2 '-5' linkage, and that the linkages may contain reversed polarity such as a 3 '-5' to 5 '-3' or a 2 '-5' to 5 '-2'. Various salt, mixed salt and free acid forms are also included. Numerous U.S. patents teach how to make and use nucleotides containing modified phosphates and include, but are not limited to, 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361 and 5,625,050, each of which is incorporated herein by reference.

It will be appreciated that a nucleotide analogue need only contain a single modification, but may also contain multiple modifications within a moiety or between different moieties.

Nucleotide substitutes are molecules that have similar functional properties as nucleotides, but do not contain a phosphate moiety, such as Peptide Nucleic Acids (PNA). Nucleotide substitutes are molecules that recognize nucleic acids in Watson-Crick (Watson-Crick) or Hoogsteen fashion, but are linked together by moieties other than phosphate moieties. Nucleotide substitutions are capable of forming structures conforming to the duplex type when interacting with an appropriate target nucleic acid.

Nucleotide substitutions are nucleotides or nucleotide analogs in which the phosphate moiety and/or sugar moiety have been replaced. Nucleotide substitutions do not contain the standard phosphorus atom. The substitution of the phosphate may be, for example, a short chain alkyl or cycloalkyl internucleoside linkage, a mixed heteroatom and alkyl or cycloalkyl internucleoside linkage, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include substitutions having the following composition: morpholino linkages (a portion formed from the sugar portion of a nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, methylacetyl (formacetyl) and thiometacetyl backbones, methylene methylacetyl and thiometacetyl backbones, olefin-containing backbones, sulfamic acid backbones, methylene imino and methylene hydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S and CH ₂A fraction of the ingredient. A number of U.S. patents disclose how to make and use these types of phosphoric acid substitutes, including, but not limited to, 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, each of which is incorporated herein by reference.

It will also be appreciated that in nucleotide substitutions, both the sugar and phosphate moieties of the nucleotide may be replaced, for example by amide type linkages (aminoethylglycine) (PNA). U.S. Pat. Nos. 5,539,082, 5,714,331 and 5,719,262, each of which is incorporated herein by reference, teach the manufacture and use of PNA molecules. (see also Nielsen et al, Science, 1991, 254, 1497-.

It is also possible to attach other types of molecules (conjugates) to the nucleotides or nucleotide analogs to enhance, for example, cellular uptake. The conjugate may be chemically linked to a nucleotide or nucleotide analog. Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, bioorg. Med. chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-trithiol (Manoharan et al, Ann. N.Y.Acad. Sci., 1992, 660, 306-309; Manoharan et al, bioorg. Med. chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20, 533-538), fatty chains such as dodecanediol or undecane residues (Saison-Behmoars et al, EMJ. Manohuas, 1991, 10, 1111; FErnovat. 1118, 1990, cetyl alcohol phosphonate et al, cetyl alcohol phosphonate, DL. K-259-33-DL. dl. cetyl alcohol, DL. K et al, Sjorang, Sjoranol et al, Sjoranol, Sjo, tetrahedron lett, 1995, 36, 3651-; shear et al, Nucl. acids Res., 1990, 18, 3777-. Many U.S. patents teach the preparation of such conjugates, including, but not limited to, U.S. patent nos.4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,138,045, 5,545,730, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,545,730, 366972, 365972, 5,545,730, 365972, and each of these patents are incorporated herein by reference.

The Watson-Crick interaction is at least one interaction on the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The watson-crick face of a nucleotide, nucleotide analog or nucleotide substitution includes the C2, N1, and C6 positions of a purine-based nucleotide, nucleotide analog or nucleotide substitution, and the C2, N3, C4 positions of a pyrimidine-based nucleotide, nucleotide analog or nucleotide substitution.

Hoogsteen interaction is an interaction that occurs on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of double-stranded DNA. The Hoogsteen face includes reactive groups (NH) at the N7 and C6 positions of purine nucleotides₂Or O).

i. Nucleic acid sequences

Provided herein are a variety of sequences, some of which are available from Genbank at www.pubmed.gov. Those skilled in the art know how to distinguish between and among sequences and how to adjust the compositions and methods associated with a particular sequence to accommodate other related sequences. Primers and/or probes may be designed for any sequence given the information disclosed herein and known in the art.

hybridization/Selective hybridization

The term hybridization generally refers to a sequence-driven interaction between at least two nucleic acid molecules, e.g., primers or probes, and a gene. Sequence-driven interactions refer to interactions that occur in a nucleotide-specific manner between two nucleotides or nucleotide analogs or nucleotide derivatives. For example, the interaction of G with C or a with T is a sequence driven interaction. Typically, sequence-driven interactions occur on the Watson-Crick face or Hoogsteen face of a nucleotide. Hybridization of two nucleic acids is affected by many conditions and parameters known to those skilled in the art. For example, salt concentration, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.

The parameters for selective hybridization between two nucleic acid molecules are well known to those skilled in the art. For example, in certain embodiments, selective hybridization conditions may be defined as stringent hybridization conditions. For example, stringency of hybridization is controlled by the temperature and salt concentration of either or both of the hybridization and wash steps. For example, hybridization conditions to achieve selective hybridization can include hybridization in a high ionic strength solution (6X SSC or 6X SSPE), at a temperature about 12-25 ℃ below Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners), followed by washing at a combination of temperature and salt concentration selected such that the washing temperature is about 5 ℃ to about 20 ℃ below Tm. Temperature and salt conditions can be readily determined empirically in preliminary experiments in which a reference DNA sample immobilized on a filter membrane is hybridized with labeled target nucleic acid and then washed under conditions of varying stringency. For DNA-RNA and RNA-RNA hybridization, the hybridization temperature is generally higher. Stringency can be achieved using conditions described above or as known in the art. (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., "Molecular Cloning: A Laboratory Manual (second edition), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al, Methods enzymol. 1987: 154: 367, 1987, to which reference is made at least to the materials involved in nucleic acid hybridization). DNA hybridization conditions of the preferred stringency can be 6X SSC or 6X SSPE in about 68 degrees C (in aqueous solution) hybridization, then at about 68 degrees C washing. Stringency of hybridization and washing can be reduced, if desired, according to the reduction in complementarity desired, and will depend, inter alia, on the abundance of G-C or A-T in any region in which search variability is sought. Likewise, stringency of hybridization and washing can be increased, if desired, according to the increase in homology desired, and, in addition, depending on the abundance of G-C or A-T in any region where high homology is desired, as is known in the art.

Another method for determining selective hybridization isThe amount (percentage) of one nucleic acid bound to another nucleic acid is examined. For example, in certain embodiments, the selective hybridization conditions will be conditions under which at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% of the constrained nucleic acid binds to the unconstrained nucleic acid. Typically, the unlimited primer will be in excess, e.g., 10 or 100 or 1000 fold. This type of analysis can be performed with both constrained and unconstrained primers compared to their Ks_dE.g.10-fold or 100-fold or 1000-fold lower, or only one nucleic acid molecule 10-fold or 100-fold or 1000-fold lower, or one or both nucleic acid molecules have a higher K than that of the other_dUnder the conditions of (1).

Another method of determining selective hybridization is by examining the percentage of primers that achieve enzymatic manipulation under conditions that require hybridization to facilitate the desired enzymatic manipulation. For example, in certain embodiments, the selective hybridization conditions are conditions under which at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% of the primers are enzymatically manipulated under conditions that facilitate enzymatic manipulation, e.g., if the enzymatic manipulation is DNA extension, the selective hybridization conditions will be conditions under which at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% of the primer molecules are extended. Preferred conditions also include conditions suggested by the manufacturer, or specified in the art as being suitable for the enzyme to perform the operation.

As with homology, it is understood that various methods for determining the level of hybridization between two nucleic acid molecules are disclosed herein. It will be appreciated that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated, it is sufficient to satisfy the parameters of any method. For example, if 80% hybridization is desired, hybridization is considered disclosed herein as long as it occurs within the parameters required for any one of these methods.

It is understood that a person skilled in the art understands that a composition or method as disclosed herein is a composition or method if it, taken together or alone, meets any of these criteria for determining hybridization.

Functional nucleic acids

Functional nucleic acids are nucleic acid molecules that have a specific function, e.g., bind to a target molecule or catalyze a specific reaction. Functional nucleic acid molecules are classified into the following categories, which are not meant to be limiting. For example, functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, RNAi and external guide sequences. Functional nucleic acid molecules can act as influencers, inhibitors, modulators, and stimulators of a particular activity possessed by a target molecule, or functional nucleic acid molecules can possess novel activities independent of any other molecule.

The functional nucleic acid molecule is capable of interacting with any macromolecule, such as DNA, RNA, a polypeptide, or a hydrocarbon chain. Thus, a functional nucleic acid can interact with the mRNA, genomic DNA, or polypeptide of any of the targeting signals or receptors thereof disclosed herein. Functional nucleic acids are typically designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other cases, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather on the formation of a tertiary structure that allows specific recognition to occur.

Antisense molecules are designed to interact with a target nucleic acid molecule through regular or irregular base pairing. The interaction of the antisense molecule with the target molecule is designed to facilitate degradation of the target molecule by, for example, RNAseH mediated degradation of the RNA-DNA hybrid. Alternatively, antisense molecules are designed to disrupt a processing function that would normally occur on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. About passing discovery of targetThere are numerous approaches to optimizing antisense efficacy by the most accessible regions of the molecule. Exemplary methods are in vitro selection experiments and DNA modification studies using DMS and DEPC. Preferably, the dissociation constant (k) for binding of the antisense molecule to the target molecule _d) Less than or equal to 10^-6、10^-8、10^-10Or 10^-12. Representative examples of methods and techniques that facilitate the design and use of antisense molecules can be found in the following non-limiting list of U.S. patents: 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.

Aptamers (aptamers) are molecules that preferably interact in a specific way with a target molecule. Typical aptamers are small nucleic acids 15-50 bases in length, folded into defined secondary and tertiary structures, such as stem-loop structures or G-quartets. Aptamers can bind small molecules such as ATP (U.S. Pat. No. 5,631,146) and theophylline (U.S. Pat. No. 5,580,737), as well as macromolecules such as reverse transcriptase 1 (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). The aptamers can bind very tightly to the target molecule, having a size of less than 10^-12K of (a)_d. Preferably, the aptamer binds to the target molecule_dLess than 10^-6、10^-8、10^-10Or 10^-12. Aptamers can bind to target molecules with a very high degree of specificity. For example, aptamers have been isolated that differ by more than a factor of 10000 in binding affinity between a target molecule and another molecule that differs only at a single position of the molecule (U.S. Pat. No. 5,543,293). Preferably, k of the aptamer with the target molecule _dK to background binding molecule_dAt least 10, 100, 1000, 10,000, or 100,000 times lower. Preferably, when comparing e.g. polypeptides, the background molecule is a different polypeptide. Representative examples of how aptamers can be made and used to bind a variety of different target molecules can be found in the following non-limiting list of U.S. patents:5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776 and 6,051,698.

Ribozymes are nucleic acid molecules that are capable of catalyzing an intramolecular or intermolecular chemical reaction. Thus, ribozymes are catalytic nucleic acids. Preferably, the ribozyme catalyzes an intermolecular reaction. Based on ribozymes found in natural systems, there are many different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions, such as hammerhead ribozymes (e.g., without limitation, U.S. Pat. Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621, 5,989,908, 5,998,193, 5,998,203, WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO9718312 by Ludwig and Sproat), hairpin ribozymes (e.g., without limitation, U.S. Pat. Nos. 5,115, 5,031,631, 5,683,683,712, 5,188,867, 5,856,869, and Tetrakis,595 (e.g, and U.g., without limitation, U.S. Pat. Nos. No. 5,339,595,595,107), and 5,595 (e.S. Pat. Nos. No. 5,595). There are also many ribozymes that are not found in natural systems, but which have been engineered to re-catalyze specific reactions (e.g., without limitation, the following U.S. Pat. Nos.: 5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, but more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to a target substrate, followed by cleavage. This recognition is often based primarily on regular or irregular base pair interactions. This property makes ribozymes particularly good candidates for target-specific cleavage of nucleic acids, since the recognition of the target substrate is based on the sequence of the target substrate. Representative examples of how ribozymes can be made and used to catalyze a variety of different reactions can be found in the following non-limiting list of U.S. patents: 5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

Functional nucleic acid molecules that form triplexes are molecules that are capable of interacting with double-stranded or single-stranded nucleic acids. When a triplex molecule interacts with a target region, a structure called a triplex is formed in which the three strands of DNA form a complex depending on watson-crick and Hoogsteen base pairing. Triplex molecules are preferred because they are capable of binding to the target region with high affinity and specificity. Preferably k, wherein the triplex forming molecule binds to the target molecule_dLess than 10^-6、10^-8、10^-10Or 10^-12. Representative examples of how triplex forming molecules can be made and used to bind a variety of different target molecules can be found in the following non-limiting list of U.S. patents: 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566 and 5,962,426.

External Guide Sequences (EGSs) are molecules that bind to a target nucleic acid molecule to form a complex, which complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target selected RNA molecules. RNAse P helps to process transport RNA (tRNA) in the cell. By using EGS to cause target RNA-EGS complexes mimic the native tRNA substrate, bacterial RNAse P can be recruited to cleave virtually any RNA sequence. (Yale, WO 92/03566, and Forster and Altaian, Science 238: 407-.

Likewise, eukaryotic EGS/RNAse P-directed RNA cleavage can be used to cleave a desired target within a eukaryotic cell. (Yuan et al, Proc. Natl. Acad. Sci. USA 89: 8006-8010 (1992); Yale WO 93/22434; Yale WO 95/24489; Yuan and Altaian, EMBO J14: 159-. Representative examples of how EGS molecules can be made and used to facilitate cleavage of a variety of different target molecules can be found in the following non-limiting list of U.S. patents: 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248 and 5,877,162.

Gene expression can also be effectively silenced in a highly specific manner by RNA interference (RNAi). This silencing was originally observed with the addition of double stranded RNA (dsRNA) (Fire, A. et al, (1998) Nature, 391, 806811) (Napoli, C et al, (1990) plantaCell 2, 279289) (Hannon, GJ. (2002) Nature, 418, 244251). Once the dsRNA enters the cell, it is cleaved by the RNase III-like enzyme Dicer into double-stranded small interfering RNAs (siRNAs) 21-23 nucleotides in length with a 2 nucleotide overhang at the 3' end (Elbashir, S.M. et al, (2001) Genes Dev., 15: 188) -200 (Bernstein, E. et al, (2001) Nature, 409, 363366) (Hammond, S.M. et al, (2000) Nature, 404: 293-296). In an ATP-dependent step, siRNAs are integrated into a multi-subunit protein complex, commonly referred to as the RNAi-induced silencing complex (RISC), which directs the siRNAs to target RNA sequences (Nykanen, A. et al, (2001) Cell, 107: 309321). The siRNA duplex is cleaved at some point and it appears that the antisense strand remains bound to RISC and directs the degradation of complementary mRNA sequences by a combination of endonucleases and exonucleases (Martinez, J., et al, (2002) Cell, 110: 563-. However, the action of the siRNA or its use is not limited to any type of mechanism.

Nucleic acids useful for RNAi or RNA interference are also disclosed. RNAi is believed to involve a two-step mechanism of RNA interference (RNAi): an initiation step and an effector step. For example, in a first step, input double-stranded (ds) RNA (siRNA) is processed into small fragments, such as "leader sequences" of 21-23 nucleotides. RNA replication appears to be able to occur throughout the animal. Typically, then, the guide RNAs can be integrated into a protein RNA complex capable of degrading RNA, a nuclease complex, which has been termed the RNA-induced silencing complex (RISC). In the second effector step, the RISC complex acts to destroy mRNAs recognized by the guide RNA through base pairing interactions. RNAi involves the introduction of double-stranded RNA into a cell by any means, thereby triggering an event that causes degradation of the target RNA. RNAi is a form of post-transcriptional gene silencing. RNA hairpins capable of functioning in RNAi are disclosed. For a description of the manufacture and use of RNAi molecules see, e.g., Hammond et al, Nature Rev Gen 2: 110-119 (2001); sharp, Genes Dev 15: 485- & ltSUB & gt 490 (2001); waterhouse et al, Proc. Natl. Acad. Sci. USA 95 (23): 13959-.

RNAi has been shown to function in a variety of cells, including mammalian cells. In order to function in mammalian cells, it is preferred that the RNA molecules to be used as targeting sequences within the RISC complex be relatively short. For example, less than or equal to 50 or 40 or 30 or 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. These RNA molecules may also have overhangs at the 3 'or 5' end relative to the target RNA to be cleaved. These overhangs may be at least or less than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nucleotides in length. RNAi plays a role in mammalian stem cells, such as mouse ES cells.

Short interfering RNAs (sirnas) are double-stranded RNAs capable of inducing sequence-specific post-transcriptional gene silencing, thereby reducing or even inhibiting gene expression. In one example, siRNA triggers specific degradation of homologous RNA molecules, such as mRNAs, in regions of sequence identity between the siRNA and the target RNA. For example, WO 02/44321, which is incorporated herein by reference, discloses that siRNAs are capable of sequence-specific degradation of target mRNAs when paired with 3' overhanging terminal bases. In mammalian cells, sequence-specific gene silencing can be achieved using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir, S.M. et al, (2001) Nature, 411: 494-498) (Ui-Tei, K. et al, (2000) FEBS Lett 479: 79-82). siRNA may be chemically or synthesized in vitro, or may be the result of the processing of short double-stranded hairpin rnas (shrnas) into siRNAs within the cell. Synthetic siRNAs are typically designed using algorithms and conventional DNA/RNA synthesizers. Suppliers include Ambion (Tex.), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands). siRNAs can also be synthesized in vitro using kits, such as Ambion's SILENCER siRNA construction kit. Disclosed herein are any sirnas designed as described above, based on the sequences of inflammatory mediators disclosed herein.

More often, siRNA production from a vector is performed by transcription of shRNA. Kits for producing vectors containing shRNA are readily available, such as the GeneSuppressor construction kit by Imgenex and BLOCK-iT inducible RNAi plasmid and lentiviral vectors by Invitrogen. Disclosed herein are any shRNA designed as described above, based on the sequence of the inflammatory mediators disclosed herein.

A vector

The transformation vector may be any nucleotide construct (e.g., a plasmid) used to deliver the gene into a cell, or as part of an overall strategy for delivering the gene, e.g., as part of a recombinant retrovirus or adenovirus (Ram et al, Cancer Res.53: 83-88, (1993)).

A plasmid or viral vector, as used herein, is an agent that transports a disclosed nucleic acid, e.g., a nucleic acid encoding scFvs, into a cell without degradation, and comprises a promoter that produces gene expression in the cell into which it is delivered. Viral vectors are, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus, Sindbis virus (Sindbis) and other RNA viruses, including those having an HIV backbone. Also preferred are any virus families that share properties with these viruses that make them suitable for use as vectors. Retroviruses include the moloney murine leukemia virus MMLV, as well as retroviruses that exhibit the desirable properties of MMLV as a vector. Retroviral vectors are commonly used because they are capable of carrying a larger genetic load, i.e., transgene or marker gene, than other viral vectors. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to handle, have high titers, and can be delivered in aerosol formulations and can infect non-dividing cells. Poxvirus vectors are large, have several loci for insertion of genes, are thermostable, and can be stored at room temperature. A preferred embodiment is a viral vector that has been engineered to suppress the immune response of a host organism elicited by a viral antigen. Preferred vectors of this type will carry coding regions for interleukin 8 or 10.

The viral vector may have a higher processing ability (ability to introduce a gene) than a chemical or physical method of introducing a gene into a cell. Typically, viral vectors contain non-structural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and encapsidation, and promoters that control transcription and replication of the viral genome. When engineered into a vector, the virus is typically depleted of one or more early genes, and a gene or gene/promoter cassette is inserted into the viral genome in place of the depleted viral DNA. Constructs of this type can carry up to about 8kb of exogenous genetic material. The necessary functions of the removed early genes are typically provided by cell lines that have been engineered to express the gene products of the early genes in trans.

v. retroviral vector

Retroviruses are animal viruses belonging to the family retroviridae, including any type, subfamily, genus or tropism. Retroviral vectors are generally described by Verma, I.M. in "Retroviral vectors for Gene transformation" in Microbiology-1985 (Retroviral vector for gene transfer. in Microbiology-1985, American Society for Microbiology, pp.229-232, Washington, (1985)), incorporated herein by reference. Examples of methods for gene therapy using retroviruses are described in U.S. Pat. Nos.4,868,116 and 4,980,286, PCT applications WO 90/02806 and WO 89/07136, and Mullgan (Science 260: 926-932(1993)), the teachings of which are incorporated herein by reference.

Retroviruses are essentially packages in which a nucleic acid cargo is packaged. The accompanying nucleic acid cargo carries a packaging signal which ensures that the replicated progeny molecules will be efficiently packaged into the packaging envelope. In addition to the packaging signal, many molecules in cis are required for replication and packaging of the replicated virus. Typically, the retroviral genome contains gag, pol and env genes, which are involved in the production of the protein coat. In general, it is the gag, pol and env genes that are replaced by the foreign DNA to be transferred into the target cell. Retroviruses generally contain: a packaging signal for integration into the packaging capsid, sequences that provide signals for initiation of the gag transcription unit, elements necessary for reverse transcription including a primer binding site for binding to a reverse transcribed tRNA primer, terminal repeat sequences that direct the switching of RNA strands during DNA synthesis, purine-rich 5 'to 3' LTR sequences that serve as priming sites for second strand synthesis in DNA synthesis, and specific sequences near the ends of the LTRs that enable insertion of a retroviral DNA state insert into the host genome. Removal of the gag, pol and env genes allows an approximately 8kb foreign sequence to be inserted into the viral genome, become reverse transcribed, and be packaged into a new retroviral particle upon replication. Depending on the size of each transcript, the amount of nucleic acid is sufficient for delivery of one to multiple genes. Preferably, the insert contains, along with the other genes, a positive or negative selectable marker.

Since the replication machinery and packaging proteins have been removed in most retroviral vectors (gag, pol and env), vectors are typically generated by placing them in a packaging cell line. Packaging cell lines are cell lines that have been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When vectors carrying the selected DNA are transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles by a mechanism provided in cis by the helper cell. The genomes of the mechanisms are not packaged because they lack the necessary signals.

Adenovirus vectors

The construction of replication-defective adenoviruses has been described (Berkner et al, J.virology 61: 1213-1220 (1987); Massie et al, mol.cell.biol.6: 2872-2883 (1986); Haj-Ahmad et al, J.virology 57: 267-274 (1986); Davidson et al, J.virology 61: 1226-1239 (1987); Zhang "Generation and identification of recombinant adenoviruses by lipolysis-mediated transfection and PCRalanylisis" (Generation and identification of recombinant adenoviruses by liposome-mediated transfection and PCR analysis), Biotechnicques 15: 868-872 (1993)). The advantage of using these viruses as vectors is that their ability to spread to other cell types is limited because they can replicate in the originally infected cell, but cannot form new infectious virions. It has been shown that recombinant adenoviruses are delivered directly in vivo to the airway epithelium, hepatocytes, high efficiency Gene transfer has been achieved following vascular endothelium, CNS parenchyma and many other tissue sites (Morsy, J.Clin.Invest.92: 1580-1586 (1993); Kirshenbaum, J.Clin.Invest.92: 381-387 (1993); Rosesser, J.Clin.Invest.92: 1085-1092 (1993); Moullier, Nature Genetics 4: 154-159 (1993); La Salle, Science 259: 988-990 (1993); Gomez-Foix, J.biol.chem.267: 25129-25134 (1992); Rich, Human Therapy 4: 461-476 (1993); Zabner, Nature Genetics 6: 75-83 (1994); Guzman, Circulation Research 73: 1993); the Gene Therapy 1207 (1993); Gene Therapy J.10: 75-83; Gene Therapy J.1993); J.75-75-83 (1993); gradient 3575: 75-75, J.75-12, 1993); Eur J.75: 75-04, J.103, J.1993); J.103, J.. The recombinant adenovirus is internalized by binding to a specific Cell surface receptor and the virus is then internalized by receptor-mediated endocytosis in the same manner as wild-type or replication-defective adenoviruses (Chardonnet and Dales, Virology 40: 462-477 (1970); Brown and Burlingham, J.virology 12: 386-396 (1973); Svensson and Persson, J.virology 55: 442-449 (1985); Seth et al, J.virology.51: 650-655 (1984); Seth et al, mol.cell.biol.4: 8-1533 (1984); Varga et al, J.virology 65: 6061-6070 (1991); Wickham et al, 73: 309-319 (1993)).

The viral vector may be an adenovirus-based vector in which the E1 gene has been removed, these viral particles being produced in a cell line, such as the human 293 cell line. In another preferred embodiment, both the E1 and E3 genes are removed from the adenovirus genome.

Adeno-associated virus vectors

Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it is capable of infecting many cell types and is not pathogenic to humans. AAV-type vectors can transport approximately 4 to 5kb, and wild-type AAV is known to stably insert into chromosome 19.

Adeno-associated viruses (AAV) are members of the parvoviridae family, which is characterized by a single-stranded linear DNA genome, and an icosahedral-shaped small capsid measuring approximately 20nm in diameter. AAV was first described as a contaminant of tissue cultures for the culture of simian virus 15, a simian adenovirus, and was found to be dependent on adenovirus for measurable replication. This has led to its name, adeno-associated virus, and its classification in the genus dependent virus (dependenvirus) (reviewed in Hoggan, m.d. prog Med Virol 12(1970) 211-39). AAV is a common contaminant of adenoviral samples and has been isolated from human viral samples (AAV2, AAV3, AAV5), simian virus 15 infected cell samples (AAV1, AAV4), and avian (AAAV) (Bossis, i. and Chiorini, j.a.j Virol 77(2003) 6799-. DNA spanning the entire rep-cap ORFs of AAV7 and AAV8 was amplified by PCR from heart tissue of rhesus monkeys (Gao, G.P. et al, Proc Natl Acad Sci USA 99(2002) 11854-9). All cloned AAV isolates, except AAVs 1 and 6, showed serological differences. 9 isolates have been cloned and a recombinant virus stock is generated from each isolated virus.

AAV2 is the best characterized adeno-associated virus and will be discussed in terms of AAV prototypes. The genome of AAV2 consists of 4780 nucleotides of linear single-stranded DNA. Both poles of the DNA are packaged with AAV at equal efficiency. The AAV2 genome contains 2 Open Reading Frames (ORFs), named rep and cap. The rep ORF encodes a non-structural protein that is essential for viral DNA replication, packaging and AAV integration. The cap ORF encodes the viral capsid protein. The rep ORF is transcribed from the promoters located in map units P5 and P19. The rep transcript contains an intron near the 3' end of the rep ORF, which can be alternatively spliced. Thus, the Rep ORF is expressed as 4 partially overlapping proteins, referred to by their molecular weights as Rep78, 68, 52 and 40. The cap ORF is expressed from a single promoter located at P40. By alternative splicing and using an alternative ACG start codon, cap is expressed as capsid protein VP 1-3, which ranges in size from 65-86 kDa. VP3 is the most abundant capsid protein, accounting for 80% of the AAV2 capsid. All viral transcripts terminate at the polyA signal at graph unit 96.

During infection with productive AAV2, unspliced mRNAs encoding Rep78, starting from the p5 promoter, were the first detectable viral transcripts. During infection, expression increased from P5, P19 and P40 to 1: 3: 18, respectively. The level of spliced transcripts increased to 50% for the P5, P19 products and 90% for the RNA expressed in P40 (Mouw, M.B. and Pintel, DJ.J Virol 74(2000) 9878-88).

The AAV2 genome ends on both sides with Inverted Terminal Repeats (ITRs) of 145 nucleotides (nt). 125 nt of ITR constitutes a palindromic sequence, which contains 2 internal palindromic sequences of 21 nt each. The ITR can fold back on itself to create a T-type hairpin of only 7 unpaired bases. The stem of the ITR contains the Rep Binding Site (RBS), and the terminal dissociation site (TRS), a sequence that is cleaved site-and strand-specifically by Rep. The ITRs are essential for replication, integration of the AAV2 genome, and contain packaging signals.

The single-stranded AAV2 genome is packaged into an icosahedral-shaped capsid without an outer membrane, approximately 20-25nm in diameter. The virus particles were composed of 26% DNA and 74% protein, and had a density of 1.41g/cm³. AAV2 particles were extremely stable, able to withstand heating at 60 ℃ for 1 hour, extreme pH, and extraction with organic solvents.

The Rep proteins are involved in almost every step of AAV2 replication, including AAV2 genomic replication, integration, and packaging. Rep78 and Rep68 have ATPase, 3 '-5' helicase, ligase, and cleavage activity, and specifically bind to DNA. Rep52 and Rep40 were shown to be involved in the encapsulation process and to encode ATPase and 3 '-5' helicase activities. Mutation analysis indicates the domain structure of Rep 78. The 225 amino acids of the N-terminus are involved in DNA binding, DNA cleavage and ligation. Rep78 and Rep68 recognized ITRs and the GCTC repeat motif in linearly truncated forms of ITRs with similar efficiency (Chiorini, J.A. et al, J Virol 68(1994) 7448-57). Rep78 and Rep68 have sequence and strand specific endonuclease activity that cleaves ITRs at terminal dissociation sites (TRSs). The endonuclease activity of Rep depends on the hydrolysis of nucleoside triphosphates and the presence of metal cations. Rep78 and 68 are also capable of binding and cleaving single-stranded DNA in an NTP-independent manner. In addition, Rep78 catalyzes the religation of single-stranded DNA substrates derived from the replication origin of AAV2, i.e., sequences containing Rep binding and terminal dissociation elements.

The central region of AAV2Rep78, representing the N-terminus of Rep52 and Rep40, contains ATPase and 3 '-5' helicase activities as well as nuclear localization signals. Helicase activity unravels DNA-DNA and DNA-RNA duplexes, but not RNA-RNA. ATPase activity is constitutive and independent of DNA substrate. The C-terminus of Rep78 contains a potential zinc finger domain that can be used to inhibit the cellular serine/threonine kinase activity of PKA and its homologue PRKX by pseudosubstrate inhibition. Rep68 results from the translation of a spliced mRNA encoding the N-terminal 529 amino acids of Rep78 fused to the unique 7 amino acids (aa) of Rep68, Rep68 not inhibiting PKA or PRKX. In addition to these biochemical activities, Rep can affect intracellular conditions through protein-protein interactions. Rep78 binds to a variety of cellular proteins, including transcription factors such as SP-1, high mobility group non-histone protein 1(HMG-1), and oncostatin p 53. Overexpression of Rep leads to pleiotropic effects. Rep78 disrupts cell cycle progression and inhibits transformation of cellular and viral oncogenes. In susceptible cell lines, overexpression of Rep leads to apoptosis and cell death. Several activities of Rep78 contribute to cytotoxicity, including its constitutive ATPase activity, interfering with cellular gene expression and protein interactions.

The first step in AAV infection is binding to the cell surface. Receptors and co-receptors for AAV2 include heparan sulfate proteoglycans, fibroblast growth factor receptor-1, and α v β 5 integrin, but require N-linked 2, 3-linked sialic acid for binding and transduction of AAV5 (Walters, r.w. et al, J Biol Chem 276(2001) 20610-6). In HeLa cells, fluorescently labeled AAV2 particles were shown to enter cells via receptor-mediated endocytosis in clathrin-coated pits. Within 10 minutes post infection, more than 60% of the bound virus was internalized. It was observed that labeled AAV particles had escaped from endosomes, transported through the cytoplasm to the nucleus, and accumulated around the nucleus before entering the nucleus, possibly through the Nuclear Pore Complex (NPC). AAV2 particles have been detected in the nucleus, indicating that uncoating occurs in the nucleus (Bartlett et al, J Virol 74(2000) 2777-85; Sanlioglu et al, J Virol 74(2000) 9184-96). AAV5 was internalized primarily by clathrin-coated vesicles in HeLa cells, but also in small uncoated pockets. AAV particles are also transported intercellularly via the Golgi apparatus (Bantel-Schaal, U.S. et al, J Virol 76(2002) 2340-9). Indicating that at least a portion of AAV5 uncoating occurred prior to entry into the nucleus, as intact AAV5 particles could not be detected in the nucleus (Bantel-Schaal et al, 2002). After dehulling, the single-stranded genome is converted into duplex DNA either by leader strand synthesis or by annealing of input DNA of opposite polarity. Replication of AAV occurs in the nucleus.

AAV is capable of efficient productive replication during coinfection with a helper virus, such as adenovirus, herpes simplex virus, or cytomegalovirus. The helper viral functions provided by adenoviruses have been studied in great detail. Early adenoviral gene products of E2A, E4, and VA sufficient to allow replication of recombinant AAV were found in human embryonic kidney 293 cells that constitutively express the adenoviral genes E1A and E1B. Allen et al reported that efficient production of rAAV was possible in 293 cells transfected with only the E4orf6 expression plasmid (Allen, J.M. et al, MolTher1(2000) 88-95). E1A stimulates entry into the S phase and inactivates the pRB restriction site at the G1/S boundary by interacting with proteins of the pRB family leading to the release of E2F, inducing extra-phase DNA synthesis (reviewed in Ben-Israel, h. and Kleinberger, t.frontbiosci 7(2002) D1369-95). This results in the induction or activation of enzymes involved in nucleotide synthesis and DNA replication. Since extraterm DNA synthesis is a strong apoptosis signal, anti-apoptotic function is required. E1B-19k is a homolog of Bcl-2, E1B-55k is an antagonist of p 53. Both proteins have anti-apoptotic functions. E4orf6 complexed with E1B-55k, resulting in degradation of p 53. It has also been reported to cause S phase arrest (Ben-Israel and Kleinberger, 2002). E2A encodes a single-stranded DNA binding protein that does not appear to be essential for DNA replication, but affects gene expression (Chang and Shenk, J Virol 64(1990) 2103-9). The VA transcription unit affects the stability and translation of AAV2RNA (Janik et al, Virology 168(1989) 320-9). E1A has a more direct effect on AAV2 gene expression. The cellular transcription factor YY-1 binds to and represses the viral P5 promoter. E1A relieved this transcriptional blockade. Late Ad gene products have not been found to be essential for replication of AAV 2. The primary function of helper viruses appears to be to create a cellular environment with active DNA replication mechanisms and blocked pro-apoptotic functions to allow high levels of AAV replication, rather than direct involvement in AAV replication.

viral vectors for large payloads

Molecular genetic experiments using large human herpesviruses provide a means by which large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et al, Nature genetics 8: 33-41, 1994; Cotter and Robertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses, Herpes Simplex Virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver > 150kb of human heterologous DNA fragments into specific cells. EBV recombinants are able to maintain large stretches of DNA in infected B cells as episomal DNA. Individual clones carrying up to 330kb of human genomic insert showed genetic stability. Maintenance of these episomes requires a specific EBV nucleoprotein, EBNA1, which is constitutively expressed during infection with EBV. Furthermore, these vectors can be used for transfection, where large amounts of protein can be produced in vitro for short periods of time. The herpes virus amplicon system was also used to package DNA fragments > 220kb and to infect cells that stably maintain DNA as episomes.

Other useful systems include, for example, replicative and host-restricted non-replicative vaccinia virus vectors.

Non-nucleic acid based systems

The disclosed compositions can be delivered to a target cell in a variety of ways. For example, the composition may be delivered by electroporation, or by lipofection, or by calcium phosphate precipitation. The choice of delivery mechanism will depend in part on the type of target cell and whether delivery is occurring, for example, in vivo or in vitro.

Thus, for example, the composition may comprise lipids, such as liposomes, for example cationic liposomes (e.g. DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can also contain proteins to facilitate targeting to specific cells, if desired. Administration of a composition comprising the compound and cationic liposomes can be to blood flowing into a target organ, or inhaled into the respiratory tract to reach target cells in the respiratory tract. For liposomes, see, e.g., Brigham et al, am.j.resp.cell.mol.biol.1: 95-100 (1989); feigner et al, proc.natl.acad.sci USA 84: 7413-7417 (1987); U.S. patent No.4,897,355. Furthermore, the compounds may be administered as components of microcapsules capable of targeting a particular cell type, such as macrophages, or where the diffusion of the compound or the delivery of the compound in the microcapsules is designed to a particular rate or dose.

In the above-described methods (i.e., gene transduction or transfection) that involve the administration and uptake of exogenous DNA into the cells of a subject, the delivery of the composition to the cells may be by a variety of different mechanisms. For example, administration can be by liposomes, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to standard procedures in the art. In addition, the disclosed nucleic acids or vectors can be delivered into the body by electroporation, a technique available from Genetronics, inc. (San Diego, CA), and by using the sonophoration machine (ImaRx Pharmaceutical corp., Tucson, AZ).

The material may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). They can be targeted to specific cell types by antibodies, receptors, or receptor ligands. The following references are examples of the use of this technique to target specific proteins to tumor tissue (Senter et al, Bioconjugate chem., 2: 447-451, (1991); Bagshawe, K.D., Br.J.cancer, 60: 275-281, (1989); Bagshawe et al, Br.J.cancer, 58: 700-703, (1988); Senter et al, Bioconjugate chem., 4: 3-9, (1993); Battelli, et al, cancer Immunother, 35: 421-425, (1992); Pietrsz and McKenzie, ImmunoLoevews, 129: 57-80, (1992); and Roffler et al, biochem. rmacol 20642: 2-2065, (1991)). These techniques can be used for a variety of other specific cell types. Mediators such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated DNA targeting via cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviruses target murine glioma cells in vivo. The following references are examples of the use of this technique to target specific proteins to tumor tissue (Hughes et al, Cancer Research, 49: 6214-. Generally, receptors are involved in the endocytic pathway either constitutively or ligand-induced. These receptor clusters in clathrin-coated pits enter the cell through clathrin-coated vesicles, pass through acidified endosomes in which the receptors are classified, and then either recycle to the cell surface for intracellular storage or degrade in lysosomes. Internalization pathways serve various functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and modulation of receptor levels. Many receptors participate in more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and concentration of ligand. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10: 6, 399-409 (1991)).

Nucleic acids that are delivered to a cell and integrated into the host cell genome typically contain integration sequences. These sequences are often virus-related sequences, particularly when using virus-based systems. These viral integration systems may also be integrated into the nucleic acid to be delivered using non-nucleic acid based delivery systems, such as liposomes, so that the nucleic acid contained in the delivery system may be integrated into the host genome.

Other common techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. Generally, these systems rely on the sequence flanking the nucleic acid to be expressed having sufficient homology to a target sequence in the host cell genome such that recombination between the vector nucleic acid and the target nucleic acid occurs, resulting in integration of the delivered nucleic acid into the host genome. The systems and methods necessary to promote homologous recombination are known to those skilled in the art.

x. in Vivo/Ex Vivo (Ex Vivo)

As noted above, the compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the cells of a subject in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA by a gene gun, endocytosis, etc.).

If ex vivo methods are used, the cells or tissues can be removed and maintained in vitro according to standard protocols well known in the art. The composition may be introduced into the cell by any gene transfer mechanism, such as calcium phosphate mediated gene delivery, electroporation, microinjection, or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or allelic transplanted back into the subject according to standard methods for cell or tissue type. Standard methods for transplanting or infusing various cells into a subject are known.

xi. expression System

The nucleic acid delivered into the cell typically contains an expression control system. For example, genes inserted into viral and retroviral systems often contain promoters and/or enhancers to help control expression of the desired gene product. Promoters are generally DNA sequences that function at a relatively fixed position at the start site of transcription. Promoters contain core elements required for substantial interaction of RNA polymerase and transcription factors, and may contain upstream and response elements.

a. Viral promoters and enhancers

Preferred promoters for controlling transcription of the vector in mammalian host cells can be obtained from various sources, such as the viral genome, for example: polyoma virus, simian virus 40(SV40), adenovirus, retrovirus, hepatitis B virus and most preferably cytomegalovirus, or a promoter from a heterologous mammal, such as the beta-actin promoter. The early and late promoters of the SV40 virus are readily available as a restriction fragment of SV40, which also contains the viral origin of replication of SV40 (Fiers et al, Nature, 273: 113 (1978)). The immediate early promoter of human cytomegalovirus is readily available as a HindIII E restriction fragment (Greenway, P.J., et al, Gene 18: 355-360 (1982)). Of course, promoters from host cells or related species may also be used herein.

Enhancers generally refer to DNA sequences that do not function at a fixed distance from the transcription start site, but can be in the 5 '(Laimins, L.et al, Proc. Natl.Acad.Sci.78: 993(1981)) or 3' orientation (Lusky, M.L.et al, mol.cell Bio.3: 1108(1983)) of the transcription unit. Furthermore, enhancers may be internal to introns (Banerji, J.L. et al, Cell 33: 729(1983)), as well as in the coding sequence itself (Osborne, T.F. et al, mol.cell Bio.4: 1293 (1984)). They are usually between 10 and 300bp in length, and they act in cis. Enhancers function to enhance transcription from adjacent promoters. Enhancers also typically contain response elements that mediate the regulation of transcription. Promoters may also contain response elements that mediate transcriptional regulation. Enhancers generally determine the regulation of gene expression. Although many enhancer sequences from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are currently known, one will typically use enhancers from eukaryotic cell viruses for universal expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma virus enhancer on the late side of the replication origin, and adenovirus enhancers.

Promoters and/or enhancers can be specifically activated by light or specific chemical events that trigger their function. The system can be modulated with agents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to radiation, such as gamma irradiation, or alkylating chemotherapeutic drugs.

In certain embodiments, the promoter and/or enhancer regions may function as constitutive promoters and/or enhancers to maximize expression of the region of the transcriptional unit that is to be transcribed. In certain constructs, the promoter and/or enhancer region is active in all eukaryotic cell types, even though it is only expressed at a particular time in a particular cell type. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, cytomegalovirus (full-length promoter) and retroviral vector LTR.

It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors for selective expression in specific cell types, such as melanoma cells. Glial Fibrillary Acidic Protein (GFAP) promoters have been used to selectively express genes in cells of glial origin.

Expression vectors for use in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells) may also contain sequences necessary to terminate transcription that may affect expression of the mRNA. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding the tissue factor protein. The 3' untranslated region also contains a transcription termination site. Preferably, the transcriptional unit also contains a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs has been established. Preferably, a homologous polyadenylation signal is used in the transgene construct. In some transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the unit to be transcribed contains other standard sequences, alone or in combination with the above sequences, to increase expression from the construct or stability of the construct.

b. Marking

The viral vector may contain a nucleic acid sequence encoding a marker product. The marker product is used to determine whether the gene is delivered into the cell and is being expressed once delivered. Preferred marker genes are the E.coli lacZ gene, which encodes beta-galactosidase, and green fluorescent protein.

In certain embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for use in mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin analog G418, hygromycin and puromycin. When such a selectable marker is successfully transferred into a mammalian host cell, the transformed mammalian host cell will survive if placed under selective pressure. There are two widely used different classes of selectivity schemes. The first is based on the metabolism of the cell and the use of mutant cells lacking the ability to grow independently of the addition of culture mediumIs described. Two examples are CHO DHFR^-Cell and mouse LTK^-A cell. These cells lack the ability to grow without the addition of nutrients such as thymine or hypoxanthine. Because these cells lack certain genes necessary for an intact nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in the supplemented medium. An alternative to supplementing the medium is to introduce the complete DHFR or TK gene into cells lacking the corresponding gene, thereby altering their growth requirements. Individual cells that have not been transformed with the DHFR or TK gene will not survive in unsupplemented media.

The second category is dominant screening, which refers to screening protocols that are used in any type of cell and do not require the use of mutated cell lines. These protocols typically use drugs to stop the growth of the host cell. Those cells with the novel gene will exhibit drug resistance conferred by the protein and will therefore survive the selection. Examples of such dominant screens use the drugs neomycin (Southern P. and Berg, P., J.Molec.appl.Genet.1: 327(1982)), mycophenolic acid (Mullgan, R.C. and Berg, P.science 209: 1422(1980)) or hygromycin (Sugden, B. et al, mol.cell.biol.5: 410-. These three examples use bacterial genes under eukaryotic cell control to confer resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puromycin.

Pilot signal

Cell migration is involved in a variety of morphogenetic processes, including the formation of blood vessels and neural networks (Lauffenburger and Horwitz, 1996, Ridley et al, 2003). In order to perform these developmental procedures, migrating cells must reorganize their actin cytoskeleton in response to positive and negative targeting signals present in the extracellular environment. The effect of these signals on cell migration is dictated by the complement of transmembrane receptors on the cell surface, as well as the diverse intracellular signaling cascades activated by specific signals.

The formation of neural and vascular networks has common molecular signals, which reduces the complex task of extending long distances to the simpler task of navigating a series of short segments based on these specific signals in the extracellular environment. The pilot signals are divided into four types: attractants and repellents, which may function as either short-range (cell or matrix-associated) or long-range (diffusible). The intermediate target is typically the source of a long-range attractive signal that attracts axons, or a short-range or long-range repulsive signal that repels axons that have entered the target or prevents them from all entering. Between intermediate targets, axons and blood vessels are often guided through tissue passages by attractive signals that cells create along the tissue passages and by repulsive signals that prevent them from entering the surrounding tissue.

As used herein, a "targeting signal" is a molecule that can act to attract or repel the navigation or formation of neurons or blood vessels. Guidance signals, such as axon guidance signals, are generally classified as "attractive" or "repulsive". However, this is a simplification, as different axons respond differently to a given signal. In addition, the same axonal growth cone may change its response to a given signal depending on timing, previous experience with the same or other signals, and the environment in which the signal is found. Thus, in one aspect, the targeting signal can be an attractive targeting signal for a particular cell. In another aspect, the targeting signal can be a repulsive targeting signal for a particular cell. The "targeting signal" disclosed herein may be a protein that acts on a cellular receptor extracellularly. However, molecules, including nucleic acids and small molecules, that are capable of acting extracellularly or intracellularly to attract or repel neurons or vascular navigation are also disclosed. Thus, for example, where a ligand directed to a signaling receptor is disclosed herein, molecules capable of modulating the activity or expression of the receptor are also disclosed. Thus, for example, compositions, such as functional nucleic acids, are disclosed that are capable of altering gene expression of a signal-directing receptor or signaling molecule thereof disclosed herein. In one aspect, these molecules affect the same cellular receptors and intracellular signaling pathways as the traditional protein targeting molecules disclosed herein. Alternatively, these molecules can be identified by the screening methods disclosed herein.

The guidance signal can be characterized by the ability to guide axons. Growing axons have a highly motile structure at the growing tip, known as the growth cone, and are able to "sniff" signals in the extracellular environment indicating to which way the axon is growing. These signals, known as pilot signals, may be fixed in one location or may be diffusible; they can attract or repel axons. For axons, the growth cone contains receptors that recognize these targeting signals and interpret the signals as a chemotactic response. The general theoretical framework is that when the growth cone "senses" a targeting signal, the receptor activates various signaling molecules in the growth cone, ultimately affecting the cytoskeleton. If the axonal growth cone senses a gradient of the pilot signal, intracellular signaling within the growth cone occurs asymmetrically, such that cytoskeletal changes occur asymmetrically, turning the growth cone toward or away from the pilot signal.

The combination of genetic and biochemical approaches has led to the discovery of several important classes of guide molecules and their receptors. The targeting factor and its receptors DCC and UNC5 are secreted molecules that act to attract or repel axons. Slits are secreted proteins that reject nerve growth cones, usually by binding to receptors of the robo (roundabout) class. Ephrins (Ephrins) are cell surface receptors that activate Eph receptors on the surface of other cells. This interaction may be attractive or repulsive. In some cases, ephrin may also serve as a receptor, conducting signals to the expressing cells, with Ephs serving as a ligand. The signal is transmitted to the ephrin and Eph-bearing cells and is called "bidirectional signaling". Many types of brain signaling proteins are primarily axon repellents and activate complexes of cell surface receptors known as Plexins (Plexins) and Neuropilins (Neuropilins). In addition, many other types of extracellular molecules are used by growth cones to navigate properly, including developmental morphogens, such as BMPs, Wnts, Hedgehogs, and FGFs; extracellular matrix and adhesion molecules such as NCAM, L1 and laminin (laminin); growth factors such as NGF, and neurotransmitters and modulators such as GABA. Thus, as disclosed herein, the repulsive signal can be, for example, a ligand for a roundabout receptor or a ligand for a targeting factor receptor.

xi. Unc5 and guide factor

The targeting factor was identified as a chemoattractant, which directs axons to the midline by binding to receptors of the DCC (deleted in colorectal cancer) family. The homing factor is also implicated in axonal repulsion, an effect mediated by the action of receptors of the Unc5 family alone or in combination with DCC receptors. In addition, DCC-Unc5 heterodimers can mediate rejection over a longer range than Unc5 receptor alone. Targeting factor 1 also modulates vascular targeting with Unc5b, one of the four mammalian Unc5 receptors. Unc5b is expressed in endothelial apical cells. In mice, loss of Unc5b resulted in abnormal extension of the apical cell filamentous prosthetic foot and excessive branching of many blood vessels. Treatment of cultured endothelial cells or growing blood vessels with targeting factor 1 in vivo induces the retraction of filamentous prosthetic feet. The role of Unc5b in mediating endothelial cell rejection was demonstrated by analysis of developing internodal blood vessels (ISVs) in zebrafish embryos.

Targeting factors constitute a family of phylogenetically conserved targeting signals associated with extracellular matrix molecular layer laminins. Four secretion targeting factors have been identified in vertebrates: targeting factor-1 in chicken, mouse, zebrafish and human; targeting factor-2 in chicken; targeting factor-3 in mice and humans; and targeting factor-4 in mice and humans. All targeting factors are structurally related to the short arm of laminin and contain laminin VI and V domains. All targeting factors also contain a positively charged C-terminal domain, called NTR module. Targeting factors-1, -2 and-3 are more closely related to the gamma chain of laminin. In contrast, targeting factor-4 is more closely associated with the beta chain of laminin.

Two families of guidance factor receptors have been identified which indicate the direction of migration. Both families belong to the immunoglobulin (Ig) superfamily of receptors. In vertebrates, there are two members of the family of Deletions (DCCs) in colon cancer, DCCs and neogenin (neogenin), which contain 6 extracellular fibronectin type III repeats, as well as 4 Ig domains and 3 intracellular homology regions (P1, P2, and P3), which mediate interactions with other receptors such as UNC5b (P1) and Robol (P3). The UNC5 family has 4 members, UNC5a (UNC5H1), UNC5b (UNC5H2), UNC5C (UNC5H3), containing two extracellular igs and two thrombospondin type I (Tsp1) domains, as well as intracellular ZU-5, DCC binding and C-terminal death domains. Functionally, the DCC family mediates attraction to leader-1, while the UNC5 family mediates repulsion by forming a leader-1 dependent complex with DCC. Members of both families have been shown to act as dependent receptors and induce apoptosis in the absence and absence of ligand.

Semaphorin and neuropilin/plexin

As disclosed herein, certain semaphorins can act through plexins to increase vascular permeability. Thus, in certain instances of the disclosed compositions and methods, the repulsive guidance cue is not a semaphorin. In certain instances of the disclosed compositions and methods, the repulsive guidance cue is not a ligand for plexin or neuropilin.

However, as disclosed herein, semaphorin 3E acts through plexin D1 to inhibit vascular permeability. Thus, in some cases, the repulsive guidance cue can be semaphorin 3E. In some cases, the repulsive guidance cue can be a ligand of plexin D1.

Semaphorins are secreted targeting signals that are capable of long-range diffusion (class 3), but in some cases have limited diffusion, or are membrane-bound and function as short-range targeting signals. Semaphorin is most widely known as a repellent, but semaphorin 3A (Sema3A) may also act as a chemoattractant, depending on the intracellular cyclic nucleotide levels. Semaphorins signal through the following multimeric receptor complexes: membrane-bound semaphorin binds to plexin, but secreted class 3 semaphorin binds to neuropilin, which acts as a non-signaling co-receptor with plexin. The exception to this rule is secreted Sema3E, which binds directly to plexin D1(Plxnd 1). In addition, membrane anchored Sema7A stimulates axonal extension by activating integrins. Semaphorins and their receptors also regulate the targeting and branching of blood vessels. Endothelial cells express various neuropilin and plexin receptors. Sema3A inhibited the formation of endothelial pseudopodia and blood vessels. Neuropilin 2 is expressed in veins and lymphatic vessels, while neuropilin 1 is widely expressed in developing vascular systems. Neuropilins are also involved in angioplasty, but this may reflect their role in modulating VEGF, not brain signaling, since neuropilins are also receptors for specific VEGF isoforms (VEGF 165) and modulate the activity of VEGF receptors. In addition, VEGF 165 competes with Sema3A for binding to neuropilin.

As disclosed herein, semaphorin 3E acts through plexin D1 to inhibit vascular permeability. Thus, in some cases, the repulsive guidance cue can be semaphorin 3E. In some cases, the repulsive guidance cue can be a ligand of plexin D1.

Ephrin and Ephs

Another important class of short-range axon-guiding molecules are the Eph receptor tyrosine kinases and their ephrin ligands. The 13 Eph receptors in mammals are classified into the A (EphA1-8) and B (EphB1-4 and EphB6) subfamilies. The 8 ephrin receptors include ephrin a1-5, which is bound to the membrane by a glycosyl-phosphatidylinositol anchor, and ephrin B11-3, which contains transmembrane and cytoplasmic regions. Ephrin a ligand binds to EphA receptor and ephrin B ligand binds to EphB receptor; only moderate cross-reactions were observed between families; for example, EphA4 binds to certain class B ephrins. Eph receptors and ephrins initiate bidirectional signaling in cells expressing either Eph receptors (forward signaling) or ephrinB ligands (reverse signaling). Ephrin was first identified as a repulsive axon-guiding molecule by a topographic projection (retinal projection) study, and subsequently implicated as a negative and positive signal in other wiring processes. Eph-ephrin signaling also controls the development of blood vessels. Some of these targeting molecules belong to the first discovered factors that are selectively expressed in arteries or veins. Historically, it was speculated that hemodynamic pressure differentials regulate the differentiation of high pressure vessels into arteries and low pressure vessels into veins. However, expression analysis and loss of function studies in mice indicate that EphB4 and ephrin B2 are expressed in developing veins and arteries, respectively, and are critical for their maintenance. These studies indicate that both repulsive ephrin B2-EphB4 signaling, both forward and reverse, can prevent the mixing of venous and arterial endothelial cells, ensure the assembly of "similar" endothelial cells, and demarcate the artery-vein boundary. The repulsive ephrin-Eph signal provides a short range guidance signal for vessel navigation through tissue boundaries. For example, ephrin B2 excludes ISVs expressing EphB3/EphB4 from entering the somite. However, ephrin-Eph interactions may also provide attractive signals and induce capillary sprouting in other cases. For example, the near-secretory expression of ephrin-B ligands and EphBs on adjacent endothelial cells or smooth muscle cells in the same vessel can provide a bidirectional signal for establishing contact-dependent communication and promote assembly, sprouting, and maturation of the vessel. For example, ephrin a ligands may also act as positive regulators of vascular morphogenesis.

EphA 2/ephrin a1 signaling has been shown to inhibit VEGF-induced retinal vascular permeability, and has been implicated in the treatment of neovascular and vascular permeability abnormalities in diabetic retinopathy (Ojima et al, 2006). Thus, in certain instances of the disclosed compositions and methods for inhibiting vascular permeability, the repulsive signal is not a ligand of the Eph or ephrin receptor. In other instances, the disclosed compositions comprise at least one targeting signal in addition to a ligand for an Eph or ephrin receptor.

Slits and roundabout

A notable example of a repulsive guidance cue is the Slit family of extracellular matrix proteins. Slit was originally identified on the midline of Drosophila embryos in genetic screening for defects in axonal guidance (Seeger et al, 1993; Kidd et al, 1998; Battey et al, 1999; Kidd et al, 1999). Subsequently, three evolutionarily conserved Slit genes were cloned in vertebrates, which encoded proteins that reject axons (Brose et al, 1999; Li et al, 1999) and promote dendritic branching of sensory axons (Wang et al, 1999).

Genetic and biochemical studies have demonstrated that transmembrane proteins of the Robo family act as receptors for Slit proteins. Like slit, robo was also found in genetic screening for axonal guidance defects in Drosophila (Seeger et al, 1993). 4 Robos have been identified in vertebrates, of which Robo1-3 is predominantly expressed in the nervous system (Marillet et al, 2002). In contrast, Robo4, also known as Magic Roundabiut, is expressed exclusively in the vascular system of embryonic mice (Park et al, 2003), the placental arteries (Huminiecki et al, 2002), and the tumor endothelium of various human malignancies (Huminiecki et al, 2002; Seth et al, 2005). Robo4 is further distinguished from Robo1-3 by its different sequence: the extracellular domain of neuronal Robos contains 5 immunoglobulin (Ig) domains and 3 fibronectin type III (FNIII) repeats, while Robo4 contains two Ig domains and two FNIII repeats (Huminiecki et al, 2002; Park et al, 2003). In addition, Robo1-3 has 4 Conserved Cytoplasmic (CC) motifs, CC0, CC1, CC2, and CC3(Kidd et al, 1998; Zalen et al, 1998), with only CC0 and CC2 being present in Robo4 (Huminiecki et al, 2002; Park et al, 2003).

The ability of Robo to facilitate targeting decisions in the nervous system depends on the activation of specific biochemical programs downstream of Slit-stimulated receptors. Analysis of Slit-dependent rejection in drosophila, nematodes (c. elegans) and mammals has identified key mediators of Robo signaling in the nervous system. In Drosophila, Abelson (abl) tyrosine kinase and actin-binding protein Enabled (Ena) are involved in regulating Robo rejection activity (Bashaw et al, 2000). Other studies in Drosophila have identified Rac GTPase Activating Protein (GAP), which is involved in Robo-mediated tracheal cell and axon rejection (Lundstrom et al, 2004; Hu et al, 2005). In nematodes, a direct function of Ena in regulating Slit signaling has been revealed from genetic analysis (Yu et al, 2002). In mammalian neurons, the Robo1 interacting protein srGAP1 is essential for Slit-dependent rejection of precursor cell migration from the anterior inferior ventricular septum region (Wong et al, 2001). These mechanistic studies not only begin to elucidate the signaling pathway downstream of neuronal Robos, but such studies have provided an explanation for the repulsive activity of receptors.

Contrary to the nervous system, little is known about Slit-Robo signaling in the vasculature, and although there is some evidence that Slit-Robo signaling inhibits migration of neuronal and non-neuronal cell types, including endothelial cells (Wu et al, 1999; Zhu et al, 1999; Wu et al, 2001; Park et al, 2003; Seth et al, 2005), several recent reports suggest that Robos can promote angiogenesis in both Slit-dependent and Slit-independent ways. For example, it has been reported that Slit2 stimulation of Robo1 induces migration and tube formation in vitro and promotes tumor angiogenesis in vivo (Feng et al, 2004). Furthermore, recent studies have shown that blocking the activity of Robo4 with the extracellular domain of soluble Robo4 inhibits migration and tube formation in vitro, consistent with a positive effect of Robo4 during angiogenesis. Furthermore, this study reported that Slit proteins do not bind to Robo4, suggesting that the receptor has an unknown ligand (suctinget al, 2004). This notion of angiogenesis promotion by Robo4 has also emerged from recent data showing that overexpression of Robo4 increases endothelial cell adhesion and migration independent of Slit (Kaur et al, 2006). These seemingly inconsistent observations underscore the need to determine the functional significance and mechanism of Slit-Robo signaling in endothelial cells.

As disclosed herein, Slit2 is a ligand for Robo4, Slit2-Robo4 signaling negatively regulates cell motility and inhibits vascular permeability. In particular, the teachings provided herein establish that Slit2 triggers repulsive signaling in endothelial cells by activating Robo4, defining a novel signaling cascade responsible for this activity. As described herein, Slit2 activation of Robo4 inhibits Rac activation, thereby inhibiting Rac-initiated or mediated cell motility and cell spreading. The teachings provided herein also establish a Slit 2-dependent association between Robo4 and connexin paxillin, and the experimental data detailed herein provide biochemical and cellular biological evidence that this interaction is critical for Robo 4-dependent inhibition of cell migration, spreading and Rac activation. Specifically, as taught herein, activation of Robo4 initiates activation of GIT1 by paxillin, which in turn, GIT1 inhibits ARF 6. Activation of Robo4 protects the barrier function of the endothelium, blocks VEGF signaling downstream of VEGF receptors, and reduces vascular leakage and pathological angiogenesis. Importantly, Robo4 activation not only blocks VEGF signaling, but also inhibits signaling from a variety of angiogenic, permeability, and inflammatory factors, including thrombin and bFGF. As also disclosed herein, Robo 4-paxillin signaling is essential for proper development of embryonic blood vessels in zebrafish.

These disclosed relationships and results relating to the activation of Robo4 allow for the generation of novel targets for modulation and cellular manipulation as described herein. As used herein, "modulating" includes altering the activity of a target and "manipulating" as used herein includes altering the state of a cell.

Permeability of blood vessel

Diseases and disorders characterized by adverse vascular permeability include, for example, edema associated with brain tumors, ascites associated with malignancies, megger's syndrome, pneumonia, nephrotic syndrome, pericardial and pleural effusion. Thus, methods of treating or preventing these or any other diseases associated with increased vascular permeability or edema are provided. For example, inhibiting the formation of edema would be beneficial to the overall outcome of patients in conditions such as inflammation, allergic disease, cancer, cerebral stroke, myocardial infarction, cardiopulmonary insufficiency, renal failure, and retinopathy. Furthermore, because edema is a common consequence of tissue hypoxia, it can also be concluded that inhibition of vascular leakage represents a potential method of treating tissue hypoxia. For example, blood flow interrupted by pathological conditions (e.g., thrombosis) or medical interventions (e.g., cardioplegia, organ transplantation, and angioplasty) can be treated acutely and prophylactically with vascular leak inhibitors.

Ischemia/reperfusion injury following stroke and myocardial infarction is also characterized by vascular permeability and edema. Inadequate tissue perfusion leads to vasogenic edema following permanent ischemia, which occurs due to increased vascular permeability. Tissue perfusion is a measure of the arrival of oxygenated blood at a given tissue due to the patency of the artery and the flow of blood in the artery. Tissue vascularization may be disrupted by occlusion or, alternatively, may be due to loss of blood flow resulting from leakage or bleeding of blood vessels upstream of the affected site. Inadequate tissue perfusion during acute myocardial infarction, cerebral stroke, surgical revascularization procedures, and other conditions in which tissue vascularization is disrupted is a critical factor in the outcome of a patient's condition. Edema can cause various types of injury, including vascular collapse and impaired electrical function, particularly in the heart. However, subsequent reperfusion can also cause similar damage in some patients, resulting in therapeutic paradox. While it is necessary to clear an obstructed vessel or repair or replace a damaged vessel, subsequent reperfusion, in some cases, can lead to further injury. Likewise, during bypass surgery, it is necessary to stop the heart and hang the patient on the heart pump. For example, some patients who have undergone bypass surgery may, in fact, experience a worsening of the condition ("post-pump syndrome"), which may be the result of ischemia during the cessation of cardiac function during surgery. Arterial occlusion may cause reduced blood flow, but even after the occlusion is removed to open the artery, further tissue damage may result if tissue reperfusion cannot occur. For example, the destruction of a blood clot can trigger a cascade of events that result in the loss of tissue perfusion, rather than obtaining perfusion.

Other diseases and disorders characterized by adverse vascular permeability include, for example, infectious and non-infectious diseases that can lead to cytokine storms. Cytokine storms can be contributed by a variety of infectious and non-infectious diseases, including, for example, Graft Versus Host Disease (GVHD), Adult Respiratory Distress Syndrome (ARDS), septicemia, avian influenza, smallpox, and Systemic Inflammatory Response Syndrome (SIRS).

Pathological angiogenesis

Angiogenesis and angiogenesis-related diseases are closely affected by cell proliferation. The term "angiogenesis" as used herein refers to the generation of new blood vessels in a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only under very specific defined conditions. For example, angiogenesis is commonly observed in wound healing, fetal and embryonic development, and formation of the corpus luteum, endometrium and placenta. The term "endothelium" as defined herein refers to a thin layer of flattened cells that line the serosal cavity, lymphatic vessels, and blood vessels. These cells are defined herein as "endothelial cells". The term "endothelial inhibitory activity" refers to the ability of a molecule to inhibit angiogenesis in its entirety. Inhibition of endothelial cell proliferation also results in inhibition of angiogenesis.

Both controlled and uncontrolled angiogenesis are considered to start in the same way. Endothelial cells and pericytes, surrounded by the basal membrane, form capillaries. Angiogenesis begins as the basal membrane is eroded by enzymes released by endothelial cells and leukocytes. Endothelial cells lining the lumen of the blood vessel then protrude through the basement membrane. Angiogenic stimulants induce endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" that is drawn from the parent vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating new blood vessels.

New blood vessels may also be formed in part by angiogenesis. Angiogenesis differs from angiogenesis by the source of endothelial cells. Angiogenesis involves the recruitment of endothelial progenitor cells known as angioblasts. These hemangioblasts may be from the circulation or from tissue. Angiogenesis is regulated by similar signaling pathways as angiogenesis. Thus, as used herein, the term "angiogenesis" is used interchangeably with angiogenesis, such that methods of modulating angiogenesis may also modulate angiogenesis.

Pathological angiogenesis, which may be characterized by permanent dysregulation or unregulated angiogenesis, occurs in a variety of disease states, tumor metastasis and abnormal growth of endothelial cells and supports the pathological damage observed in these conditions. A number of disease states in which pathological angiogenesis exists have been grouped together as angiogenesis-dependent, angiogenesis-associated, angiogenesis-related diseases. These diseases are the result of abnormal or adverse cell proliferation, particularly endothelial cell proliferation.

Diseases and processes mediated by abnormal or unwanted endothelial cell proliferation include, but are not limited to, hemangiomas, solid tumors, leukemias, tumor metastases, telangiectatic psoriatic scleroderma, pyogenic granulomas, myocardial angiogenesis, plaque neovascularization, coronary collaterals, ischemic limb angiogenesis, keratopathy, rubeosis iridis, neovascular glaucoma, Diabetic Retinopathy (DR), retrolental fibroplasia, nonproliferative Diabetic Macular Edema (DME), arthritis, diabetic neovascularization, age-related macular degeneration (AMD), retinopathy of prematurity (ROP), Ischemic Retinal Vein Occlusion (IRVO), wound healing, peptic ulcers, bone fractures, keloids, angiogenesis, hematopoiesis, ovulation, menstruation, and placentation.

Composition comprising a metal oxide and a metal oxide

Provided herein are compositions for inhibiting vascular permeability and pathological angiogenesis in a tissue.

In one embodiment, such a composition comprises a ligand of Unc5 or a receptor for deletion in colorectal cancer (DCC). In one such embodiment, the ligand of Unc5 or DCC may be any composition or molecule capable of acting through Unc5 or DCC receptor to inhibit Rac activation by VEGF. The term "act through a receptor" as used herein means that binding of the composition to the receptor promotes the activity of the receptor. For example, the composition may contain a ligand for Unc5 or DCC, which act through Unc5 or DCC receptor to activate the inhibition of ARF6 by Git 1. In another example, the composition may contain a ligand of Unc5 or DCC, which act through Unc5 or DCC receptor to activate the activation of Git1 by paxillin. In another example, the compositions described herein may contain compositions or molecules that mimic the activation of Git1 by Unc5 or DCC receptor activating paxillin.

In one embodiment, the compositions described herein comprise a ligand of Unc5, wherein the ligand is a homing factor, such as human homing factor 1, homing factor 2, homing factor 4, homing factor G1, or homing factor G2, and rodent (e.g., mouse or rat) homing factor 1, homing factor 3, homing factor 4, homing factor G1, or homing factor G2, or a fragment or variant thereof, that binds to and activates Unc5b to inhibit ARF 6. For example, the targeting factor ligand may comprise a sequence selected from SEQ ID NOs: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25, or a variant or fragment of these amino acid sequences that binds Unc5 b. Such amino acid sequence fragments are at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In another embodiment, the homing factor ligand of Unc5b may comprise a ligand sequence identical to a sequence selected from SEQ ID NOs: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25 or an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% sequence identity to a Unc5 b-binding fragment thereof.

In another embodiment, the compositions described herein may contain ligands for Eph. In one such embodiment, the composition comprises a ligand capable of acting through the Eph receptor to inhibit Eph activation of Rac by VEGF. In another such embodiment, the composition contains a ligand for Eph that is capable of acting through the Eph receptor to activate the inhibition of ARF6 by Git 1. In another embodiment, the compositions of the present description may contain any composition or molecule capable of acting through an Eph receptor to activate Eph's activation of Git 1. In another embodiment, the compositions described herein may contain any composition or molecule that mimics the Eph receptor to activate activation of Git1 by paxillin.

In another embodiment, the compositions provided herein contain a ligand for the Robo4 receptor. In one such embodiment, the ligand of Robo4 can be any composition or molecule capable of negative regulation of cell movement through the action of Robo 4. In another such embodiment, the ligand of Robo4 can be any composition or molecule capable of acting through Robo4 to inhibit vascular permeability. In another such embodiment, the ligand of Robo4 may be any composition or molecule capable of acting through Robo4 to inhibit the activation of Rac by VEGF. In another embodiment, the compositions described herein comprise a ligand of the Robo4 receptor, wherein the ligand is capable of acting through Robo4 to initiate activation of GIT1 by the paxillin. In another embodiment, the compositions described herein include a ligand of the Robo4 receptor, wherein the ligand is capable of acting through Robo4 to activate the inhibition of ARF6 by Git 1. In another embodiment, the compositions described herein comprise a ligand for the Robo4 receptor, wherein the manner in which the ligand functions through Robo4 results in one or more of the following effects: protecting endothelial barrier function, blocking VEGF signaling downstream of VEGF receptor, inhibiting vascular leakage, inhibiting pathological angiogenesis, and inhibiting signaling of various angiogenesis, permeability, and inflammatory factors.

When a composition of the invention comprises a ligand of Robo4, the ligand is any composition or molecule that binds the extracellular domain of Robo 4. Alternatively, the ligand of Robo4 can be any composition or molecule that acts through the Robo4 receptor to inhibit the activation of Rac by VEGF. Furthermore, the ligand of Robo4 can be any composition or molecule that acts through the Robo4 receptor to activate the inhibition of ARF6 by Git 1. Furthermore, the ligand of Robo4 can be any composition or molecule that acts through Robo4 receptor to activate the activation of Git1 by paxillin. In another instance, the ligand of Robo4 can be any composition or molecule that mimics the Robo4 receptor to activate activation of Git1 by paxillin. In one embodiment, the ligand of Robo4 included in the compositions of the present description comprises an isolated polypeptide of about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 amino acids in length.

When the compositions described herein comprise a ligand to Robo4, such ligand can be a Slit, such as Slit2, or a fragment or variant thereof that binds and activates Robo 4. In particular embodiments, the manner in which Slit ligands, or fragments or variants thereof, bind to and activate Robo4 results in one or more of the following effects: inhibition of ARF 6; protection of endothelial barrier function; block VEGF signaling downstream of VEGF receptors; inhibiting vascular leakage; inhibiting pathological angiogenesis; and signal inhibition by various angiogenic, permeability, and inflammatory factors. For example, a ligand of Robo4 may comprise an amino acid sequence selected from SEQ ID NOs: 3. SEQ ID NO: 5. SEQ ID NO: 7. and SEQ ID NO: 36 to SEQ ID NO: 47 or a fragment thereof of Robo 4. For example, a fragment of such an amino acid sequence is at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. The ligand of Robo4 may comprise an amino acid sequence identical to a sequence selected from SEQ ID NOs: 3. SEQ ID NO: 5. SEQ ID NO: 7. and SEQ ID NO: 36 to SEQ ID NO: 47 or a fragment of or a binding Robo4 thereof has an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% sequence identity. A fragment of a Slit may contain the N-terminal region of the Slit. For example, the ligand of Robo4 may contain amino acids 1-1132 (SEQ ID NO: 36) of Slit1, amino acids 1-1121 (SEQ ID NO: 37) of Slit2, amino acids 1-1118 (SEQ ID NO: 38) of Slit3, or the amino acids depicted in FIG. 23 and depicted in SEQ ID NO: 39 to SEQ ID NO: 47, or an N-terminal fragment thereof as described in detail in seq id no. In particular embodiments, the ligand of Robo4 may comprise a sequence consisting essentially of a sequence selected from SEQ ID NOs: 36 to SEQ ID NO: 47 or a pharmaceutically acceptable salt thereof. In certain embodiments, as in SEQ ID NO: 39 to SEQ ID NO: 47, the Slit fragment comprised in the composition of the invention does not comprise the most N-terminal amino acid. For example, the amino acid sequence may lack about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 of the N-terminal amino acids of a native Slit. In other embodiments, a Slit fragment may not comprise about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids of the most C-terminal end of a native Slit.

For example, the ligand of Robo4 may comprise a polypeptide consisting essentially of amino acids 281-511 of Slit1 (SEQ ID NO: 15) or amino acids 271-504 of Slit2 (SEQ ID NO: 16). Thus, the ligand of Robo4 may comprise SEQ ID NO: 15 or SEQ ID NO: 16 or a fragment thereof of Robo 4. The ligand of Robo4 may comprise a sequence identical to SEQ ID NO: 15 or SEQ ID NO: 16 or a fragment thereof of Robo4 has an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% sequence identity.

In another embodiment, the composition of the invention may contain a fragment of Robo4 capable of activating paxillin activating Git 1. Accordingly, provided herein are isolated polypeptides comprising the paxillin binding sequence of Robo4, wherein the polypeptide does not comprise full-length Robo 4. In one such embodiment, the paxillin binding sequence may comprise the amino acid sequence of SEQ ID NO: 27 or a fragment or variant thereof. For example, a fragment may be at least about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. Amino acid sequence SEQ ID NO: 27 may comprise a sequence identical to SEQ ID NO: 27 or a fragment thereof that binds to a dockerin has an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% sequence identity.

In another embodiment, a composition described herein contains an isolated polypeptide comprising the Paxillin Binding Sequence (PBS) of Robo4, wherein the polypeptide is defined by the formula:

R¹-PBS-R²

wherein R is¹And R²Independently of each other H, acyl, NH₂Amino acids or peptides, wherein the polypeptide does not comprise full length Robo 4. PBS can be composed of a sequence identical to SEQ ID NO: 27 or a fragment thereof of at least 10 residues in length, having an amino acid sequence homology of at least 80%.

Also provided herein are isolated nucleic acids encoding any of the polypeptides disclosed herein. Thus, isolated nucleic acids encoding polypeptides comprising the paxillin binding sequence of Robo4 are provided, wherein the polypeptides do not contain full-length Robo 4. Also provided are nucleic acid molecules comprising SEQ ID NO: 2 or a fragment thereof of at least 30 residues in length, wherein said nucleic acid does not encode full-length Robo 4.

Pharmaceutical composition

The compositions described herein, such as the ligands, proteins, and peptides disclosed herein, can be formulated in pharmaceutical compositions. For example, such compositions can be combined with a pharmaceutically acceptable carrier to provide a dosage form suitable for therapeutic administration. As used herein, "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable, i.e., the substance can be administered to a subject with the composition (e.g., a desired ligand, protein, peptide, nucleic acid, small molecule therapeutic, etc.) without causing any undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the pharmaceutical composition in which it is contained. The carrier should naturally be selected to minimize any degradation of the active ingredient in the subject and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

The substance may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). They can be targeted to specific cell types by antibodies, receptors, or receptor ligands. The following references are examples of the use of this technique to target specific proteins to tumor tissue: (Senter et al, Bioconjugate chem., 2: 447-containing materials 451, (1991); Bagshawe, K.D., Br.J. cancer, 60: 275-containing materials 281, (1989); Bagshawe et al, Br.J. cancer, 58: 700-containing materials 703, (1988); Senter et al, Bioconjugate chem., 4: 3-9, (1993); Battelli, et al, cancer Immunol. Immunotherr., 35: 421-containing materials 425, (1992); Pietesz and McKenzie, immunolog. reviews, 129: 57-80, (1992); and Roffler et al, biochem. 2065, 42: 2062-containing materials 2065, (1991)). Mediators such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drugs targeting colon cancer), receptor-mediated DNA targeting via cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviruses target murine glioma cells in vivo. The following references are examples of the use of this technique to target specific proteins to tumor tissue (Hughes et al, cancer research, 49: 6214-. Generally, receptors participate in the endocytic pathway either constitutively or induced by ligands. These receptor clusters in clathrin-coated pits enter the cell through clathrin-coated vesicles, where the receptors are sorted by acidified endosomes, and then either recycle to the cell surface, become stored intracellularly, or degrade in lysosomes. The internalization pathway has a variety of different functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and modulation of receptor levels. Many receptors participate in more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNAand Cell Biology 10: 6, 399-409 (1991)).

Suitable carriers and their formulation are described in Remington, authored by a.r. gennaro: pharmaceutical sciences and practices, 19th edition (Remington: The Science and practice of Pharmacy (19th ed.), Mack Publishing Company, Easton, PA 1995). Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to impart isotonicity to the formulation. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextran solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition being administered.

Pharmaceutical carriers are known to those skilled in the art. These are most typically present in standard carriers for administration to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

The pharmaceutical compositions may contain, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surfactants, and the like. The pharmaceutical compositions may also contain one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, ethanol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose solution, dextrose and sodium chloride, lactated ringer's solution, or fixed oils. Intravenous media include fluid and nutrient supplements, electrolyte supplements (such as those based on ringer's dextrose solution), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, sachets or tablets. Thickeners, perfumes, diluents, emulsifiers, dispersing aids or binders may be desirable.

Certain compositions may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, and phosphoric acids, as well as organic acids such as formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic, and fumaric acids, or with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, and trialkyl and acyl amines and substituted ethanolamines.

Method

Provided herein are methods of screening or evaluating agents that inhibit vascular permeability or pathological angiogenesis. In one embodiment, the method comprises determining the ability of the agent to affect Robo 4-mediated activation of Git 1. For example, Robo4 mediated activation of Git1 can be determined by the following steps comprising: contacting a first cell expressing Robo4 with a candidate agent, contacting a second cell substantially identical to the first cell but substantially lacking Robo4 with the candidate agent, and analyzing activation of Git1 in the first and second cells, wherein detection of higher Git1 activation in the first cell as compared to the second cell indicates Robo 4-mediated Git1 activation of the agent.

As disclosed herein, Robo 4-mediated activation of Git1 results in ARF6 inactivation. ARF6 is involved in VEGF-mediated Rac activation, Rac activates Pak, Pak activates MEK, MEK activates ERK, ERK promotes vascular permeability. Thus, as disclosed herein, Git1 activation can be analyzed by detecting whether any component of the signaling pathway is activated or inactivated. Thus, Robo4 mediated activation of Git1 can be assayed by detecting ARF6 inactivation, Rac inactivation, Pak inactivation, MEK inactivation, or ERK inactivation. It should be understood that any other known or newly discovered method of monitoring such signaling pathways may be used in the disclosed methods.

Also provided are methods of screening or evaluating agents capable of inhibiting vascular permeability comprising determining the ability of the agent to inhibit ARF6, Rac, Pak, MEK, or ERK. For example, Robo 4-mediated inhibition of ARF6, Rac, Pak, MEK or ERK is determined by the following steps comprising: contacting a first cell expressing Robo4 with a candidate agent, contacting a second cell substantially identical to the first cell but substantially lacking Robo4 with the candidate agent, and analyzing the inhibition of ARF6, Rac, Pak, MEK, ERK, or a combination thereof in the first and second cells, wherein a lower activation of ARF6, Rac, Pak, MEK, or ERK detected in the first cell as compared to the second cell indicates Robo 4-mediated inhibition of ARF6, Rac, Pak, MEK, or ERK by the agent.

Activation of signalling proteins such as Rac, Pak, MEK, ERK can be analysed by detecting phosphorylation of the protein. Cell-based and cell-free assays for detecting protein phosphorylation are well known in the art, including the use of antibodies including, for example, anti-phosphoserine: (AB 1603) (Chemicon, Temecula, CA), threonine phosphate resistance ((R))AB1607) and phosphotyrosine (anti-phosphotyrosine: (B) ((R))AB 1599). Site-specific antibodies specific for the phosphorylated form of DDX-3 can also be generated. Methods of producing and using such antibodies are well known in the art.

The assay methods disclosed herein can be performed in the substantial absence of VEGF, TNF, thrombin, or histamine. Alternatively, the disclosed assay methods can be performed in the presence of a biologically active amount of VEGF, TNF, thrombin, or histamine.

"activity" of a protein includes, for example, transcription, translation, intracellular translocation, secretion, phosphorylation by kinases, cleavage by proteases, homophilic and heterophilic binding to other proteins, ubiquitination.

In one embodiment, the screening method described herein is a screening assay, such as a high throughput screening assay. Thus, the contacting step can be a cell-based or cell-free assay. For example, vascular endothelial cells may be contacted with a candidate agent in vivo, ex vivo, or in vitro. The cells may be in monolayer culture, but preferably constitute the epithelium. The cells may be assayed in vitro or in situ, or protein extracts of the cells may be assayed in vitro to detect activated (e.g., phosphorylated) Rac, Pak, MEK, ERX. Endothelial cells can also be engineered to express a reporter gene construct, wherein the cells are contacted with a candidate agent and expression of the reporter gene is assessed. The use of other such cell-based and cell-free assays is also contemplated herein.

For example, the effect of small molecules, amino acids, or nucleic acid mimetics on vascular permeability or pathological angiogenesis can be assessed in endothelial cells expressing Robo4 and compared to endothelial cells lacking Robo 4.

In general, candidate agents can be identified from natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the art of drug discovery and development will appreciate that the exact source of the extract or compound to be tested is not critical to the screening procedure of the present invention. Thus, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant, fungal, prokaryotic, or animal based extracts, fermentation broths, and synthetic compounds, as well as modifications of existing compounds. There are a number of methods available for random or directed synthesis (e.g., semi-synthesis or total synthesis) to produce any number of chemical compounds, including but not limited to carbohydrate, lipid, peptide, polypeptide and nucleic acid based compounds. Libraries of synthetic compounds are commercially available, for example from brandon associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are commercially available from a number of sources, including Biotics (suslex, UK), Xenova (Slough, UK), Harbor Branch oceanggraphics Institute (ft.pierce, Fla.) and PharmaMar, u.s.a. (Cambridge, Mass.). Furthermore, natural and synthetically produced libraries can be generated, if desired, according to methods known in the art, for example by standard extraction and fractionation methods. In addition, any library or compound is readily modified, if desired, using standard chemical, physical or biochemical methods. Furthermore, it is readily understood by those skilled in the art of drug discovery and development that methods for deduplicating (e.g., taxonomic deduplicating, biological deduplicating and chemical deduplicating, or any combination thereof) or eliminating duplicates or duplications of substances whose effects are known should be used whenever possible.

When the crude extract is found to have the desired activity, it is necessary to further fractionate the positive lead extract in order to isolate the chemical components that cause the observed effect. Thus, the goal of the extraction, fractionation and purification process is to carefully characterize and identify chemical entities with vascular permeability stimulating or inhibiting activity in the crude extract. The same assays described herein for detecting activity in mixtures of compounds can be used to purify the active ingredient and test derivatives thereof. Methods for fractionating and purifying such heterologous extracts are known in the art. If desired, compounds which have been shown to be therapeutically useful agents may be chemically modified according to methods known in the art. The compounds identified as being of therapeutic value can then be analyzed using animal models for diseases or conditions in which modulation of vascular permeability is desired.

Also provided herein are methods for inhibiting vascular permeability in a subject. As described in detail herein, activation of Robo4 inhibits vascular permeability, inhibits activation of Rac by VEGF, preserves endothelial cell barrier function, blocks VEGF signaling downstream of VEGF receptors, inhibits vascular leakage, and inhibits a variety of angiogenic, permeability, and inflammatory factors. As identified herein, activation of Robo4 signaling accomplishes such an effect by initiating the activation of GIT1 by the paxillin, which in turn leads to the inhibition of ARF6 by GIT 1. Thus, in one embodiment, the methods of inhibiting vascular permeability provided herein comprise administering a therapeutically effective amount of a ligand of Robo4, wherein such ligand results in the inhibition of ARF6 by GIT 1. In another embodiment, the administered ligand is a Slit protein described herein. In particular embodiments, vascular permeability experienced by a subject and treated by administration of a therapeutically effective amount of Robo4 ligand is associated with a disease state selected from infectious and non-infectious diseases that can lead to cytokine storm, including, for example, Graft Versus Host Disease (GVHD), Adult Respiratory Distress Syndrome (ARDS), sepsis, avian influenza, smallpox, and Systemic Inflammatory Response Syndrome (SIRS), ischemia/reperfusion injury following stroke and myocardial infarction, edema associated with brain tumors, ascites associated with malignancies, Magazine syndrome, pneumonia, nephrotic syndrome, pericardial and pleural effusion, inflammation, allergic diseases, cancer, cerebral stroke, myocardial infarction, cardiopulmonary insufficiency, renal failure, and retinopathy.

Provided herein are methods of inhibiting pathological angiogenesis in a subject. As described in detail herein, activation of Robo4 inhibits the effects of a variety of inflammatory, permeability, and angiogenic factors. As also determined herein, activation of Robo4 signaling initiates activation of GIT1 by paxillin, which in turn leads to inhibition of ARF6 by GIT 1. Thus, in one embodiment, the methods provided herein for inhibiting pathological angiogenesis comprise administering a therapeutically effective amount of a ligand of Robo4, wherein such ligand results in the inhibition of ARF6 by GIT 1. In another embodiment, the administered ligand is a Slit protein described herein. In particular embodiments, the pathological angiogenesis experienced by a subject and treated by administering a therapeutically effective amount of a Robo4 ligand is associated with a disease state selected from the group consisting of: hemangioma, solid tumor, leukemia, tumor metastasis, telangiectatic psoriasis scleroderma, pyogenic granuloma, myocardial angiogenesis, plaque neovascularization, coronary collaterals, ischemic limb angiogenesis, corneal disease, rubeosis iridis, neovascular glaucoma, Diabetic Retinopathy (DR), retrocrystallic fibroblastic disease, non-proliferative Diabetic Macular Edema (DME), arthritis, diabetic neovascularization, age-related macular degeneration (AMD), retinopathy of prematurity (ROP), Ischemic Retinal Vein Occlusion (IRVO), wound healing, peptic ulcer, fracture, keloid, angiogenesis, hematopoiesis, ovulation, menstruation, and placentation.

In another embodiment, a method of treating or preventing avian influenza is provided, wherein the method comprises identifying a subject having or at risk of developing said avian influenza and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Adult Respiratory Distress Syndrome (ARDS) is provided, wherein the method comprises identifying a subject having or at risk of developing said ARDS, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Systemic Inflammatory Response Syndrome (SIRS) is provided, wherein the method comprises identifying a subject having or at risk of developing the SIRS, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, there is provided a method of treating or preventing Graft Versus Host Disease (GVHD), wherein the method comprises identifying a subject having or at risk of having said RDS, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing tumor formation or growth is provided, wherein the method comprises identifying a subject having or at risk of having said tumor formation or growth, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Respiratory Distress Syndrome (RDS) is provided, wherein the method comprises identifying a subject having or at risk of having said RDS, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Ischemic Retinal Vein Occlusion (IRVO) in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the IRVO, and administering to the subject a therapeutically effective amount of a ligand of roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing non-proliferative Diabetic Macular Edema (DME) in a subject is provided, wherein the method comprises identifying a subject having or at risk of developing said DME, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing retinopathy of prematurity (ROP) is provided, wherein the method comprises identifying a subject having or at risk of developing the ROP, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Diabetic Retinopathy (DR) in a subject is provided, wherein the method comprises identifying a subject having or at risk of developing said DR and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing wet macular degeneration (AMD) in a subject is provided, wherein the method comprises identifying a subject having or at risk of developing said AMD, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing ischemia in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the ischemia, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing a hemorrhagic stroke in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the hemorrhagic stroke and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing reperfusion injury, such as that observed in myocardial infarction and stroke, in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the reperfusion injury, and administering to the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing a cutaneous vascular defect or abnormality in a subject is provided, wherein the method comprises identifying a subject having the defect or at risk of having the defect, and administering to the skin of the subject a therapeutically effective amount of a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing avian influenza is provided, wherein the method comprises identifying a subject having or at risk of developing said avian influenza and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Adult Respiratory Distress Syndrome (ARDS) is provided, wherein the method comprises identifying a subject having or at risk of developing said ARDS, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Systemic Inflammatory Response Syndrome (SIRS) is provided, wherein the method comprises identifying a subject having or at risk of developing the SIRS, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Graft Versus Host Disease (GVHD) is provided, wherein the method comprises identifying a subject having or at risk of developing said GVHD and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand for the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing tumor formation or growth is provided, wherein the method comprises identifying a subject having or at risk of having said tumor formation or growth, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Respiratory Distress Syndrome (RDS) is provided, wherein the method comprises identifying a subject having or at risk of having said RDS, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Ischemic Retinal Vein Occlusion (IRVO) in a subject is provided, wherein the method comprises identifying a subject having or at risk of having said IRVO, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing non-proliferative Diabetic Macular Edema (DME) in a subject is provided, wherein the method includes identifying a subject having or at risk of developing the DME, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing retinopathy of prematurity (ROP) is provided, wherein the method comprises identifying a subject having or at risk of developing the ROP, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing Diabetic Retinopathy (DR) in a subject is provided, wherein the method comprises identifying a subject having or at risk of developing said DR and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing wet macular degeneration (AMD) in a subject is provided, wherein the method comprises identifying a subject having the AMD or at risk of developing the AMD, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing ischemia in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the ischemia, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing a hemorrhagic stroke in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the hemorrhagic stroke and administering to the subject a therapeutically effective amount of an repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing reperfusion injury, such as that observed in myocardial infarction and stroke, in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the reperfusion injury, and administering to the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of roundabout-4(Robo4) receptor.

In another embodiment, a method of treating or preventing a cutaneous vascular defect or abnormality in a subject is provided, wherein the method comprises identifying a subject having or at risk of having the spot, and administering to the skin of the subject a therapeutically effective amount of a repulsive guidance cue, such as a ligand of the roundabout-4(Robo4) receptor.

Ligands suitable for use in conjunction with the methods described herein include, for example, the ligands described herein. For example, in particular embodiments, compositions described herein that relate to Robo receptors, including the Robo4 receptor, and compositions that relate to the Unc5 or the Deleted (DCC) receptor in colorectal cancer, may be used as ligands in the methods of the invention. More specifically, for example, the slit compounds described herein can be used as ligands for activating Robo4 and achieving the therapeutic benefits of the methods described herein.

In some cases, the subject is identified by medical diagnosis. For example, subjects with diabetic retinopathy and macular degeneration may be identified by observing excess blood vessels in the eye. Acute lung injury can be diagnosed by pulmonary edema in the absence of congestive heart failure. Ischemic stroke can be diagnosed by neuroimaging and imaging (MRI and CT). Other known or newly discovered medical assays may be used to identify a subject for use in the methods of the present disclosure.

In addition, subjects can be identified by genetic susceptibility. For example, genes have been identified that predispose patients to age-related macular degeneration (Klein RJ et al, 2005; Yang Z et al, 2006; Dewan A et al, 2006). Similarly, genetic mutations have been identified that predispose patients to vascular malformations in the brain (Plummer NW et al, 2005). Other known or newly discovered genetic assays may be used to identify subjects for use in the methods of the present disclosure.

The nucleic acid and polypeptide molecules disclosed herein, as well as any compositions necessary to perform the methods of the present disclosure, may be prepared using any method known to those of skill in the art for the particular agent or compound, unless specifically indicated otherwise.

For example, nucleic acids, such as oligonucleotides to be used as primers, may be prepared using standard chemical synthesis methods, or may be produced using enzymatic methods or any other known method. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see, e.g., Sambrook et al, Molecular Cloning: Laboratory Manual, second edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 5, 6), to purely synthetic methods, such as by the cyanoethyl phosphoramidite method using a Milligen or Beckman System 1Plus DNA synthesizer (e.g., Milligen-Biosearch, Burlington, MA model 8700 automated synthesizer or ABI 380B). Synthetic methods that can be used to prepare oligonucleotides are also described by Ikuta et al, ann.rev.biochem.53: 323-356(1984) (phosphotriester and phosphite triester Methods) and Narang et al, Methods enzymol, 65: 610-620(1980) (phosphotriester method). Protein nucleic acid molecules can be prepared using known methods, for example, by Nielsen et al, bioconjugate. 3-7 (1994).

One method of producing the disclosed proteins described herein is by linking two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using existing laboratory equipment using Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (t-butyloxycarbonyl) chemistry (Applied Biosystems, Inc., Foster City, Calif.). One skilled in the art will readily recognize that, for example, peptides or polypeptides corresponding to the proteins of the present disclosure may be synthesized by standard chemical reactions. For example, a peptide or polypeptide may be synthesized and not cleaved from the synthetic resin, but another fragment of the peptide or protein may be synthesized and then cleaved from the resin, thereby exposing a functionally blocked end group on the other fragment. These two fragments may be covalently linked at their carboxy and amino termini, respectively, by Peptide bonds, by Peptide condensation reactions to form antibodies or fragments thereof (Grant GA (1992) < Synthetic Peptides: User guides >, (W.H.Freeman and Co., N.Y. (1992); Bodansky M and Trost B., eds. (1993) < Principles of Peptide Synthesis >, (Principles of Peptide Synthesis), Springer-Verlag Inc., NY (at least incorporated herein by reference for its material relevant to Peptide Synthesis.) alternatively, the Peptides or polypeptides may be independently synthesized in vivo as described herein.

For example, enzymatic ligation of cloned or synthetic peptide fragments allows for the ligation of relatively short peptide fragments to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen L et al, Biochemistry, 30: 4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be used to synthetically construct large peptides or polypeptides from shorter peptide fragments. The method consists of a two-step Chemical reaction (Dawson et al, Synthesis of proteins by Natural Chemical Ligation, Science 266: 776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide-thioester with another unprotected peptide segment containing a Cys residue at the amino terminus, giving a thioester-linked intermediate as the initial covalent product. Without changing the reaction conditions, the intermediate undergoes a spontaneous, rapid intramolecular reaction, forming a natural peptide bond at the ligation site (Baggiolini M et al, (1992) FEBS Lett.307: 97-101; Clark-Lewis I et al, J.biol.chem., 269: 75 (1994); Clark-Lewis I et al, Biochemistry, 30: 3128 (1991); Rajaratham K et al, Biochemistry 33: 6623-30 (1994)).

Alternatively, unprotected peptide fragments are chemically linked, since the bond formed between the peptide fragments by chemical linking is a non-natural (non-peptide) bond (Schnolzer, M et al, Science, 256: 221 (1992)). This technique has been used to synthesize analogues of Protein domains, as well as a large number of relatively pure proteins with full biological activity (Delisle Milton RC et al, Techniques in Protein Chemistry (IV), academic Press, New York, pp.257-267 (1992)).

Methods for making the nucleic acids disclosed herein and for making nucleic acids that can be used to express the protein and peptide molecules described herein are disclosed. There are a variety of methods available for making these compositions, such as synthetic chemistry methods and standard molecular biology methods. It is to be understood that methods of making these and other disclosed compositions have been specifically disclosed.

Disclosed is a nucleic acid molecule produced by a method comprising: operably linking a polypeptide comprising SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26 or SEQ ID NO: 28, linked to a sequence controlling the expression of said nucleic acid.

Also disclosed are nucleic acid molecules produced by a method comprising: operably, a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26 or SEQ ID NO: 28, linked to a sequence controlling the expression of said nucleic acid.

Disclosed is a nucleic acid molecule produced by a method comprising: operably, the sequence contained is hybridized under stringent hybridization conditions with the sequence set forth in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26 or SEQ ID NO: 28, linked to a sequence controlling the expression of said nucleic acid.

Disclosed is a nucleic acid molecule produced by a method comprising: operably, the sequence contained encodes the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. or SEQ ID NO: 36 to SEQ ID NO: 47, linked to a sequence controlling the expression of said nucleic acid molecule.

Disclosed is a nucleic acid molecule produced by a method comprising: operably linking a peptide encoded by the contained sequence to the sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. or SEQ ID NO: 36 to SEQ ID NO: 47, linked to a sequence controlling the expression of said nucleic acid molecule.

Disclosed is a nucleic acid molecule produced by a method comprising: operably linking the peptide encoded by the contained sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. or SEQ ID NO: 36 to SEQ ID NO: 47, and wherein any change is conservative, is linked to a sequence that controls the expression of the nucleic acid molecule.

Therapeutic agents

The compositions disclosed herein, including pharmaceutical compositions, can be administered in a variety of ways depending on whether local or systemic treatment is desired, and depending on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, orally, parenterally (e.g., intravenously), intratracheally, ocularly, vaginally, rectally, intranasally, topically, etc., including intranasal topical administration or inhalation administration.

Parenteral administration of a composition, if employed, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Modified methods for parenteral administration include the use of sustained release or sustained release systems to maintain a constant dosage. See, for example, U.S. Pat. No.3,610,795, incorporated herein by reference.

The compositions disclosed herein can be administered prophylactically to a patient or subject at risk for vascular permeability or pathological angiogenesis. Thus, the methods can further comprise identifying an individual at risk for vascular permeability or pathological angiogenesis prior to administration of the compositions disclosed herein.

The exact amount of the composition required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the allergic disease being treated, the particular nucleic acid or vector used, the mode of administration thereof, and the like. Therefore, it is not possible to specify an exact amount for each composition. For example, effective dosages and schedules for administering the compositions can be determined empirically, and making such determinations is within the skill of the art. The dosage range of the composition administered is large enough to produce the desired effect affecting the symptomatic disease. The dosage should not be so large as to cause adverse side effects such as unwanted cross-reactions, allergic reactions, etc. In general, the dosage will vary with the age, condition, sex, extent of illness, route of administration, or whether other drugs are included in the treatment regimen of the patient, and can be determined by one skilled in the art. In the event of any contraindication, the dosage is adjusted by the individual physician. The dosage may vary and one or more doses may be administered daily for one or more days. Guidance on suitable dosages for a given type of pharmaceutical product can be found in the literature. For example, guidance in selecting an appropriate dosage of an antibody can be found in the literature for therapeutic use of Antibodies, e.g., Handbook of Monoclonal Antibodies (Handbook of Monoclonal Antibodies), major eds by Ferrone et al, Nos. Publications, Park Ridge, NJ., (1985) chapters 22 and p 303-357; smith et al, "Antibodies in Human diagnostics and therapeutics" (Antibodies in Human diagnostics and Therapy), edited by Haber et al, Raven Press, New York (1977) pp.365-389. Typical daily dosages of peptide or protein therapeutic agents alone may range from about 1 μ g/kg to up to 100mg/kg body weight per day, depending on the factors mentioned above. For example, the concentration of the ligands, proteins, peptides, and targeting signals disclosed herein can be in the range of about 1pM to 100 μ M in a subject, including about 1pM, 2pM, 3pM, 4pM, 5pM, 6pM, 7pM, 8pM, 9pM, about 10pM, about 20nM, about 30nM, about 40nM, about 50nM, about 60nM, about 70nM, about 80nM, about 90nM, or about 100nM, about 1 μ M, 2 μ M, 3 μ M, 4 μ M, 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, about 10 μ M, about 20 μ M, about 30 μ M, about 40 μ M, about 50 μ M, about 60 μ M, about 70 μ M, about 80 μ M, about 90 μ M, or about 100 μ M.

Examples

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the compositions and methods described herein in any way. In each case, unless otherwise indicated, standard materials and methods were used to perform the work described in the examples provided. All patents and literature references cited in this specification are hereby incorporated by reference in their entirety.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, Molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art (see, e.g., Maniatis, T. et al, (1982) 'Molecular Cloning: A Laboratory Manual) (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); Sambrook, J. et al (1989)' Molecular Cloning: Laboratory Manual (second edition) (Molecular Cloning: A Laboratory Manual, 2)^ndEd.) (Cold Spring Harbor Laboratory, Cold Spring Harbor, n.y.); ausubel, f.m., et al (1992), "modern methods of molecular biology" (Current Protocols in molecular biology), (j.wiley and Sons, NY); glover, D. (1985) DNA Cloning (I and II) (DNA Cloning, I and II) (Oxford Press); anand, R. (1992) Complex genomics Analysis Techniques for the Analysis of complex genomics), (Academic Press); guthrie, G. and Fink, G.R (1991) Guide to Yeast Genetics and molecular biology (Guide to Yeast Genetics and molecular biology) (Academic Press); harlow and Lane (1988) antibodies: laboratory guidelines (Antibodies: A Laboratory Manual) (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); jakoby, W.B. and Pastan, I.H, (eds.) (1979) methods in Cell culture, enzymology, Vol.58 (Cell culture. methods in enzymology) y, vol.58) (Academic Press, Inc., Harbour Brace Jovanovich (NY); nucleic Acid Hybridization (Nucleic Acid Hybridization) (B.D. Hames&S.j. higgins master edition, 1984); transcription And Translation (Transcription And Translation) (b.d. hames)&S.j. higgins master edition, 1984); culture of animal Cells (Culture of animal Cells) (r.i. freshney, Alan r.liss, inc., 1987); fixed Cells And Enzymes (Immobilized Cells And Enzymes) (IRL Press, 1986); perbal, Guide To practice of Molecular Cloning (A Practical Guide To Molecular Cloning) (1984); monograph, Methods In Enzymology (the treatise, Methods In Enzymology) (Academic Press, Inc., N.Y.); gene Transfer Vectors For Mammalian Cells (Gene Transfer Vectors For Mammalian Cells) (J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); enzymatic methods (MethodsIn Enzymology), volumes 154 and 155 (Wu et al eds.); immunochemical Methods In Cell And molecular biology (Immunochemical Methods In Cell And molecular biology) (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology (Handbook Of Experimental Immunology), volumes I-IV (eds. D.M.Weir and C.C.Blackwell, 1986); hogan et al eds (1994), mouse embryo manipulation, Laboratory Manual (second edition), Manipulating the mouse embryo ^ndEdition), Cold Spring Harbor laboratory Press, Cold Spring Harbor, n.y. A general discussion of techniques and materials for human gene mapping, including human chromosome 1 mapping, is provided, for example, in White and Lalouel (1988) ann. 259-279. The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics and immunology. (see, e.g., Maniatis et al, 1982; Sambrook et al, 1989; Ausubel et al, 1992; Glover, 1985; and, 1992; Guthrie and Fink, 1991).

Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It should be clearly understood that, although a number of publications are cited herein, these citations are not to be construed as an admission that any of these documents forms part of the common general knowledge in the art.

Example 1

Robo4 is required for in vivo vascular targeting: zebrafish have become an attractive model for analyzing vascular development over the past decade (Weinstein, 2002), and have been selected to study the biological importance of Robo4 in vivo. To inhibit Robo4 gene expression, the previously described splicing-blocking morpholino targeting the exon 10-intron 10 boundary of Robo4 pre-mRNA was used (Bedell et al, 2005). To confirm the efficacy of Robo4 morpholinos, RNA was isolated from uninjected and morpholino-injected embryos and analyzed by RT-PCR using primers flanking the target exon (fig. 8A). Injection of Robo4 morpholino resulted in complete loss of wild type RNA compared to non-injected controls, indicating no Robo4 function in morpholino (morphant) zebrafish (fig. 8B).

TG(flil:egfp)^y1Zebra fish embryos expressed green fluorescent protein under the control of an endothelial-specific flil promoter and allowed detailed in vivo observation of developing endothelium, were utilized to assess the consequences of morpholino-mediated Robo4 knock-out on vascular development (FIG. 1A; Lawson and Weinstein, 2002). At 48hpf, embryos injected with Robo4 MO exhibited wild-type formation of the primitive central axis tubes (dorsal aorta and posterior major vein), and dorsal longitudinal anastomotic and paraspinal vessels, respectively, indicating angiogenesis and vasculogenesis, were not affected by reduced levels of Robo4 (FIG. 1B, right panel). However, a significant degree of abnormality in the internodal vascular architecture was observed in Robo4 morphotypes. In wild type embryos, the internodal vessels originate in the dorsal aorta and grow towards the dorsal surface of the embryo, next to the somite border. It is this precise trajectory of the internode that determines the characteristic chevron shape of the internode vessel (fig. 1A, right). Internodal vascular graft of Robo4 morpholine-type embryosInstead, it grows in the wrong direction (FIG. 1B, right-hand side: white arrows indicate abnormal blood vessels). At 48hpf, 60% of Robo MO-injected embryos showed this defect, compared to only 5% in wild-type embryos. Importantly, Robo4 morphotypes could not be distinguished from control embryos by phase microscopy, indicating that the observed angioplastic defects were not the result of gross morphological perturbations. Taken together, these data demonstrate the need for Robo4 during zebrafish vascular development and indicate that the functional consequences of this receptor trigger a repulsive guidance cue.

Example 2

The cytoplasmic tail of Robo4 is required for vascular targeting in vivo: it was next determined whether the vascular defects observed in Robo4 morphotypes could be inhibited by reconstituting Robo 4. Robo4MO and morpholino-resistant wild type murine Robo4RNA were injected into TG (flil: egfp)^y1In the embryos, the formation of blood vessels was analyzed at 48 hpf. Robo4RNA restored a typical pattern of major vessels in approximately 60% of morpholino embryos, confirming the specificity of gene knock-out (FIGS. 1B and C, right panel).

The ability of robo4 to regulate vascular development may be a result of its ability to transmit cytoplasmic signals. To confirm this idea, Robo4MO was co-injected with a murine Robo4 mutant form (Robo4 Δ tail) lacking the portion of the receptor that interacts with cytoplasmic components, and the architecture of the blood vessels was assessed at 48 hpf. Unlike wild-type Robo4RNA, Robo4 Δ tail failed to rescue molding defects in morpholino embryos (FIGS. 1B and D, right panel). These data demonstrate that the information contained in the cytoplasmic tail of Robo4 is critical for vascular targeting during zebrafish embryogenesis. Taken together, these in vivo analyses indicate that Robo4 activity is required for accurate determination of the trajectory of internodal vessels during vertebrate vascular development.

Example 3

The Robo4 cytoplasmic tail is required for inhibition of thigmotaxis: slit2-Robo4 signaling inhibited primary endothelial cell migration toward the VEGF gradient, as well as HEK 293 cells ectopically expressing Robo4 toward serum (Park et al, 2003; Seth et al, 2005). In addition to soluble growth factors, immobilized extracellular matrix proteins such as fibronectin play an important role in cell motility (Ridley et al, 2003), and gradients of fibronectin can direct migration in a process called thigmotaxis. In fact, it has recently been shown that fibronectin is located near migrating endothelial cells in early zebrafish embryos (Jin et al, 2005). Robo4 is an observation required for proper migration of endothelial cells in vivo (FIG. 1), indicating the ability of Slit2-Robo4 signaling to modulate fibronectin-induced thigmotaxis. HEK 293 cells were transfected with Robo4 or Robo4 delta tail (fig. 2A) and subjected to chemotactic migration analysis on membranes coated with a mixture of fibronectin and Slit 2. Slit2 inhibited fibronectin-induced migration of cells expressing Robo4 but not Robo4 Δ tail, confirming that the repulsive activity of the cytoplasmic tail of Robo4 was critical for the receptor (fig. 2B).

The region of the Robo4 cytoplasmic tail required for inhibition of cell migration was next identified. HEK 293 cells were transfected with the Robo4 deletion construct (fig. 2A) and subjected to a chemotactic migration assay. Fibronectin-dependent migration of Robo4-NH2, but not Robo4-COOH expressing cells, was inhibited by Slit2 (FIG. 2C), demonstrating that the N-terminal half of the cytoplasmic tail of Robo4 is necessary and sufficient for modulating cell motility.

Example 4

The paxillin family member is the Robo4 interacting protein: the identification of regions in the Robo4 cytoplasmic tail that confer functional activity allows for the search for cytoplasmic components that can modulate Robo4 signaling. Yeast two-hybrid screening of a human aortic cDNA library using the N-terminal half of the tail region of Robo4 as bait identified Hic-5, a member of the paxillin adapter family, as a possible Robo4 interacting protein (fig. 8). To confirm this interaction, the Hic-5 plasmid was isolated and re-transformed into yeast with Robo4 or the empty vector. Only the strains co-expressing Robo4 and Hic-5 were able to grow on the auxotrophic medium and induce potent beta-galactosidase activity (FIG. 8B). To further confirm this interaction, co-immunoprecipitation experiments were performed using mammalian cells co-transfected with Hic-5 and Robo4 cytoplasmic tails. Hic-5 was found in anti-Robo 4 immunoprecipitates from HEK 293 cells expressing Robo4 and Hic-5, but not in Hic-5-expressing cells (FIG. 3A). Taken together, these data demonstrate that Hic-5 specifically interacts with the cytoplasmic tail of Robo4 in yeast and mammalian cells.

Hic-5 and its paralogs, paxillin, may exhibit cell type-specific expression (Turner, 2000; Yumiamochi et al, 2003). Thus, it was determined which of these proteins were expressed in the cell line HEK 293 cells used for the chemotactic migration assay. Western blot analysis of cell lysates from CHO-K1, HEK 293 and NIH3T3 cells with antibodies against Hic-5 or paxillin detected paxillin in all cell lines, but only Hic-5 was found in CHO-K1 and NIH3T3 cells (FIG. 3B). This not only indicates that Hic-5 and paxillin are able to interact with Robo4 to regulate cell migration, but also that paxillin may be a binding partner in HEK 293 cells. Looking at the latter idea, co-immunoprecipitation experiments were performed using mammalian cells expressing paxillin and the cytoplasmic tail of Robo 4. As observed with Hic-5, paxillin was identified in anti-Robo 4 immunoprecipitates from HEK 293 cells expressing paxillin and Robo4, but not in cells expressing paxillin alone (fig. 3C).

Since Slit2 is a physiological ligand of Robo4 (Park et al, 2003; Hohennester et al, 2006), it was determined whether Slit2 stimulation modulates the interaction between Robo4 and paxillin. HEK 293 cells expressing Robo4 were incubated in the presence and absence of Slit 2. Endogenous paxillin was detected in Robo4 immunoprecipitates in the presence of Slit 2. In sharp contrast, in the absence of Slit2, no paxillin was detected in the immunoprecipitates (fig. 3E). Thus, engagement of Robo4 with Slit2 stimulated its binding to paxillin.

Example 5

Identification of paxillin interaction motif of Robo 4: to accurately determine the region of Robo4 required for interaction with paxillin, a series of GST-Robo4 fusion proteins spanning the entire length of the cytoplasmic tail were generated (FIG. 4A). In vitro binding analysis with purified recombinant paxillin demonstrated that the amino-terminal half of the tail region of Robo4 (494-731) was necessary and sufficient for direct interaction with paxillin (FIG. 4B). 4 additional GST-Robo4 fusion proteins containing an approximately 70 amino acid fragment of the amino-terminal half of the cytoplasmic tail were then generated (FIG. 4C). In vitro binding assays showed that the paxillin selectively interacts with the fragment of the tail region of Robo4 located between the CC0 and CC2 motifs (604-674; FIG. 4D). To determine whether this Robo4 region was necessary for interaction with the paxillin, amino acids 604-674 were deleted from the cytoplasmic tail and the mutant GST-Robo4 fusion protein was analyzed for in vitro binding. Although the interaction with paxillin was reduced, binding to the known Robo4 binding protein Mena was also reduced, indicating that elimination of amino acid at position 604-674 affected the conformation of the tail region of Robo 4. To avoid this problem, a small deletion was made within this 70 amino acid stretch and an additional in vitro binding assay was performed. In this way, a mutant GST-Robo4 fusion protein lacking 36 amino acids (604-639; FIG. 9) was identified which lost binding to the paxillin but retained binding to Mena (FIG. 4E). To this end, this Robo4 region is referred to as the Paxillin Interaction Motif (PIM).

Example 6

Paxillin interaction motifs are required for Robo 4-dependent chemotactic inhibition: it was next determined whether the paxillin interaction motif of Robo4 is important for the functional activity of the receptor. A mutant form lacking amino acids 604-639 of full-length Robo4 (Robo 4. DELTA. PIM) was generated by site-directed mutagenesis and used in the chemotactic migration assay. Robo4 Δ PIM was unable to mediate Slit2 directed inhibition of migration towards fibronectin gradients (FIG. 4F), demonstrating that the region necessary for paxillin binding in the tail region of Robo4 is also desirable for Robo 4-dependent inhibition of cell migration.

Example 7

Slit2-Robo4 signaling inhibited cell spreading and adhesion-dependent Rac activation: the ability of immobilized Slit2 to inhibit the migration of cells expressing Robo4 on fibronectin may result from negative regulation of adhesion and/or spreading on this ECM protein. To determine whether Slit2-Robo4 signaling affected these processes, HEK 293 cells were transfected with Robo4 or empty vector (pcDNA3) and analyzed for adhesion and spreading on fibronectin. Although cells expressing Robo4 adhered normally to the fibronectin and Slit2 coated coverslips, their spreading was significantly reduced compared to cells transfected with pcDNA3 (FIG. 5A). These data indicate that Slit2-Robo4 signaling regulates intracellular pathways that control cell spreading.

The ability of cells to spread on ECM proteins such as fibronectin is regulated by the activation of small GTPases of the Rho family, including Rho, Cdc42 and Rac migration (Nobes and Hall, 1995; Nobes and Hall, 1998). Among these proteins, Rac plays a fundamental role in promoting actin polymerization leading to cell spreading and migration (Nobes and Hall, 1995; Nobes and Hall, 1998). This established relationship between Rac and cell spreading suggests that Slit2-Robo4 signaling may inhibit adhesion-dependent activation of Rac. To assess this possibility, HEK 293 cells were transfected with either Robo4 or pcDNA3, plated on dishes coated with fibronectin and Slit2, and assayed for Rac-GTP levels using a GST-PBD pull down assay. Cells expressing Robo4 showed significantly lower adhesion-stimulated Rac activation compared to cells transfected with pcDNA3 (FIGS. 5B and C). To confirm the specificity of this effect, Cdc42 activation was also detected in cells expressing Robo4, which were also exposed to Slit2 (fig. 11A). This result is supported by the observation that Robo4 does not interact with the Robo1 binding protein srGAP1, a known Cdc42 GTPase activating protein (FIG. 11B). Taken together, these data demonstrate that Slit2-Robo4 signaling specifically inhibits adhesion-induced Rac activation.

Example 8

Paxillin interaction motifs are required for Robo 4-dependent inhibition of cell spreading and Rac activation: next, it was evaluated whether Robo4 Δ PIM was able to inhibit fibronectin-induced cell spreading and Rac activation. HEK 293 cells were transfected with Robo4 Δ PIM, plated on fibronectin and Slit2 coated surfaces, and subjected to spreading or Rac analysis. This mutant form of the receptor failed to inhibit cell spreading and adhesion-dependent Rac activation (fig. 5D, E and F), demonstrating that the paxillin interaction motif is essential for the functional activity of Robo4 in vitro.

To confirm that the Robo 4-dependent inhibition of cell spreading was primarily due to inhibition of Rac activation, HEK 293 cells were co-transfected with Robo4 and the dominant active form of Rac-Rac (G12V) and subjected to spreading analysis. Cells expressing Rac (G12V) were resistant to Robo 4-dependent inhibition of cell spreading (fig. 5G), demonstrating that Slit2-Robo4 signaling blocks spreading by inhibiting Rac activity.

Example 9

Slit2 inhibited VEGF-induced Rac activation in primary human endothelial cells: slit2 inhibited VEGF-stimulated migration of several primary human endothelial cell lines (Park et al, 2003), and Rac plays a fundamental role in VEGF-induced cell motility (Soga et al, 2001 a; Soga et al, 2001 b). Thus, it was determined whether Slit2-Robo4 signaling could inhibit the activation of Rac in an endogenous context. Human Umbilical Vein Endothelial Cells (HUVECs) were stimulated with VEGF in the presence and absence of Slit2, and GTP-Rac levels were analyzed with GST-PBD pull down analysis. Treatment with Slit2 completely inhibited VEGF-stimulated Rac activation (fig. 5H and I), demonstrating that endogenous Slit2-Robo4 signaling regulates Rac activation.

Example 10

Lim4 of paxillin is required for interaction with Robo4 and Robo 4-dependent inhibition of cell spreading: although Robo4 Δ PIM maintained its interaction with Mena (fig. 4E), it is likely that this mutation interfered with Robo4 interaction with proteins other than paxillin. To specifically address this issue, mutants of paxillin were generated that had disrupted binding to Robo 4. Paxillin is a regulatory protein consisting of an N-terminal leucine/aspartate (LD) repeat and a C-terminal Lim domain (fig. 6A). Analysis of clones recovered from the yeast two-hybrid screen (see fig. 9A) showed that Lim domains, particularly Lim3 and Lim4, are important for interaction with Robo 4. To confirm this idea, co-immunoprecipitation experiments were performed using HEK 293 cells co-transfected with paxillin-LD or paxillin-Lim at the tail region of Robo 4. paxillin-Lim was found in Robo4 co-immunoprecipitates, but paxillin-LD was not found (fig. 6B), confirming that the Lim domain of paxillin is necessary and sufficient for interaction with Robo 4. To elucidate which Lim domain is required for binding to Robo4, serial deletions were made from the carboxy terminus of paxillin, co-transfected with the tail region of Robo4 into HEK 293 cells, and co-immunoprecipitation experiments were performed. The Lim4 domain deletion of paxillin completely abolished binding to Robo4 (fig. 6C), demonstrating that this region of paxillin is critical for its ability to interact with Robo 4.

Delineation of the Robo4 binding site on paxillin allows a direct assessment of the role of paxillin in Robo 4-dependent inhibition of cell spreading. Endogenous pilin was knocked out using siRNA in HEK 293 cells and reconstituted with either wild-type chicken pilin (Ch-pilin) or Ch-pilin Δ Lim4 (fig. 6D). These cells were then spread for analysis on fibronectin and Slit2 coated coverslips. Cells expressing Ch-paxillin Δ Lim4 were resistant to Robo 4-dependent inhibition of cell spreading, while cells expressing Ch-paxillin showed a characteristic decrease in cell area (fig. 6E). These data demonstrate that interaction of paxillin with Robo4 enables Slit2-Robo4 signaling to inhibit cell spreading.

Example 11

The paxillin interaction motif is required for vascular targeting in vivo: the requirement for the paxillin interaction motif of Robo4 during zebrafish vascular development was assessed. As described previously, robo4MO was injected into TG (flil: egfp)^y1EmbryoCausing a disturbance of the internode blood vessels (see fig. 1B). Co-injection of robo4 Δ PIM RNA deepened the defects caused by robo4MO, while wild-type robo4RNA suppressed these defects (fig. 7A). Neither Robo4 Δ tail nor Robo4 Δ PIMRNA was able to rescue the angioplastic defect in morpholino (morphant) embryos, confirming that the 36 amino acid paxilin interaction motif is a key signaling module in the Robo4 cytoplasmic tail. Furthermore, these data indicate that interaction between paxillin and Robo4 is necessary for proper modeling of zebrafish vasculature.

Example 12

We determined that Robo4 interacts with paxillin and inhibits prominent activity, which prompted us to determine whether Robo4 affected the Arf6 pathway. Cells expressing α IIb-Robo4: β 3 were plated on fibronectin alone, or fibronectin and fibrinogen, and analyzed for levels of Arf6-GTP using GST-GGA3 affinity precipitation techniques. Although fibronectin stimulated the activation of Arf6, fibrinogen decreased the level of Arf6-GTP in cells expressing α IIb-Robo4: β 3 (fig. 16A). This result confirms that Robo4 signaling inhibits the activation of Arf6 and indicates that the ability of Robo4 to block Rac activity results from its regulation of Arf 6.

Next, we analyzed the need for the paxillin-GIT 1 complex in Robo 4-dependent inhibitory highlighted activity. Paxillin Binding Sequence (PBS) on GIT1 was found on the carboxy terminus of the protein and has been shown to prevent interaction of GIT1 and paxillin (Uemura et al, 2006). Cells were transfected with α IIb-Robo 4:. beta.3 with empty vector or GIT1-PBS and spreading assays were performed on fibronectin, or fibronectin and fibrinogen. As described previously, cells expressing α IIb-Robo 4:. beta.3 showed a decrease in cell area when plated on fibrinogen, but this phenomenon of area disappeared in cells transfected with GIT1-PBS (FIG. 16B). We repeated this experiment in cells expressing full-length Robo4 plated on fibronectin or fibronectin and Slit2, and similar to the chimeric receptor experiment, GIT1-PBS prevented Slit 2-dependent reduction in cell area (fig. 16C). These data confirm that a functional paxillin complex is required for Slit2-Robo4 signaling.

To determine whether Slit2-Robo4 signaling inhibited prominent activity by inactivating Arf6, we co-transfected Arf6 guanosine exchanging factor ARNP with Robo4 and performed a spreading assay. Overexpression of ARNO blocked the ability of Slit2 to reduce cell area, suggesting that the primary role of Slit2-Robo4 signaling is to prevent GTP loading of Arf6 (fig. 16C). If ARNO restores the ability of Robo4 expressing cells to spread on Slit2, we conclude that it will also reconstitute the activation of Rac in response to fibronectin. Indeed, overexpression of ARNO resulted in normal levels of GTP-Rac in cells plated on fibronectin and Slit2 (fig. 16D). Taken together, these experiments demonstrated that Slit2-Robo4 signaling inactivates Arf6, resulting in a local block of Rac activation and subsequent inhibition of membrane protrusion necessary for cell spreading and migration.

Example 13

Immunoprecipitation confirmed the interaction between Slit ligands and the Robo4 receptor: cell lysates of uninfected human embryonic kidney cells (HEK), HEK cells transfected with Slit bearing a myc epitope tag (Slit-myc), HEK cells transfected with Robo4 bearing an HA epitope tag (Robo4-HA), and HEK cells transfected with a control vector (control-HEK) were immunoprecipitated. After mixing the Slit-myc and Robo4-HA cell lysates and immunoprecipitation with anti-HA antibody, Slit-myc protein was detected by Western blotting with anti-myc antibody (FIG. 17A, lane 6). The specificity of this interaction was confirmed by the inability to detect Slit proteins using all other lysate combinations (fig. 17A, lanes 2-5). The same amount of lysate was used in each experiment. Western blot analysis of lysates of Slit-myc cells was used as a control, demonstrating that Slit proteins are consistent with previous reports, with a molecular weight of approximately 210kD (FIG. 17A, lane 1). The lower band shown in lanes 2-6 of FIG. 17A corresponds to an immunoglobulin heavy chain.

Conditioned media from untransfected HEK cells (HEK CM), HEK cells transfected with Slit bearing a myc epitope tag (Slit-myc CM), HEK cells transfected with the N-terminal soluble extracellular domain of Robo4 bearing an HA epitope tag (NRobo4-HA CM), and HEK cells transfected with a control vector (control-HEK CM) were also immunoprecipitated. The full-length Slit-myc protein (210kD) and its C-terminal proteolytic fragment (70kD) were detected in Slit-myc CM by anti-myc antibody (FIG. 17B, lane 1). After mixing Slit-myc and Robo4-HA conditioned media and immunoprecipitation with anti-HA antibody, Slit-myc protein was also detected by Western blotting (FIG. 17B, lane 6). The specificity of this interaction was confirmed by the inability to detect Slit protein using all other combinations of conditioned media.

As shown in fig. 17C-17F, Slit proteins bound to the plasma membrane of cells expressing Robo 4. Binding of Slit-myc protein can be detected using anti-myc antibodies and anti-mouse antibodies to which Alexa 594 binds. As can be seen in fig. 17D and 17F, binding was detected on the surface of Robo4-HEK cells (fig. 17F) but not on the surface of control-HEK cells (fig. 17D).

Example 14

Robo4 knockout mice: to determine the functional significance of Robo4 in vivo, knockout mice were generated using standard techniques. To produce knockout mice, homologous recombination was used, with exons 1 to 5 of the gene expressing Robo4 replaced with an Alkaline Phosphatase (AP) reporter gene. The allele Robo4^APThe exons of the immunoglobulin (IgG) repeat sequences encoding the extracellular domain of Robo4 that are predicted to be required for interaction with Slit proteins are missing. Mixing Robo4^+/APAnimals were intercrossed to produce mice homozygous for the target allele. A schematic representation of the genomic structure of the mice is provided in figure 25. Robo4^AP/APThe animals were viable and fertile, exhibiting normal formation of the vascular system. These data indicate that Robo4 is not essential for sprouting angiogenesis in developing mice and indicate that there is an alternative function of Robo4 signaling in mammalian endothelium. In these animals, the presence of alkaline phosphatase activity throughout all vascular bed endothelium of developing embryonic and adult mice was detected, confirming that Robo4^APThe allele is Robo4 expression of a useful marker.

Example 15

Activation of Robo4 stabilizes mature vessels: the central region of the murine retinal vascular plexus, which contains exclusively stalk cells, is an example of a differentiated/stable phenotypic characteristic of mature, cavitated blood vessels. Therefore, we conclude that expression of Robo4 in stalk cells may maintain this phenotype by inhibiting processes stimulated by pro-angiogenic factors such as VEGF-A. The effect of Robo4 signaling on VEGF-A stimulated processes was evaluated using a VEGF-A endothelial cell migration assay and a VEGF-A tube formation assay. Both assays are routinely used to study angiogenesis in vitro.

For endothelial cell migration and tube formation assays, a cell from Robo4 was isolated^+/+And Robo4^AP/APLung endothelial cells of mice, and their identity was confirmed using immunocytochemistry and flow cytometry. These cells were then used in VEGF-A dependent endothelial cell migration and tube formation assays. The molecule of Slit2 used in these analyses was Slit2N (SEQ ID NO: 39). As shown in FIGS. 19A and 19B, Slit2 suppressed Robo4^+/+Migration of endothelial cells and tube formation. However, in Robo4^AP/APIn endothelial cells, the inhibitory activity of Slit2 was lost. These results demonstrate that Slit2 inhibits endothelial cell migration and tube formation in a Robo 4-dependent manner, and indicate that activation of Robo4 by Slit2 can be used to stabilize the vascular endothelium of mature vessels.

Example 16

Activation of Robo4 preserved endothelial barrier function: in mature vascular beds, endothelial cells behave not independently of each other; instead, they form a monolayer that prevents the movement of proteins, fluids and cells from the endothelial lumen into the surrounding tissue. This barrier function was simulated in vitro using the Transwell assay to analyze the transport of horseradish peroxidase (HRP) through a slave Robo4^+/+And Robo4^AP/APConfluent cell monolayers of endothelial cells obtained from mouse lungs. Stimulation of Robo4 with the known permeability-inducing factor VEGF-A ^+/+And Robo4^AP/APEndothelial cells, enhanced HRP accumulation in the lower portion of the Transwell chamber. However, as shown in FIG. 19C, cell monolayers were pretreated with Slit2 protein (Slit2N (SEQ ID NO: 39)) in Robo4^+/+This effect is prevented in endothelial cells, but is shown in Robo4^AP/APNot in endothelial cells.

Next, the effect of Slit2 on endothelial barrier function in vivo was evaluated. By injecting Evans blue into Robo4^+/+And Robo4^AP/APIn the tail vein of mice, the Miles assay was performed. VEGF-A was then injected into the dermis in the presence and absence of Slit2 protein (Slit2N (SEQ ID NO: 39)). Similar to in vitro assays, in Robo4^+/+VEGF-A stimulated Evans blue leakage into the dermis in mice could be prevented by the simultaneous administration of Slit2 protein, but in Robo4^AP/APNone of the mice (shown in figure 19D). These observations were extended to evaluate the ability of Slit2 to inhibit VEGF-a-induced retinal endothelial hyperpermeability. In particular, it is found in Robo4^+/+Intravitreal injection of VEGF-A in mice induced leakage of Evans blue from retinal blood vessels. However, in Robo4^+/+In mice, such VEGF-A-induced leakage of Evans blue from retinal vessels was inhibited by co-injection of the Slit2 protein Slit2N (SEQ ID NO: 39)) (FIG. 19E). The experiment is described in Robo4 ^AP/APReplicates were found in the retinas of mice, and Robo4 was found^AP/APIt was resistant to treatment with Slit2N (SEQ ID NO: 39). These data confirm that Robo4 mediates Slit 2-dependent inhibition of VEGF-A induced endothelial hyperpermeability in vitro and in vivo.

Example 17

Robo4 blocks VEGF signaling downstream of VEGF receptors: the ability of VEGF-a to promote angiogenesis and permeability is dependent on the activation of VEGFR2, which occurs through autophosphorylation following ligand binding. Subsequently, many non-receptor tyrosine kinases, serine/threonine kinases, and small GTPases are activated in a spatially and temporally specific manner, performing VEGF-a signaling. To determine that Slit2-Robo4 signaling crossed the VEGF-A-VEGFR2 pathway, VEGF-A and Slit2 stimulated phosphorylation of VEGFR2 was analyzed using Slit2N (SEQ ID NO: 39). Slit2N (SEQ ID NO: 39) had NO effect on VEGF-A-induced VEGFR2 phosphorylation (FIG. 19F), indicating that the Slit2-Robo4 pathway must intersect VEGF-A signaling downstream of the receptor. Attention was then focused on the Src family of non-receptor tyrosine kinases, Fyn Yes and Src, as they have well-documented functions in mediating VEGF-A induced angiogenesis and permeability (Eliceiri et al, 2002; Eliceiri et al, 1999). Treatment of endothelial cells with Slit2N (SEQ ID NO: 39) reduced VEGF-A stimulated c-Src phosphorylation (FIG. 19G). Recently, several reports have shown that activation of Src-dependent Rho family small GTPase, Rac1, is essential for VEGF-A induced endothelial cell migration and permeability (Gavard et al, 2006; Garrett et al, 2007). Treatment of the endothelial cell monolayer with Slit2N (SEQ ID NO: 39) prevented VEGF-A dependent Rac1 activation (FIG. 19H). These biochemical experiments indicate that the Slit2-Robo4 pathway inhibits VEGF-A-induced endothelial cell migration and hyperpermeability by inhibiting the Src-Rac1 signaling axis.

Example 18

Activation of Robo4 reduced vascular leakage and pathological angiogenesis in CNV and OIR models: a murine model of oxygen-induced retinopathy (OIR), which mimics the ischemia-induced angiogenesis observed in diabetic retinopathy and retinopathy of prematurity, was used to study the effect of Robo4 signaling on retinal vascular disease. In this model, P7 mice were maintained in a 75% oxygen environment for 5 days, then returned to 25% oxygen for an additional 5 days. Sensing oxygen deficiency initiates a rapid increase in VEGF-A expression in the retina, leading to pathological angiogenesis (Ozaki et al, 2000; Werdich et al, 2004). Using the model pair Robo4^+/+Mouse and Robo4^AP/APMice were evaluated. At Robo4^+/+Intravitreal administration of Slit2N (SEQ ID NO: 39) significantly reduced angiogenesis in mice, but at Robo4^AP/APNone of the mice (fig. 20A-20E, in which the arrows indicate the areas of pathological angiogenesis). Further, Robo4 after exposure to high oxygen conditions^AP/APMouse and Robo4^+/+Mice, in contrast, showed more aggressive angiogenesis (see, e.g., fig. 20A and 20C).

In addition to the described OIR model, laser-induced choroidal neovascularization mimics age-related macular degeneration, and is commonly used to study pathological angiogenesis in mice (Lima et al, 2005). In this model, the Bruch membrane is destroyed with a laser, which causes the underlying choroidal vasculature to penetrate the subretinal pigment epithelium. To discern the effect of Robo4 signaling on this pathological process, Robo4, 8-12 weeks old, was studied ^+/+And Robo4^AP/APMice were laser-induced choroidal neovascularization and then intravitreally injected with Slit2N (SEQ ID NO: 39). Similar to the results obtained in the mouse model of oxygen-induced retinopathy, intravitreal administration of Slit2N at Robo4^+/+Angiogenesis was reduced in mice, but in Robo4^AP/APNone of the mice (see fig. 20F-fig. 20J). Taken together, the oxygen-induced retinopathy and choroidal neovascularization models suggest that two vascular beds with distinct characteristics, one being a tight blood-brain barrier and the other being a porous endothelium, are protected from pathological damage by activation of Slit2-Robo4 signaling.

Example 19

Robo4 inhibits signaling from a variety of factors that destabilize mature vessels: the effect of activation of Robo4 by Slit2 molecule on bFGF, angiogenic and thrombin activities, and endothelial permeability factor was evaluated. As shown in FIG. 21, Slit2N (SEQ ID NO: 39) blocked bFGF-induced endothelial tube formation and thrombin-induced permeability. These studies demonstrated that Slit-Robo4 signaling is able to inhibit signaling induced by a variety of angiogenic and permeability factors, and support the concept that the Slit-Robo4 pathway protects mature vascular beds from a variety of angiogenic, permeability factors, and cytokines.

To strengthen the understanding that Robo4 signaling protects the vasculature from the effects of various angiogenic, permeability factors and cytokines, the effect of Slit2N (SEQ ID NO: 39) on Robo4 activation was evaluated in a mouse model of acute lung injury. In this model, mice were administered the bacterial endotoxin LPS by intratracheal administration. Exposure to bacterial endotoxin results in a cytokine storm, causing catastrophic destabilization of the pulmonary vascular bed and producing non-cardiogenic pulmonary edema (Matthay et al, 2005). After intratracheal administration of LPS, mice were treated with Slit2N (SEQ ID NO: 39) or a mock preparation, the latter being a pseudoprotein extract used as a control. As shown in FIG. 22, the concentrations of inflammatory cells and proteins in bronchoalveolar lavage (BAL) fluid from mice treated with Slit2N (SEQ ID NO: 39) were significantly lower than in mice treated with the mock preparation. These results confirm that under these circumstances, activating Robo4 provides a potent vascular stabilizing effect and suggest that Slit2-Robo4 is a potent vascular stabilizing pathway that functions to protect the integrity of mature endothelium and maintain vascular homeostasis against extreme forms of cytokine storm.

Example 20

Administration of Slit2 protein reduced mortality in a mouse avian influenza model: in the following examples, the effect of Slit proteins on the survival of mice infected with avian influenza virus was analyzed. A total of 120 female BALB/c mice were inoculated intranasally with 50. mu.l of a 1: 400 dilution of avian influenza strain H5N 1/Duck/Mn/1525/81. The mice used in this example were obtained from Charles River and the average body weight was in the range of 18-20 grams. Referring to table 2, mice were randomly divided into six cages of 20 mice each, and each group was treated daily for 5 days. Survival (death) and body weight were observed during and after treatment.

TABLE 2

Number of mice/cage	Group number	Whether the infection is	Compound (I)	Dosage form	Treatment schedule
						20	1	Is that	PSS	Volume of 50. mu.l	Qd X4 or 5 (if possible 5), i.v. starting 4 days before exposure to the virus.
20	2	Is that	SLIT "mimetics" 1	Mu.l SLIT/mimic + 34.375. mu.l PSS per mouse	Same as 1#
						20	3	Is that	SLIT 'mimic' 2	Mu.l SLIT/mimic + 48.44. mu.l PSS per mouse	Same as 1#
20	4	Is that	SLIT-concentration 1	Mu.l of 800. mu.g/ml SLIT + 34.375. mu.l PSS per mouse	Same as 1#
						20	5	Is that	SLIT-concentration 2	1.5625. mu.l/mouse of 800. mu.g/ml SLIT + 48.44. mu.l PSS	Same as 1#
20	6	Is that	Ribavirin	75 mg/kg/day	0.1ml I.P.BID X5 days

Briefly, as shown in table 2, group 1 was treated with a Physiological Saline Solution (PSS) negative control. Groups 2 and 3 were treated with mock preparations. Groups 4 and 5 were treated with different concentrations of Slit protein (Slit2N (SEQ ID NO: 39)). As a positive control, 20 mice in 6 groups were treated intraperitoneally with ribavirin at 75 mg/kg/day, adjusted to a total volume of 0.1ml with PSS.

The results of the analysis are shown in fig. 24 and detailed in table 3. After 23 days, mice treated with Slit protein in groups 4 and 5 had lower mortality compared to mice that did not receive Slit protein in groups 1, 2, and 3. Mice from group 4, treated with 12.5 μ g Slit per dose, had a survival rate of 25%. Mice from group 5, treated with 1.25 μ g Slit per dose, had a survival rate of 50%. In contrast to the survivability of groups 4 and 5, only 5% (1/20) of the negative control mice treated with PSS in group 1 survived for 23 days.

Table 3 shows that the average body weight of survivors in groups 1, 2 and 3 was significantly lower than that of Slit-treated survivors in groups 4 and 5 at 14 days after inoculation. In addition, 10/20 mice in the 5 groups with lower Slit treatment concentrations survived at day 21 with an average body weight of 17.6 grams, which was almost as high as the initial average body weight of 17.7 grams. Therefore, those infected mice treated with Slit protein were able to maintain their body weight better than untreated mice.

TABLE 3

Table 3 (continuation)

Example 21

Fragments of Slit protein function to activate Robo 4: fig. 23 shows various constructs of Slit2 protein. As already described herein, the 150kD protein Slit2N (SEQ ID NO: 39) has been found to be effective in vitro and in vivo models, including Miles analysis, retinal permeability analysis, tube formation and endothelial cell migration, and in OIR and CNV models of ocular disease. Furthermore, as shown in FIG. 23, the (40kD) protein SlitD1(SEQ ID NO: 42) and Slit2N (SEQ ID NO: 39) constructs showed similar activity in VEGF-induced endothelial cell migration assay as full-length Slit2(SEQ ID NO: 40).

Materials and methods

Reagent: HEK 293 and COS-7 cells and all IMAGE clones were from ATCC. SP6 and T7 Message Machine kits were from Ambion. HUVEC, EBM-2 and bulletin kits were from Cambrex. The yeast two-hybrid plasmid and reagents were from Clontech. FBS is from Hyclone. anti-HA affinity matrix, Fugene6 and protease inhibitor cocktail were from Roche. Goat anti-mouse HRP and goat anti-rabbit HRP secondary antibodies were from jacksonn immunoresearch. anti-V5 antibody, DAPI, DMEM, Lipofectamine 2000, penicillin-streptomycin, Superscript III kit, Trizol, and TrypLE Express were from Invitrogen. anti-Flag M2, phosphorylase inhibitor cocktail, soybean trypsin inhibitor and fatty acid free Bovine Serum Albumin (BSA) were from Sigma. Human fibronectin is from Biomedical Technologies and Invitrogen. Costar Transwells and Amiconultra-15 concentration columns were from Fisher. Rosetta2 E.coli from Novagen. glutathione-Sepharose 4B, parent pGEX-4T1 and ECL PLUS from Amersham-Pharmacia. Coomassie blue and PVDF were from BioRad. The Quick change site directed mutagenesis kit is from Stratagene. Normal rat IgG agarose conjugate was from Santa Cruz. Robo4 morpholino was from Gene Tools. The oligonucleotides used for PCR were from the University of Utah Core Facility. Alexa 564-phalloidin, anti-GFP and goat anti-rabbit Alex488 were from Molecular Probes. The low melting agarose was from NuSieve. The T7 in vitro transcription/translation kit was from Promega.

Molecular biology: robo4-HA, Slit2-Myc-His, and chicken pilin plasmids have been previously described (Park et al, 2003; Nishiya et al, 2005). Robo4-NH2 was amplified from Robo4-HA and cloned into pcDNA3-HA in EcoRV/NotI. Robo4-COOH was amplified from Robo4-HA by overlap extension PCR and cloned into pcDNA3-HA in EcoRV/NotI. The amino-terminal half of the cytoplasmic tail of human Robo4 (AA 465-723) was amplified by PCR and cloned into pGBKT7 (EcoRI/BamHI). The murine Robo4 fragment was amplified by PCR and cloned into BamHI/EcoRI of pGEX-4T 1. Murine Hic-5, Mena and paxillin (including deletions) were amplified by PCR from the IMAGE clone and cloned into EcoRV/NotI from pcDNA 3-V5. GST-Robo4 Δ PIM and full-length Robo4 Δ PIM were generated by site-directed mutagenesis of the relevant wild-type constructs using Quick Change. The integrity of all constructs was confirmed by sequencing at the University of Utah Core Facility.

Embryo culture and zebra fish stock: zebrafish, Danio reio, were maintained according to standard procedures (Westerfield, 2000). The developmental stage was performed using standard morphological characteristics of embryos produced by incubation at 28.5 ℃ (Kimmel et al, 1995). Tg (fli: EGFP) used in this study ^y1Transgenic zebrafish lines are described in Lawson and Weinstein, 2002. The imaged embryos were treated with 0.2mM 1-phenyl-2-thiourea (PTU) after 24hpf to prevent pigmentation.

Antisense deletion of robo 4: the anti-sense Morpholino Oligonucleotide (MO) (5'-tttttagcgtacctatgagcagtt-3', SEQ ID NO: 28) directed against the exon 10/intron 10 splice site of robo4 was dissolved in 1 XDanieau's buffer at a concentration of 5ng/nl, respectively. Prior to injection, morpholino was heated at 65 ℃ for 5 minutes, briefly cooled, mixed with negligible amounts of dye to monitor the efficiency of injection, and approximately 1nl was injected into the flowing yolk of embryos at the 1-2 cell stage.

Reverse Transcription (RT) PCR: RNA was extracted from 20 uninjected and 20 robo4 MO-injected embryos using Trizol reagent, followed by cDNA synthesis using Superscript III, primed with a mixture of random hexamer and oligo dT primers. Robo4 was amplified from cDNA by PCR using the forward primer (5'-caacaccagacacttacgagtgcc-3', SEQ ID NO: 29) in exon 8 and the reverse primer (5'-ttcgaaggccagaattctcctggc-3', SEQ ID NO: 30) in exon 12, using the following parameters: (94 ℃ C. for 4 minutes, 94 ℃ C. for 30 seconds, 58 ℃ C. for 30 seconds, 68 ℃ C. for 45 seconds, 68 ℃ C. for 1 minute). To identify the linear range of the PCR reaction, the cDNA was amplified for 23, 25, 27 and 30 cycles. Beta-actin was amplified from all samples using forward (5 ' -cccaaggccaacagggaaaa, SEQ ID NO: 31) and reverse (5'-ggtgcccatctcctgctcaa-3', SEQ ID NO: 32) primers to control the input amount of cDNA.

Whole embryo indirect immunofluorescence: briefly, age-matched 24 and 48hpf embryos were dehaired and fixed in 4% PFA/4% sucrose/PBS overnight at 4 ℃. Embryos were then washed in PBS/0.1% Tween-20, dehydrated with anhydrous methanol, rehydrated in PBS-Tween 20, further permeabilized in PBS/1% Triton-X, rinsed in PBS/1% Triton-X/2% BSA, blocked in PBS/1% Triton-X/2% BSA/10% sheep serum/1% DMSO at room temperature, and then incubated overnight at 4 ℃ in blocking solution containing IgG purified anti-GFP (1: 400 dilution). The following day, embryos were washed vigorously in PBS/1% Triton-X/2% BSA and then incubated overnight at 4 ℃ in blocking solution containing goat anti-rabbit Alexa 488-conjugated secondary antibody (1: 200 dilution). The next day, embryos were washed extensively in PBS/1% Triton-X/2% BSA, then embedded in PBS containing 1% low melting agarose, photographed under a Leica confocal microscope, and processed using Adobe Photoshop software.

Cell culture: HEK 293 and COS-7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in EGM-2 supplemented with 10% FBS. HUVECs between passage 2 and 5 were used routinely.

Transfection: HEK293 and COS-7 cells were transfected with Fugene6 or Lipofectamine2000 according to the manufacturer's protocol.

Preparation of concentrated Slit2 protein: COS-7 cells were transiently transfected with empty pSECTAG2 or pSECTAG 2:hSlit 2. After 48 hours, the cells were washed twice with PBS and incubated with 6ml of salt extraction buffer (10mM HEPES, pH 7.5, 1M NaCl and 1 Xproteinase inhibitor) for 15 minutes at 25 ℃. The salt extraction was repeated and the sample was centrifuged at 10,000rpm for 20 minutes to pellet the cell debris. The supernatant was applied to an Amicon Ultra-15 concentration column/cut-off of 100kDa and centrifuged until 12ml of salt extract had reduced to about 500. mu.l. The concentrated protein preparations were analyzed by coomassie blue staining and stored at 4 ℃ for up to one week. Using this protocol, Slit2 was typically obtained at a concentration of 20-50. mu.g/ml. The same protocol was performed for cell fluid transfected with empty vector (pSECTAG2) except that concentrated protein was prepared from cells transfected with Slit2 plasmid. The resulting preparation, referred to as the "mock" preparation, was used as a control in all experiments to analyze the effect of Slit 2.

Thigmotaxis analysis: transfected HEK293 cells were removed from the tissue culture dish with TrypLE Express, washed once with DMEM or EBM-2 containing 0.1% trypsin inhibitor, 0.2% fatty acid-free BSA and twice with relevant medium containing 0.2% BSA. Washed cells were counted and resuspended at 0.3X10 ⁵Individual cells/ml. Mix 1.5x10⁵The individual cells were loaded into the upper chamber of a 12 μm Costar transwells pre-coated on the lower surface with 5 μ g/ml fibronectin. The effect of Slit2 on thigmotaxis was analyzed by co-coating with 0.5 μ g/ml Slit2 or an equivalent amount of mock preparation. Cell migration was allowed to proceed for 6 hours, and then cells on the upper surface of the transwell were removed with a cotton swab. Cells on the lower surface were fixed with 4% formaldehyde for 5 minutes and washed three times with PBS. For HEK 293 cells, the number of GFP positive cells (HEK 293) on the lower surface was counted by counting 6 fields of 10X under an inverted fluorescence microscope. For data presentation and subsequent statistical analysis, the number of migrating cells on fibronectin/mimetic coated membranes was considered to be 100%. Independent experiments were performed in at least two replicates.

Yeast two-hybrid analysis: pGBKT 7:hRobo4465-723 was transformed into yeast strain PJ694A, yielding PJ694A-Robo 4. The human aortic cDNA library was cloned into the capture plasmid pACT2 and then transformed into PJ694A-Robo 4. Co-transformed yeast strains were plated on SD-Leu-Trp (-LT) to analyze transformation efficiency and on SD-Leu-Trp-His-Ade (-LTHA) to identify putative interacting proteins. Yeast strains capable of growing on SD-LTHA were then tested for β -galactosidase expression by filter lift analysis (filter lift assay). The capture plasmid was isolated from a yeast strain capable of growing on SD-LTHA and expressing beta-galactosidase, and sequenced at the University of Utah Core Facility.

And (3) immunoprecipitation: cell lysates were prepared in 50mM Tris-Cl, pH 7.4, 50mM NaCl, 1mM DTT, 0.5% Triton X-100, phosphatase, and protease inhibitor, centrifuged at 14K for 20 minutes to pellet insoluble material, clarified with normal IgG bound to agarose beads for 60 minutes, and incubated with relevant antibody bound to agarose beads for 2 hours at 4 ℃. The pellet was washed thoroughly in lysis buffer and resuspended in 2 Xsample buffer (125mM Tris-Cl pH 6.8, 4% SDS, 20% glycerol, 0.04% bromophenol blue and 1.4M 2-mercaptoethanol).

GST Pull Down assay: rosetta2 E.coli with pGEX-4T1: mRobo4 was grown to an OD600 of 0.6 and then induced with 0.3mM IPTG. After 3-4 hours at 30 ℃ at 220rpm, cells were lysed in 20mM Tris-Cl pH 7.4, 1% Triton X-100, 1. mu.g/ml lysozyme, 1mM DTT and protease inhibitor by sonication. The GST-fusion protein was captured on glutathione-Sepharose 4B, washed once with lysozyme-free lysis buffer, and then washed twice with binding/washing buffer (50mM Tris-Cl pH 7.4, 150mM NaCl, 1mM DTT, 1% Triton X-100, 0.1% BSA and protease inhibitor). GST-fusion proteins were incubated with 60nM purified recombinant paxillin overnight at 4 deg.C, washed extensively in binding/washing buffer, and resuspended in 2 Xsample buffer.

Western blotting: the immunoprecipitates and GST-fusion proteins were incubated at 100 ℃ for 2 minutes, separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes. PVDF membrane and 5% skim milk powder containing PBS + 0.1% Tween20(PBST) (PBST-M) at 25 degrees C temperature in 60 minutes. Blocked membranes were incubated with primary antibodies (1: 2000 anti-Flag M2; 1: 10,000 diluted anti-HA; 1: 500 anti-Hic-5; 1: 10,000 anti-paxillin; 1: 1,000 anti-Rac and 1: 500 anti-Cdc 42) in PBST-M at 25 ℃ for 60 minutes, or at 4 ℃ overnight. The membrane was washed in PBST for 3X10 minutes and then incubated with a secondary antibody (1: 10,000 goat anti-mouse or goat anti-rabbit horseradish peroxidase) for 60 minutes at 25 ℃. Membranes were washed in PBST for 3x10 minutes and visualized with ECL PLUS.

In vitro transcription/translation: Mena-V5 was synthesized using the T7Quick Coupled in vitro transcription/translation system according to the manufacturer's instructions.

Spreading analysis: transfected HEK 293 cells were plated onto coverslips coated with 5. mu.g/ml fibronectin. After incubation at 5% CO2 and 37 ℃ for 30 minutes, cells were washed 3 times with ice-cold PBS and fixed with 3.7% formaldehyde for 10 minutes at room temperature. Cells were then permeabilized with 0.2% TritonX-100 for 3 minutes, washed 3 times with PBS + 0.1% Tween20(PBST), and incubated with 10. mu.g/ml rhodamine-phalloidin for 1 hour at room temperature. After washing 3 times with PBS-T, the coverslips were placed in Pro-Long Gold and analyzed by confocal microscopy. The total area of 150 cells was determined in three independent experiments using ImageJ.

siRNA-mediated knock-down of paxillin: HEK 293 cells were transfected with 100nM siRNA duplexes (5'-CCCUGACGAAAGAGAAGCCUAUU-3', SEQ ID NO: 33 and 5'-UAGGCUUCUCUUUCGUCAGGGUU-3', SEQ ID NO: 34) using LipofectAMINE 2000, according to the manufacturer's instructions. 48 hours after transfection, cells were processed for biochemical analysis or cell spreading analysis. Reconstitution of paxillin was achieved by transfection with an expression vector encoding paxillin having nucleotide sequence 5'-CCCCTACAAAAGAAAAACCAA-3' (SEQ ID NO: 35) in the siRNA target site. Knockdown and reconstitution can be observed by western blotting with paxillin antibody and quantified by densitometry.

Rac and Cdc42 activation analysis: transfected HEK 293 cells were detached from cell culture dishes, kept in suspension in DMEM + 0.2% BSA for 1 hour, and plated on bacterial culture dishes coated with 5. mu.g/ml fibronectin for 5 minutes. The cells were then washed twice with ice cold PBS and in 50mM Tris pH 7.0, 500mM NaCl, 1mM MgCl₂1mM EGTA, 1mM MDTT, 0.5% NP-40, 1X protease inhibitor, 1X phosphatase inhibitor and 20. mu.g/ml GST-PBD. The lysate was centrifuged at 14,000rpm for 5 minutes and the supernatant was incubated with 30. mu.l glutathione agarose for 30 minutes at 4 ℃. After 3 washes with lysis buffer, the bound proteins were eluted with 2X sample buffer. Rac and Cdc42 were detected by western blotting with antibodies specific for each protein. The Rac activation levels were normalized to the total Rac, with the highest value in each experiment being normalized Designated as a value of 1.

Robo4^AP/APMouse generation and genotyping: the Robo4 targeting vector was electroporated into Embryonic Stem (ES) cells. ES cells that are heterozygous for the targeted allele are injected into blastocysts and then transferred into pseudopregnant females. Chimeric males were identified by the presence of dark and light ring colors and mated with C57BL/6 females to produce ES cell-derived offspring. Genotype was confirmed by Southern blot analysis of tail DNA. PCR genotyping was performed using genomic DNA from ear punch or tail samples under the following conditions: denaturation at 94 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 60 seconds, 40 cycles. The following two primers were used to genotype Robo 4: 5 'cccttcacagacagactctcgtatttcc 3' (forward) and 5 'cccagacctacattaccttttgccg 3' (reverse), the following two primers were used for AP: 5 'ggcaacttccagaccattggcttg 3' (forward) and 5 'ggttaccactcccactgacttccctg 3' (reverse).

Embryo and expression analysis: embryo staging, in situ hybridization, Paraffin sectioning and Whole embryo PECAM-1 immunohistochemical analysis as described previously¹The process is carried out. For Northern blot analysis, 20. mu.g of total RNA was loaded per lane after separation with TRIZOL. Using the prime It II random primer labeling kit (Stratagene), a ³²P-labeled probes. Lung lysates were prepared using lysis buffer [ 1% NP-40, 150mM NaCl, 50mM Tris-Cl (pH 7.5), 1mM EDTA and protease inhibitor cocktail (Roche)]. Robo4 protein from lung lysates was detected by Western blot analysis using polyclonal anti-Robo 4 antibody as previously described.

Alkaline Phosphatase (AP) staining: embryo or tissue is treated with a solution containing 4% paraformaldehyde and 2mM MgCl₂Was fixed overnight in PBS at 4 ℃ with shaking. The samples were washed 3 times for 15 minutes each in PBST (PBS, 0.5% Tween 20). Endogenous alkaline phosphatase in a medium containing 2mM MgCl₂In PBS (5) at 65 ℃ for 90 minutes, and then in AP buffer (100mM Tris-Cl pH9.5, 100mM NaCl, 50mM MgCl)₂0.1% Tween 20, 2mM levamisole)Twice for 15 minutes each. Staining was performed in BM violet substrate (Boehringer Mannheim) for embryos and NBT/BCIP for adult tissues. Staining was stopped in PBS containing 5mM EDTA.

Whole embryo immunohistochemistry after AP staining: alkaline Phosphatase (AP) staining was performed on fixed and sectioned retinas as previously described. Staining was stopped in EDTA-5mM PBS. Retinas were washed twice in PBS, postfixed in 4% paraformaldehyde, phosphate buffered saline for 5 minutes at room temperature, and then washed 2 times in PBS. After 2 hours incubation in PBlec (PBS, pH 6.8, 1% Triton-X100, 0.1mM CaCl, 0.1mM MgCl, 0.1mM MnCl), the retinas were incubated with the antibodies overnight at 4 ℃. Pericytes were labeled with a rabbit anti-NG 2 antibody (1: 200; Chemicon) and endothelial cells were labeled with a rat anti-endomicin (clone V.7C7, supplied by Dietmar Vestweber friend; 1: 20 dilution). After 3 washes in PBS-T (PBS, pH 7.4, 1% Triton-X100), the samples were incubated with a secondary antibody conjugated to a suitable fluorescent dye Alexa Fluor 488 or 568(Molecular Probes; Invitrogen) in PBS. After washing and short post-fixation in 4% PFA, the retinas were flattened and fixed on coverslips using Mowiol/DABCO (Sigma-Aldrich). The samples were analyzed by conventional optical and fluorescence microscopy using a Zeiss Stereomicroscope Stemi SV11Bioquad equipped with a Zeiss Axiocam HRc digital camera and also by laser confocal laser scanning microscopy using a Zeiss LSM Meta 510. AP staining was visualized with a 633nm HeNe laser and reflective assembly. Digital photographs were processed using voicity (4.0 improvement) and assembled in adobe photoshop CS 2.

Immunohistochemistry: whole embryo triple immunofluorescence confocal microscopy as previously described³The process is carried out. Briefly, antibodies to PECAM, NP1, CX40, 2H3, BFABP and α SMA were used to label the skin of extremities of Robo4+/+ or Robo 4-/-embryos at E15.5.

Constructing an expression vector of the recombinant Slit fragment: the planned expression vector is depicted in fig. 23. The DNA encoding all fragments was cloned into the pSECTAG2 vector (Invitrogen) sharing the following characteristics: CMV promoter, Kozak consensus sequence, myc/his tag in-frame fusion, and the polyA sequence of bovine growth hormone. Fc fusions were generated by replacing the myc/his epitope with the Fc domain of human IgG1 in recombinant form, in which the complement activation and effector cell interaction domains had been replaced by IgG4 and IgG2 sequences, respectively (Katoh et al, 2005; Armour et al, 1999). Recombinant Slit fragments and Slit fragment-Fc fusion proteins were isolated from transiently transfected cells. The desired constructs were stably transfected into CHO cells and selected for resistance to Zeocin.

Binding and activity of Robo4 agonist on Robo 4-expressing HEK cells: stable cell lines expressing Robo4-HA (Robo4-HEK) or pcDNA3 vector alone (control-HEK) were seeded into 6-well dishes pre-coated with 100. mu.g/ml poly L-lysine. Cells were incubated with HEK CM or Slit-myc CM at 37 ℃. After 1 hour incubation with conditioned medium, followed by 3 washes in PBS, cells were fixed in 4% paraformaldehyde for 20 minutes. Cells were then washed 3 times in PBS and incubated with mouse anti-myc antibody (Santa cruz biotech) and anti-mouse Alexa 594-conjugated secondary antibody (Molecular Probes). The ability of those agonists to bind to Robo4 to inhibit migration is according to Park KW, Morrison CM, Sorensen LK et al, "Robo 4 is a vascular-specific receptor for inhibiting endothelial cell migration," (Robo4 is a vascular-specific receptor for inhibiting endothelial cell migration) Dev Biol 2003; 261(1): 251-67.

Isolation of murine lung endothelial cells: isolation of murine endothelial cells has been previously performed⁴A description is given. Sheep anti-rat IgG Dynal beads (Dynal Biotech) were conjugated with anti-PECAM-1 or anti-ICAM-2 monoclonal antibody (BD Pharmingen) at 5. mu.g antibody per 100. mu.L of beads. Beads were pre-coated and stored at 4 ℃ (4x 10)⁸beads/mL PBS containing 0.1% BSA) for up to 2 weeks. Lungs were obtained from three adult mice. The lobes of the lung were dissected from visible bronchial and mediastinal connective tissue. Lungs were washed in 50mL of cold isolation medium (20% FBS-DMEM) to remove erythrocytes,cut with scissors and digested in 25mL of pre-warmed collagenase (2mg/mL, Worthington) for 45 minutes at 37 ℃ with gentle shaking. The digested tissue was dissociated by aspiration 12 times with a 60cc syringe attached to a 14 gauge metal cannula, then filtered through a sterile 70 μm disposable cell filter (Falcon). The suspension was centrifuged at 400Xg for 10 min at 4 ℃. The cell pellet was resuspended in 2mL cold PBS and then incubated with PECAM-1 coated beads (15. mu.L/mL cells) for 10 minutes at room temperature. The bead-bound cells were recovered using a magnetic separator, washed in isolation medium, and then resuspended in complete medium (EGM-2MV, Lonza). Cells were plated at 75-cm coated with fibronectin alone ²In tissue culture flasks, non-adherent cells were removed after overnight incubation. Adherent cells were washed with PBS and 15ml of complete medium was added. The cultured cells were fed with complete medium every other day. When the cultures reached 70% to 80% confluency, they were split with trypsin-EDTA, resuspended in 2mL PBS, and sorted a second time using ICAM-2-bound beads (15. mu.L/mL cells). Cells were washed and plated as before. Generations 2 to 5 were used for functional analysis.

Cell culture: human epidermal microvascular endothelial cells (HMVEC, Cambrex) were grown in EGM-2MV, used between 3 and 6 times.

Immunocytochemistry: prior to cell plating, an 8-well chamber slide (Lab-Tek) was used at 1.5. mu.g/cm²Coated for 2 hours. Murine lung endothelial cells were plated in complete medium EGM-2MV overnight (100,000 cells/well) at 37 ℃. Cells were then washed 3 times in PBS and fixed in 4% paraformaldehyde for 10 minutes at room temperature. After 3 additional washes in PBS, cells were washed in PBS containing 1% Triton X-100 for 15 minutes at room temperature and then in PBST (PBS containing 0.1% Triton X-100) for 3 washes. Cells were then blocked in PBS containing 2% BSA for 20 minutes at room temperature and incubated with primary antibodies in 2% BSA for 1 hour at room temperature, the primary antibodies were: rat anti-PECAM-1 (Pharmingen), rabbit anti-Von Willebrand factor (vWF) (DAKO). After incubation with primary antibody, cells were washed in PBST and Incubate for 1 hour at room temperature in 2% BSA with secondary antibodies: alexa Fluor 488 donkey anti-rat IgG and Alexa Fluor 594 donkey anti-rabbit IgG (molecular probes). Cells were washed once in PBST, once in PBS, fixed in Vectashield fixed medium (VectorLaboratories), and photographed by confocal microscopy.

Fluorescence Activated Cell Sorting (FACS): murine lung endothelial cells were detached from the culture dish by brief trypsin treatment (no more than 2 minutes) at 37 ℃. Protein cleavage was terminated by the addition of trypsin inhibitor in EBM-2+ 0.1% BSA. Cells were in FACS buffer (Ca free)²⁺And Mg²⁺PBS + 0.1% BSA) and then resuspended in 1mL FACS buffer. Expression analysis of cell surface markers was performed in two steps of immunofluorescence staining. Contacting the cells with purified monoclonal antibodies: rat anti-PECAM-1, rabbit anti-vWF were incubated at 4 ℃ for 30 min. The cells were then washed twice in FACS buffer and resuspended in 1mL FACS buffer. The cells were then incubated with a fluorescent secondary antibody: alexa Fluor 488 donkey anti-rat IgG and Alexa Fluor 594 donkey anti-rabbit IgG (molecular probes) were incubated at 4 ℃ for 30 min. The cells were washed twice again, resuspended in 1mL of FACS buffer, and analyzed by FACS.

Cell migration analysis: cells were labeled with CellTracker Green CMFDA (molecular probes) for 1 hour, washed, and then starved overnight in EBM-2 supplemented with 0.1% BSA. Cells were trypsinized, washed, and resuspended to 300,000 cells/mL. 100 μ L of the cell suspension (30,000 cells) was loaded onto an 8- μm HTS FluoroBlock filter (BD Falcon) which had been coated on both sides with 5 μ g/mL human fibronectin. The test factor was diluted in EBM-2/0.1% BSA and placed in the lower part of the chamber. After 3 hours of incubation at 37 ℃, two 5X fields per well were photographed on an inverted fluorescence microscope (Axiovert 200). The number of migrated cells was counted by counting the fluorescent cells. Robo4^+/+The baseline migration of cells was set to 1. Data are expressed as mean ± s.e. of triplicates from three independent experiments.

Tube formation analysis: tube formation analysis As previously described⁵The process is carried out. Briefly, from Robo4^+/+And Robo4^AP/APMouse isolated lung endothelial cells were plated in wells of matrigel-coated 48-well dishes and starved overnight in 0.5% serum. Cells were then stimulated with 0.48nM VEGF-A for 3.5 hours in the absence or presence of Slit2, and then photographed. Mean tube lengths were determined using ImageJ software. Data are expressed as the mean ± s.e. of two replicates of three independent experiments.

In vitro permeability analysis: will be selected from Robo4^+/+And Robo4^AP/APLung Endothelial Cells (ECs) isolated from mice were plated at 1.5. mu.g/cm²Human fibronectin was pre-coated on 3.0 μm costartranwells and grown to confluence. Cells were starved overnight, pretreated with 0.3nM Slit2 for 30-60 min, and then stimulated with 2.4nM VEGF-A for 3.5 h. Horseradish peroxidase (HRP) was added to the top of the chamber at a final concentration of 100 μ g/ml and the medium was removed from the bottom of the chamber after 30 minutes. Aliquots were mixed with 0.5mM guaiacol, 50mM Na₂HPO₄And 0.6mMH₂O₂Incubation, the formation of o-phenylenediamine is determined by measuring the absorbance at 470 nm. The fundamental permeability of the monolayer was set to 100%. Data are expressed as mean ± s.e. of triplicates from three independent experiments.

VEGF-induced retinal permeability: retinal permeability according to⁵³The description in (1) was evaluated. Briefly, 8-10 week old mice were anesthetized with Avermectin (2-2-2 tribromoethanol, 0.4 mg/g; Acros Organics, Morris Plains, N.J.). Mice were injected intraocularly with 1.4uL35.7ug/mL VEGF-A (R)&D Systems inc. minneapolis, MN) and 50ngSlit2N (SEQ ID NO: 39). An equal volume of the mock preparation was injected in the contralateral eye. Other conditions of 1.4uL saline, mock preparation or slit were applied as indicated. Six hours later, mice were injected intravenously with 60mg/mL Evans blue 50uL via the tail vein. Two hours later, the mice were sacrificed and perfused with citric acid buffered paraformaldehyde to remove intravenous evans blue . The eye was enucleated and the retina was detached. Evans blue dye was eluted in 0.3mL formamide at 70 ℃ for 18 hours. The extract was ultracentrifuged through a 5kD filter for 2 hours. The absorbance at 620nm was measured. The background absorbance was measured at 740nm and subtracted.

Adenoviral expression of Robo 4: robo4 was expressed by adenovirus as previously described.

Miles analysis: evans blue was injected into the tail vein of 6-8 week old mice and 30 minutes later, saline, or 10ng VEGF-A was injected into the dermis in the absence and presence of 100ng Slit 2. After an additional 30 minutes, a punch biopsy was performed and evans blue was eluted from the dermal tissue into formamide at 60 ℃ for 18 hours. After centrifugation, the absorbance was measured at 620 nm. The amount of dermal permeability observed in saline injected animals was set to 1. Data are expressed as the mean ± s.e. of two replicates of 5 individual mice per treatment (total 6 injections per animal).

Retinal permeability: retinal permeability as described earlier⁸Evaluation was performed. Briefly, 8-10 week old mice were anesthetized with Avermectin (2-2-2 tribromoethanol, 0.4 mg/g; Across organics, Morris Plains, N.J.). Mice were injected intraocularly with 1.4. mu.L of 35.7. mu.g/mL VEGF-A (R) &D Systems inc. minneapolis, MN) and 50ng Slit 2. An equal volume of the mimetic was injected in the contralateral eye. Other conditions were applied as indicated. Six hours later, mice were administered 50 μ L of Evans blue at 60mg/mL via the femoral vein. Two hours later, the mice were sacrificed and perfused with citric acid buffered formaldehyde to remove intravenous evans blue. The eye was enucleated and the retina was detached. Evans blue dye was eluted in 0.4mL formamide at 70 ℃ for 18 hours. The extract was ultracentrifuged through a 5kD filter for 2 hours. The absorbance at 620nm was measured. The background absorbance was measured at 740nm and subtracted. Data are expressed as mean ± s.e. of 5 individual mice per genotype.

Biochemical analysis: HMVEC in fibronectin-coated dishesGrow to confluency and starve overnight in EBM-2+ 0.2% BSA. The following day, cells were stimulated with 50ng/mLVEGF-A for 5 min, washed twice with ice cold PBS, at 50mM Tris pH 7.4, 150mM NaCl, 10mM MgCl₂1mM DTT, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1X protease inhibitor, 1X phosphatase inhibitor. Lysates were mixed with 2 Xsample buffer, separated by SDS-PAGE, and probed with 1: 1000 diluted antibodies against phosphorylated VEGFR2, phosphorylated p42/44, and phosphorylated src (CellSignaling). For Rac activation assays, crude membrane preparations were generated ⁹And GTP-Rac was precipitated with 20. mu.g/ml GST-PBD. After 3 washes with lysis buffer, the bound proteins were eluted with 2X sample buffer. Rac1 was detected by western blotting using monoclonal antibodies (BDBiosciences).

Oxygen-induced retinopathy: briefly, P7 pups and lactating mothers were placed in 75% oxygen, which was maintained by Pro-OX oxygen controller (BioSpherix, Redfield, NY). Pups were removed at P12 and injected intra-ocularly with Slit2N (SEQ ID NO: 39) agonist or mock preparation, which was used as a control condition. Mice were sacrificed at P17 and 1ml of 50mg/ml FITC-dextran (Sigma, St. Louis, Mo.) was perfused through the left ventricle. The eyes were enucleated and fixed in 4% paraformaldehyde for 30 minutes to produce retinal accessory tablets. Photographs were taken with an Axiovert 200 fluorescence microscope (Carl Zeiss, Thornwood, NY). Neovascularization was quantified using AxioVision software which calculated the amount of vascularization in each region (Carl Zeiss, Thornwood, NY). Data are expressed as mean ± s.e. of 5 individual mice per genotype.

Laser-induced choroidal neovascularization: 2-3 month old mice were anesthetized with Avermectin (2-2-2 tribromoethanol, 0.4 mg/g; Acros Organics, Morris Plains, NJ) and mydriasis with 1% tropicamide (Alcon, Fort Worth, TX). With an IridexOcuLight GL 532nm laser photocoagulator (Iridex, Mountain View, CA) with a slit lamp delivery system, 3 burns were generated from 3, 6, and 9 o' clock directions at 3 disc diameters from the optic disc with the following parameters: 150mW power, 75um spot size and 0.1 second duration. Bubbles are generated when a laser is used, and rupture of the Bruch film is an important factor in obtaining CNV; therefore, only burns that produce blisters were included in this study. Immediately and 3 days after laser treatment, mice were injected intravitreally with 50ng Slit2N (SEQ ID NO: 39). An equivalent volume of the mock preparation was administered by intravitreal injection in the other eye. After 1 week of laser treatment, mice were sacrificed to produce choroidal subplates. CNV was stained with biotin-bound isolectin (Sigma, st.louis, MO) and texas red-bound streptavidin (Sigma, st.louis, MO). The subplates were examined using a Zeiss LSM 510 confocal microscope (Zeiss, Thornwood, NY) and CNV was quantified using ImageJ software (NIH, Bethesda, MD).

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Sequence listing

<110> Utah University Research Foundation (University of Utah Research Foundation)

<120> compositions and methods for treating pathological angiogenesis and vascular permeability

(COMPOSITIONS AND METHODS FOR TREATING PATHOLOGIC ANGIOGENESIS AND

VASCULAR PERMEABILITY)

<130>SCT092083-00

<150>60/869526

<151>2006-12-11

<160>47

<170>PatentIn version 3.4

<210>1

<211>1007

<212>PRT

<213>Homo sapiens

<400>1

Met Gly Ser Gly Gly Asp Ser Leu Leu Gly Gly Arg Gly Ser Leu Pro

1 5 10 15

Leu Leu Leu Leu Leu Ile Met Gly Gly Met Ala Gln Asp Ser Pro Pro

20 25 30

Gln Ile Leu Val His Pro Gln Asp Gln Leu Phe Gln Gly Pro Gly Pro

35 40 45

Ala Arg Met Ser Cys Gln Ala Ser Gly Gln Pro Pro Pro Thr Ile Arg

50 55 60

Trp Leu Leu Asn Gly Gln Pro Leu Ser Met Val Pro Pro Asp Pro His

65 70 75 80

His Leu Leu Pro Asp Gly Thr Leu Leu Leu Leu Gln Pro Pro Ala Arg

85 90 95

Gly His Ala His Asp Gly Gln Ala Leu Ser Thr Asp Leu Gly Val Tyr

100 105 110

Thr Cys Glu Ala Ser Asn Arg Leu Gly Thr Ala Val Ser Arg Gly Ala

115 120 125

Arg Leu Ser Val Ala Val Leu Arg Glu Asp Phe Gln Ile Gln Pro Arg

130 135 140

Asp Met Val Ala Val Val Gly Glu Gln Phe Thr Leu Glu Cys Gly Pro

145 150 155 160

Pro Trp Gly His Pro Glu Pro Thr Val Ser Trp Trp Lys Asp Gly Lys

165 170 175

Pro Leu Ala Leu Gln Pro Gly Arg His Thr Val Ser Gly Gly Ser Leu

180 185 190

Leu Met Ala Arg Ala Glu Lys Ser Asp Glu Gly Thr Tyr Met Cys Val

195 200 205

Ala Thr Asn Ser Ala Gly His Arg Glu Ser Arg Ala Ala Arg Val Ser

210 215 220

Ile Gln Glu Pro Gln Asp Tyr Thr Glu Pro Val Glu Leu Leu Ala Val

225 230 235 240

Arg Ile Gln Leu Glu Asn Val Thr Leu Leu Asn Pro Asp Pro Ala Glu

245 250 255

Gly Pro Lys Pro Arg Pro Ala Val Trp Leu Ser Trp Lys Val Ser Gly

260 265 270

Pro Ala Ala Pro Ala Gln Ser Tyr Thr Ala Leu Phe Arg Thr Gln Thr

275 280 285

Ala Pro Gly Gly Gln Gly Ala Pro Trp Ala Glu Glu Leu Leu Ala Gly

290 295 300

Trp Gln Ser Ala Glu Leu Gly Gly Leu His Trp Gly Gln Asp Tyr Glu

305 310 315 320

Phe Lys Val Arg Pro Ser Ser Gly Arg Ala Arg Gly Pro Asp Ser Asn

325 330 335

Val Leu Leu Leu Arg Leu Pro Glu Lys Val Pro Ser Ala Pro Pro Gln

340 345 350

Glu Val Thr Leu Lys Pro Gly Asn Gly Thr Val Phe Val Ser Trp Val

355 360 365

Pro Pro Pro Ala Glu Asn His Asn Gly Ile Ile Arg Gly Tyr Gln Val

370 375 380

Trp Ser Leu Gly Asn Thr Ser Leu Pro Pro Ala Asn Trp Thr Val Val

385 390 395 400

Gly Glu Gln Thr Gln Leu Glu Ile Ala Thr His Met Pro Gly Ser Tyr

405 410 415

Cys Val Gln Val Ala Ala Val Thr Gly Ala Gly Ala Gly Glu Pro Ser

420 425 430

Arg Pro Val Cys Leu Leu Leu Glu Gln Ala Met Glu Arg Ala Thr Gln

435 440 445

Glu Pro Ser Glu His Gly Pro Trp Thr Leu Glu Gln Leu Arg Ala Thr

450 455 460

Leu Lys Arg Pro Glu Val Ile Ala Thr Cys Gly Val Ala Leu Trp Leu

465 470 475 480

Leu Leu Leu Gly Thr Ala Val Cys Ile His Arg Arg Arg Arg Ala Arg

485 490 495

Val His Leu Gly Pro Gly Leu Tyr Arg Tyr Thr Ser Glu Asp Ala Ile

500 505 510

Leu Lys His Arg Met Asp His Ser Asp Ser Gln Trp Leu Ala Asp Thr

515 520 525

Trp Arg Ser Thr Ser Gly Ser Arg Asp Leu Ser Ser Ser Ser Ser Leu

530 535 540

Ser Ser Arg Leu Gly Ala Asp Ala Arg Asp Pro Leu Asp Cys Arg Arg

545 550 555 560

Ser Leu Leu Ser Trp Asp Ser Arg Ser Pro Gly Val Pro Leu Leu Pro

565 570 575

Asp Thr Ser Thr Phe Tyr Gly Ser Leu Ile Ala Glu Leu Pro Ser Ser

580 585 590

Thr Pro Ala Arg Pro Ser Pro Gln Val Pro Ala Val Arg Arg Leu Pro

595 600 605

Pro Gln Leu Ala Gln Leu Ser Ser Pro Cys Ser Ser Ser Asp Ser Leu

610 615 620

Cys Ser Arg Arg Gly Leu Ser Ser Pro Arg Leu Ser Leu Ala Pro Ala

625 630 635 640

Glu Ala Trp Lys Ala Lys Lys Lys Gln Glu Leu Gln His Ala Asn Ser

645 650 655

Ser Pro Leu Leu Arg Gly Ser His Ser Leu Glu Leu Arg Ala Cys Glu

660 665 670

Leu Gly Asn Arg Gly Ser Lys Asn Leu Ser Gln Ser Pro Gly Ala Val

675 680 685

Pro Gln Ala Leu Val Ala Trp Arg Ala Leu Gly Pro Lys Leu Leu Ser

690 695 700

Ser Ser Asn Glu Leu Val Thr Arg His Leu Pro Pro Ala Pro Leu Phe

705 710 715 720

Pro His Glu Thr Pro Pro Thr Gln Ser Gln Gln Thr Gln Pro Pro Val

725 730 735

Ala Pro Gln Ala Pro Ser Ser Ile Leu Leu Pro Ala Ala Pro Ile Pro

740 745 750

Ile Leu Ser Pro Cys Ser Pro Pro Ser Pro Gln Ala Ser Ser Leu Ser

755 760 765

Gly Pro Ser Pro Ala Ser Ser Arg Leu Ser Ser Ser Ser Leu Ser Ser

770 775 780

Leu Gly Glu Asp Gln Asp Ser Val Leu Thr Pro Glu Glu Val Ala Leu

785 790 795 800

Cys Leu Glu Leu Ser Glu Gly Glu Glu Thr Pro Arg Asn Ser Val Ser

805 810 815

Pro Met Pro Arg Ala Pro Ser Pro Pro Thr Thr Tyr Gly Tyr Ile Ser

820 825 830

Val Pro Thr Ala Ser Glu Phe Thr Asp Met Gly Arg Thr Gly Gly Gly

835 840 845

Val Gly Pro Lys Gly Gly Val Leu Leu Cys Pro Pro Arg Pro Cys Leu

850 855 860

Thr Pro Thr Pro Ser Glu Gly Ser Leu Ala Asn Gly Trp Gly Ser Ala

865 870 875 880

Ser Glu Asp Asn Ala Ala Ser Ala Arg Ala Ser Leu Val Ser Ser Ser

885 890 895

Asp Gly Ser Phe Leu Ala Asp Ala His Phe Ala Arg Ala Leu Ala Val

900 905 910

Ala Val Asp Ser Phe Gly Phe Gly Leu Glu Pro Arg Glu Ala Asp Cys

915 920 925

Val Phe Ile Asp Ala Ser Ser Pro Pro Ser Pro Arg Asp Glu Ile Phe

930 935 940

Leu Thr Pro Asn Leu Ser Leu Pro Leu Trp Glu Trp Arg Pro Asp Trp

945 950 955 960

Leu Glu Asp Met Glu Val Ser His Thr Gln Arg Leu Gly Arg Gly Met

965 970 975

Pro Pro Trp Pro Pro Asp Ser Gln Ile Ser Ser Gln Arg Ser Gln Leu

980 985 990

His Cys Arg Met Pro Lys Ala Gly Ala Ser Pro Val Asp Tyr Ser

995 1000 1005

<210>2

<211>3872

<212>DNA

<213>Homo sapiens

<400>2

gcggccgcga attcggcacg agcagcagga caaagtgctc gggacaagga catagggctg 60

agagtagcca tgggctctgg aggagacagc ctcctggggg gcaggggttc cctgcctctg 120

ctgctcctgc tcatcatggg aggcatggct caggactccc cgccccagat cctagtccac 180

ccccaggacc agctgttcca gggccctggc cctgccagga tgagctgcca agcctcaggc 240

cagccacctc ccaccatccg ctggttgctg aatgggcagc ccctgagcat ggtgccccca 300

gacccacacc acctcctgcc tgatgggacc cttctgctgc tacagccccc tgcccgggga 360

catgcccacg atggccaggc cctgtccaca gacctgggtg tctacacatg tgaggccagc 420

aaccggcttg gcacggcagt cagcagaggc gctcggctgt ctgtggctgt cctccgggag 480

gatttccaga tccagcctcg ggacatggtg gctgtggtgg gtgagcagtt tactctggaa 540

tgtgggccgc cctggggcca cccagagccc acagtctcat ggtggaaaga tgggaaaccc 600

ctggccctcc agcccggaag gcacacagtg tccggggggt ccctgctgat ggcaagagca 660

gagaagagtg acgaagggac ctacatgtgt gtggccacca acagcgcagg acatagggag 720

agccgcgcag cccgggtttc catccaggag ccccaggact acacggagcc tgtggagctt 780

ctggctgtgc gaattcagct ggaaaatgtg acactgctga acccggatcc tgcagagggc 840

cccaagccta gaccggcggt gtggctcagc tggaaggtca gtggccctgc tgcgcctgcc 900

caatcttaca cggccttgtt caggacccag actgccccgg gaggccaggg agctccgtgg 960

gcagaggagc tgctggccgg ctggcagagc gcagagcttg gaggcctcca ctggggccaa 1020

gactacgagt tcaaagtgag accatcctct ggccgggctc gaggccctga cagcaacgtg 1080

ctgctcctga ggctgccgga aaaagtgccc agtgccccac ctcaggaagt gactctaaag 1140

cctggcaatg gcactgtctt tgtgagctgg gtcccaccac ctgctgaaaa ccacaatggc 1200

atcatccgtg gctaccaggt ctggagcctg ggcaacacat cactgccacc agccaactgg 1260

actgtagttg gtgagcagac ccagctggaa atcgccaccc atatgccagg ctcctactgc 1320

gtgcaagtgg ctgcagtcac tggtgctgga gctggggagc ccagtagacc tgtctgcctc 1380

cttttagagc aggccatgga gcgagccacc caagaaccca gtgagcatgg tccctggacc 1440

ctggagcagc tgagggctac cttgaagcgg cctgaggtca ttgccacctg cggtgttgca 1500

ctctggctgc tgcttctggg caccgccgtg tgtatccacc gccggcgccg agctagggtg 1560

cacctgggcc caggtctgta cagatatacc agtgaggatg ccatcctaaa acacaggatg 1620

gatcacagtg actcccagtg gttggcagac acttggcgtt ccacctctgg ctctcgggac 1680

ctgagcagca gcagcagcct cagcagtcgg ctgggggcgg atgcccggga cccactagac 1740

tgtcgtcgct ccttgctctc ctgggactcc cgaagccccg gcgtgcccct gcttccagac 1800

accagcactt tttatggctc cctcatcgct gagctgccct ccagtacccc agccaggcca 1860

agtccccagg tcccagctgt caggcgcctc ccaccccagc tggcccagct ctccagcccc 1920

tgttccagct cagacagcct ctgcagccgc aggggactct cttctccccg cttgtctctg 1980

gcccctgcag aggcttggaa ggccaaaaag aagcaggagc tgcagcatgc caacagttcc 2040

ccactgctcc ggggcagcca ctccttggag ctccgggcct gtgagttagg aaatagaggt 2100

tccaagaacc tttcccaaag cccaggagct gtgccccaag ctctggttgc ctggcgggcc 2160

ctgggaccga aactcctcag ctcctcaaat gagctggtta ctcgtcatct ccctccagca 2220

cccctctttc ctcatgaaac tcccccaact cagagtcaac agacccagcc tccggtggca 2280

ccacaggctc cctcctccat cctgctgcca gcagccccca tccccatcct tagcccctgc 2340

agtcccccta gcccccaggc ctcttccctc tctggcccca gcccagcttc cagtcgcctg 2400

tccagctcct cactgtcatc cctgggggag gatcaagaca gcgtgctgac ccctgaggag 2460

gtagccctgt gcttggaact cagtgagggt gaggagactc ccaggaacag cgtctctccc 2520

atgccaaggg ctccttcacc ccccaccacc tatgggtaca tcagcgtccc aacagcctca 2580

gagttcacgg acatgggcag gactggagga ggggtggggc ccaagggggg agtcttgctg 2640

tgcccacctc ggccctgcct cacccccacc cccagcgagg gctccttagc caatggttgg 2700

ggctcagcct ctgaggacaa tgccgccagc gccagagcca gccttgtcag ctcctccgat 2760

ggctccttcc tcgctgatgc tcactttgcc cgggccctgg cagtggctgt ggatagcttt 2820

ggtttcggtc tagagcccag ggaggcagac tgcgtcttca tagatgcctc atcacctccc 2880

tccccacggg atgagatctt cctgaccccc aacctctccc tgcccctgtg ggagtggagg 2940

ccagactggt tggaagacat ggaggtcagc cacacccagc ggctgggaag ggggatgcct 3000

ccctggcccc ctgactctca gatctcttcc cagagaagtc agctccactg tcgtatgccc 3060

aaggctggtg cttctcctgt agattactcc tgaaccgtgt ccctgagact tcccagacgg 3120

gaatcagaac cacttctcct gtccacccac aagacctggg ctgtggtgtg tgggtcttgg 3180

cctgtgtttc tctgcagctg gggtccacct tcccaagcct ccagagagtt ctccctccac 3240

gattgtgaaa acaaatgaaa acaaaattag agcaaagctg acctggagcc ctcagggagc 3300

aaaacatcat ctccacctga ctcctagcca ctgctttctc ctctgtgcca tccactccca 3360

ccaccaggtt gttttggcct gaggagcagc cctgcctgct gctcttcccc caccatttgg 3420

atcacaggaa gtggaggagc cagaggtgcc tttgtggagg acagcagtgg ctgctgggag 3480

agggctgtgg aggaaggagc ttctcggagc cccctctcag ccttacctgg gcccctcctc 3540

tagagaagag ctcaactctc tcccaacctc accatggaaa gaaaataatt atgaatgcca 3600

ctgaggcact gaggccctac ctcatgccaa acaaagggtt caaggctggg tctagcgagg 3660

atgctgaagg aagggaggta tgagaccgta ggtcaaaagc accatcctcg tactgttgtc 3720

actatgagct taagaaattt gataccataa aatggtaaag acttgaaaaa aaaaaaaaaa 3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 3872

<210>3

<211>1534

<212>PRT

<213>Homo sapiens

<400>3

Met Ala Leu Thr Pro Gly Trp Gly Ser Ser Ala Gly Pro Val Arg Pro

1 5 10 15

Glu Leu Trp Leu Leu Leu Trp Ala Ala Ala Trp Arg Leu Gly Ala Ser

20 25 30

Ala Cys Pro Ala Leu Cys Thr Cys Thr Gly Thr Thr Val Asp Cys His

35 40 45

Gly Thr Gly Leu Gln Ala Ile Pro Lys Asn Ile Pro Arg Asn Thr Glu

50 55 60

Arg Leu Glu Leu Asn Gly Asn Asn Ile Thr Arg Ile His Lys Asn Asp

65 70 75 80

Phe Ala Gly Leu Lys Gln Leu Arg Val Leu Gln Leu Met Glu Asn Gln

85 90 95

Ile Gly Ala Val Glu Arg Gly Ala Phe Asp Asp Met Lys Glu Leu Glu

100 105 110

Arg Leu Arg Leu Asn Arg Asn Gln Leu His Met Leu Pro Glu Leu Leu

115 120 125

Phe Gln Asn Asn Gln Ala Leu Ser Arg Leu Asp Leu Ser Glu Asn Ala

130 135 140

Ile Gln Ala Ile Pro Arg Lys Ala Phe Arg Gly Ala Thr Asp Leu Lys

145 150 155 160

Asn Leu Gln Leu Asp Lys Asn Gln Ile Ser Cys Ile Glu Glu Gly Ala

165 170 175

Phe Arg Ala Leu Arg Gly Leu Glu Val Leu Thr Leu Asn Asn Asn Asn

180 185 190

Ile Thr Thr Ile Pro Val Ser Ser Phe Asn His Met Pro Lys Leu Arg

195 200 205

Thr Phe Arg Leu His Ser Asn His Leu Phe Cys Asp Cys His Leu Ala

210 215 220

Trp Leu Ser Gln Trp Leu Arg Gln Arg Pro Thr Ile Gly Leu Phe Thr

225 230 235 240

Gln Cys Ser Gly Pro Ala Ser Leu Arg Gly Leu Asn Val Ala Glu Val

245 250 255

Gln Lys Ser Glu Phe Ser Cys Ser Gly Gln Gly Glu Ala Gly Arg Val

260 265 270

Pro Thr Cys Thr Leu Ser Ser Gly Ser Cys Pro Ala Met Cys Thr Cys

275 280 285

Ser Asn Gly Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Ala Ile Pro

290 295 300

Ala Asn Leu Pro Glu Thr Met Thr Glu Ile Arg Leu Glu Leu Asn Gly

305 310 315 320

Ile Lys Ser Ile Pro Pro Gly Ala Phe Ser Pro Tyr Arg Lys Leu Arg

325 330 335

Arg Ile Asp Leu Ser Asn Asn Gln Ile Ala Glu Ile Ala Pro Asp Ala

340 345 350

Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys

355 360 365

Ile Thr Asp Leu Pro Arg Gly Val Phe Gly Gly Leu Tyr Thr Leu Gln

370 375 380

Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys Ile Arg Pro Asp Ala

385 390 395 400

Phe Gln Asp Leu Gln Asn Leu Ser Leu Leu Ser Leu Tyr Asp Asn Lys

405 410 415

Ile Gln Ser Leu Ala Lys Gly Thr Phe Thr Ser Leu Arg Ala Ile Gln

420 425 430

Thr Leu His Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys Asn Leu Lys

435 440 445

Trp Leu Ala Asp Phe Leu Arg Thr Asn Pro Ile Glu Thr Ser Gly Ala

450 455 460

Arg Cys Ala Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile

465 470 475 480

Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu Gln Tyr Phe Ile Pro

485 490 495

Gly Thr Glu Asp Tyr Gln Leu Asn Ser Glu Cys Asn Ser Asp Val Val

500 505 510

Cys Pro His Lys Cys Arg Cys Glu Ala Asn Val Val Glu Cys Ser Ser

515 520 525

Leu Lys Leu Thr Lys Ile Pro Glu Arg Ile Pro Gln Ser Thr Ala Glu

530 535 540

Leu Arg Leu Asn Asn Asn Glu Ile Ser Ile Leu Glu Ala Thr Gly Met

545 550 555 560

Phe Lys Lys Leu Thr His Leu Lys Lys Ile Asn Leu Ser Asn Asn Lys

565 570 575

Val Ser Glu Ile Glu Asp Gly Ala Phe Glu Gly Ala Ala Ser Val Ser

580 585 590

Glu Leu His Leu Thr Ala Asn Gln Leu Glu Ser Ile Arg Ser Gly Met

595 600 605

Phe Arg Gly Leu Asp Gly Leu Arg Thr Leu Met Leu Arg Asn Asn Arg

610 615 620

Ile Ser Cys Ile His Asn Asp Ser Phe Thr Gly Leu Arg Asn Val Arg

625 630 635 640

Leu Leu Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ser Pro Gly Ala

645 650 655

Phe Asp Thr Leu Gln Ser Leu Ser Thr Leu Asn Leu Leu Ala Asn Pro

660 665 670

Phe Asn Cys Asn Cys Gln Leu Ala Trp Leu Gly Gly Trp Leu Arg Lys

675 680 685

Arg Lys Ile Val Thr Gly Asn Pro Arg Cys Gln Asn Pro Asp Phe Leu

690 695 700

Arg Gln Ile Pro Leu Gln Asp Val Ala Phe Pro Asp Phe Arg Cys Glu

705 710 715 720

Glu Gly Gln Glu Glu Gly Gly Cys Leu Pro Arg Pro Gln Cys Pro Gln

725 730 735

Glu Cys Ala Cys Leu Asp Thr Val Val Arg Cys Ser Asn Lys His Leu

740 745 750

Arg Ala Leu Pro Lys Gly Ile Pro Lys Asn Val Thr Glu Leu Tyr Leu

755 760 765

Asp Gly Asn Gln Phe Thr Leu Val Pro Gly Gln Leu Ser Thr Phe Lys

770 775 780

Tyr Leu Gln Leu Val Asp Leu Ser Asn Asn Lys Ile Ser Ser Leu Ser

785 790 795 800

Asn Ser Ser Phe Thr Asn Met Ser Gln Leu Thr Thr Leu Ile Leu Ser

805 810 815

Tyr Asn Ala Leu Gln Cys Ile Pro Pro Leu Ala Phe Gln Gly Leu Arg

820 825 830

Ser Leu Arg Leu Leu Ser Leu His Gly Asn Asp Ile Ser Thr Leu Gln

835 840 845

Glu Gly Ile Phe Ala Asp Val Thr Ser Leu Ser His Leu Ala Ile Gly

850 855 860

Ala Asn Pro Leu Tyr Cys Asp Cys His Leu Arg Trp Leu Ser Ser Trp

865 870 875 880

Val Lys Thr Gly Tyr Lys Glu Pro Gly Ile Ala Arg Cys Ala Gly Pro

885 890 895

Gln Asp Met Glu Gly Lys Leu Leu Leu Thr Thr Pro Ala Lys Lys Phe

900 905 910

Glu Cys Gln Gly Pro Pro Thr Leu Ala Val Gln Ala Lys Cys Asp Leu

915 920 925

Cys Leu Ser Ser Pro Cys Gln Asn Gln Gly Thr Cys His Asn Asp Pro

930 935 940

Leu Glu Val Tyr Arg Cys Ala Cys Pro Ser Gly Tyr Lys Gly Arg Asp

945 950 955 960

Cys Glu Val Ser Leu Asn Ser Cys Ser Ser Gly Pro Cys Glu Asn Gly

965 970 975

Gly Thr Cys His Ala Gln Glu Gly Glu Asp Ala Pro Phe Thr Cys Ser

980 985 990

Cys Pro Thr Gly Phe Glu Gly Pro Thr Cys Gly Val Asn Thr Asp Asp

995 1000 1005

Cys Val Asp His Ala Cys Ala Asn Gly Gly Val Cys Val Asp Gly

1010 1015 1020

Val Gly Asn Tyr Thr Cys Gln Cys Pro Leu Gln Tyr Glu Gly Lys

1025 1030 1035

Ala Cys Glu Gln Leu Val Asp Leu Cys Ser Pro Asp Leu Asn Pro

1040 1045 1050

Cys Gln His Glu Ala Gln Cys Val Gly Thr Pro Asp Gly Pro Arg

1055 1060 1065

Cys Glu Cys Met Pro Gly Tyr Ala Gly Asp Asn Cys Ser Glu Asn

1070 1075 1080

Gln Asp Asp Cys Arg Asp His Arg Cys Gln Asn Gly Ala Gln Cys

1085 1090 1095

Met Asp Glu Val Asn Ser Tyr Ser Cys Leu Cys Ala Glu Gly Tyr

1100 1105 1110

Ser Gly Gln Leu Cys Glu Ile Pro Pro His Leu Pro Ala Pro Lys

1115 1120 1125

Ser Pro Cys Glu Gly Thr Glu Cys Gln Asn Gly Ala Asn Cys Val

1130 1135 1140

Asp Gln Gly Asn Arg Pro Val Cys Gln Cys Leu Pro Gly Phe Gly

1145 1150 1155

Gly Pro Glu Cys Glu Lys Leu Leu Ser Val Asn Phe Val Asp Arg

1160 1165 1170

Asp Thr Tyr Leu Gln Phe Thr Asp Leu Gln Asn Trp Pro Arg Ala

1175 1180 1185

Asn Ile Thr Leu Gln Val Ser Thr Ala Glu Asp Asn Gly Ile Leu

1190 1195 1200

Leu Tyr Asn Gly Asp Asn Asp His Ile Ala Val Glu Leu Tyr Gln

1205 1210 1215

Gly His Val Arg Val Ser Tyr Asp Pro Gly Ser Tyr Pro Ser Ser

1220 1225 1230

Ala Ile Tyr Ser Ala Glu Thr Ile Asn Asp Gly Gln Phe His Thr

1235 1240 1245

Val Glu Leu Val Ala Phe Asp Gln Met Val Asn Leu Ser Ile Asp

1250 1255 1260

Gly Gly Ser Pro Met Thr Met Asp Asn Phe Gly Lys His Tyr Thr

1265 1270 1275

Leu Asn Ser Glu Ala Pro Leu Tyr Val Gly Gly Met Pro Val Asp

1280 1285 1290

Val Asn Ser Ala Ala Phe Arg Leu Trp Gln Ile Leu Asn Gly Thr

1295 1300 1305

Gly Phe His Gly Cys Ile Arg Asn Leu Tyr Ile Asn Asn Glu Leu

1310 1315 1320

Gln Asp Phe Thr Lys Thr Gln Met Lys Pro Gly Val Val Pro Gly

1325 1330 1335

Cys Glu Pro Cys Arg Lys Leu Tyr Cys Leu His Gly Ile Cys Gln

1340 1345 1350

Pro Asn Ala Thr Pro Gly Pro Met Cys His Cys Glu Ala Gly Trp

1355 1360 1365

Val Gly Leu His Cys Asp Gln Pro Ala Asp Gly Pro Cys His Gly

1370 1375 1380

His Lys Cys Val His Gly Gln Cys Val Pro Leu Asp Ala Leu Ser

1385 1390 1395

Tyr Ser Cys Gln Cys Gln Asp Gly Tyr Ser Gly Ala Leu Cys Asn

1400 1405 1410

Gln Ala Gly Ala Leu Ala Glu Pro Cys Arg Gly Leu Gln Cys Leu

1415 1420 1425

His Gly His Cys Gln Ala Ser Gly Thr Lys Gly Ala His Cys Val

1430 1435 1440

Cys Asp Pro Gly Phe Ser Gly Glu Leu Cys Glu Gln Glu Ser Glu

1445 1450 1455

Cys Arg Gly Asp Pro Val Arg Asp Phe His Gln Val Gln Arg Gly

1460 1465 1470

Tyr Ala Ile Cys Gln Thr Thr Arg Pro Leu Ser Trp Val Glu Cys

1475 1480 1485

Arg Gly Ser Cys Pro Gly Gln Gly Cys Cys Gln Gly Leu Arg Leu

1490 1495 1500

Lys Arg Arg Lys Phe Thr Phe Glu Cys Ser Asp Gly Thr Ser Phe

1505 1510 1515

Ala Glu Glu Val Glu Lys Pro Thr Lys Cys Gly Cys Ala Leu Cys

1520 1525 1530

Ala

<210>4

<211>5094

<212>DNA

<213>Homo sapiens

<400>4

gcgaaacggc agaggagccg agccccctcc gcccaaggcg ccctccctcc gtccgcgcac 60

aggcgccgtc gcttggagga gcaaggtgcc tcccagcccg caggggcgcc gcgcgcaagc 120

ccgcgggctc ttcggtggct ctgccccggg actgcacctg gaggcggccc cggacgggga 180

tggtcagcgg ctgctgccgt ctggctcgcg agcgggacgc tgtgagggca ccatggcgct 240

gactcccggg tgggggtcct cggcggggcc ggtccggccg gagctctggc tgctgctgtg 300

ggcagccgcg tggcgcctgg gtgcctcggc gtgccccgcc ctctgcacct gcaccggaac 360

cacggtggac tgccacggca cggggctgca ggccattccc aagaatatac ctcggaacac 420

cgagcgcctg gaactcaatg gcaacaacat cactcggatc cataagaatg actttgcggg 480

gctcaagcag ctgcgggtgc tgcagctgat ggagaaccag attggagcag tggaacgtgg 540

tgcttttgat gacatgaagg agctggagcg gctgcgactg aaccgaaacc agctgcacat 600

gttaccggaa ctgctgttcc agaacaacca ggctttgtca agactggact tgagtgagaa 660

cgccatccag gccatcccca ggaaagcttt tcggggagct acggacctta aaaatttacg 720

gctggacaag aaccagatca gctgcattga ggaaggggcc ttccgtgctc tgcgggggct 780

ggaggtgctg accctgaaca acaacaatat caccaccatc cccgtgtcca gcttcaacca 840

tatgcccaag ctacggacct tccgcctgca ctccaaccac ctgttttgcg actgccacct 900

ggcctggctc tcgcagtggc tgaggcagcg gccaaccatc gggctcttca cccagtgctc 960

gggcccagcc agcctgcgtg gcctcaatgt ggcagaggtc cagaagagtg agttcagctg 1020

ctcaggccag ggagaagcgg ggcgcgtgcc cacctgcacc ctgtcctccg gctcctgccc 1080

ggccatgtgc acctgcagca atggcatcgt ggactgtcgt ggaaaaggcc tcactgccat 1140

cccggccaac ctgcccgaga ccatgacgga gatacgcctg gagctgaacg gcatcaagtc 1200

catccctcct ggagccttct caccctacag aaagctacgg aggatagacc tgagcaacaa 1260

tcagatcgct gagattgcac ccgacgcctt ccagggcctc cgctccctga actcgctggt 1320

cctctatgga aacaagatca cagacctccc ccgtggtgtg tttggaggcc tatacaccct 1380

acagctcctg ctcctgaatg ccaacaagat caactgcatc cggcccgatg ccttccagga 1440

cctgcagaac ctctcactgc tctccctgta tgacaacaag atccagagcc tcgccaaggg 1500

cactttcacc tccctgcggg ccatccagac tctgcacctg gcccagaacc ctttcatttg 1560

cgactgtaac ctcaagtggc tggcagactt cctgcgcacc aatcccatcg agacgagtgg 1620

tgcccgctgt gccagtcccc ggcgcctcgc caacaagcgc atcgggcaga tcaagagcaa 1680

gaagttccgg tgctcagcca aagagcagta cttcattcca ggcacggagg attaccagct 1740

gaacagcgag tgcaacagcg acgtggtctg tccccacaag tgccgctgtg aggccaacgt 1800

ggtggagtgc tccagcctga agctcaccaa gatccctgag cgcatccccc agtccacggc 1860

agaactgcga ttgaataaca atgagatttc catcctggag gccactggga tgtttaaaaa 1920

acttacacat ctgaagaaaa tcaatctgag caacaacaag gtgtcagaaa ttgaagatgg 1980

ggccttcgag ggcgcagcct ctgtgagcga gctgcaccta actgccaacc agctggagtc 2040

catccggagc ggcatgttcc ggggtctgga tggcttgagg accctaatgc tgcggaacaa 2100

ccgcatcagc tgcatccaca acgacagctt cacgggcctg cgcaacgtcc ggctcctctc 2160

gctctacgac aaccagatca ccaccgtatc cccaggagcc ttcgacaccc tccagtccct 2220

ctccacactg aatctcctgg ccaacccttt caactgcaac tgccagctgg cctggctagg 2280

aggctggcta cggaagcgca agatcgtgac ggggaacccg cgatgccaga accctgactt 2340

tttgcggcag attcccctgc aggacgtggc cttccctgac ttcaggtgtg aggaaggcca 2400

ggaggagggg ggctgcctgc cccgcccaca gtgcccacag gagtgcgcct gcctggacac 2460

cgtggtccga tgcagcaaca agcacctgcg ggccctgccc aagggcattc ccaagaatgt 2520

cacagaactc tatttggacg ggaaccagtt cacgctggtt ccgggacagc tgtctacctt 2580

caagtacctg cagctcgtgg acctgagcaa caacaagatc agttccttaa gcaattcctc 2640

cttcaccaac atgagccagc tgaccactct gatcctcagc tacaatgccc tgcagtgcat 2700

cccgcctttg gccttccagg gactccgctc cctgcgcctg ctgtctctcc acggcaatga 2760

catctccacc ctccaagagg gcatctttgc agacgtgacc tccctgtctc acctggccat 2820

tggtgccaac cccctatact gtgactgcca cctccgctgg ctgtccagct gggtgaagac 2880

tggctacaag gaaccgggca ttgctcgttg tgctgggccc caggacatgg agggcaagct 2940

gctcctcacc acgcctgcca agaagtttga atgccaaggt cctccaacgc tggctgtcca 3000

ggccaagtgt gatctctgct tgtccagtcc gtgccagaac cagggcacct gccacaacga 3060

cccccttgag gtgtacaggt gcgcctgccc cagcggctat aagggtcgag actgtgaggt 3120

gtccctgaac agctgttcca gtggcccctg tgaaaatggg ggcacctgcc atgcacagga 3180

gggcgaggat gccccgttca cgtgctcctg tcccaccggc tttgaaggac caacctgtgg 3240

ggtgaacaca gatgactgtg tggatcatgc ctgtgccaat gggggcgtct gtgtggatgg 3300

tgtgggcaac tacacctgcc agtgccccct gcagtatgag ggaaaggcct gtgagcagct 3360

ggtggacttg tgctctccgg atctgaaccc atgtcaacac gaggcccagt gtgtgggcac 3420

cccggatggg cccaggtgtg agtgcatgcc aggttatgca ggtgacaact gcagtgagaa 3480

ccaggatgac tgcagggacc accgctgcca gaatggggcc cagtgtatgg atgaagtcaa 3540

cagctactcc tgcctctgtg ctgagggcta cagtggacag ctctgtgaga tccctcccca 3600

tctgcctgcc cccaagagcc cctgtgaggg gactgagtgc cagaatgggg ccaactgtgt 3660

ggaccagggc aacaggcctg tgtgccagtg cctcccaggc ttcggtggcc ctgagtgtga 3720

gaagttgctc agtgtcaact ttgtggatcg ggacacttac ctgcagttca ctgacctgca 3780

aaactggcca cgggccaaca tcacgttgca ggtctccacg gcagaggaca atgggatcct 3840

tctgtacaac ggggacaacg accacattgc agttgagctg taccagggcc atgtgcgtgt 3900

cagctacgac ccaggcagct accccagctc tgccatctac agtgctgaga cgatcaacga 3960

tgggcaattc cacaccgttg agctggttgc ctttgaccag atggtgaatc tctccattga 4020

tggcgggagc cccatgacca tggacaactt tggcaaacat tacacgctca acagcgaggc 4080

gccactctat gtgggaggga tgcccgtgga tgtcaactca gctgccttcc gcctgtggca 4140

gatcctcaac ggcaccggct tccacggttg catccgaaac ctgtacatca acaacgagct 4200

gcaggacttc accaagacgc agatgaagcc aggcgtggtg ccaggctgcg aaccctgccg 4260

caagctctac tgcctgcatg gcatctgcca gcccaatgcc accccagggc ccatgtgcca 4320

ctgcgaggct ggctgggtgg gcctgcactg tgaccagccc gctgacggcc cctgccatgg 4380

ccacaagtgt gtccatgggc aatgcgtgcc cctcgacgct ctttcctaca gctgccagtg 4440

ccaggatggg tactcggggg cactgtgcaa ccaggccggg gccctggcag agccctgcag 4500

aggcctgcag tgcctgcatg gccactgcca ggcctcaggc accaaggggg cacactgtgt 4560

gtgtgacccc ggcttttcgg gcgagctgtg tgagcaagag tccgagtgcc ggggggaccc 4620

tgtccgggac tttcaccagg tccagagggg ctatgccatc tgccagacca cgcgccccct 4680

gtcatgggtg gagtgccggg gctcgtgccc aggccagggc tgctgccagg gccttcggct 4740

gaagcggagg aagttcacct ttgagtgcag cgatgggacc tcttttgccg aggaggtgga 4800

aaagcccacc aagtgtggct gtgccctctg cgcatagcgc tgggcgtgga caggccggtg 4860

agggcgggca aggggcccca gccgctgcag cagcggagac agtcgccagc agctgggctg 4920

gggtgcaggt catcacagga cggctcctgg gcagctgggc cctcctgggt ggggtggtgc 4980

cagagcagcc ttttaaaagc aaattgcgcc atagctgggg gcagcggggg tgggcgaggc 5040

ctgagctgcg ggctgccctc tccggaagtc cttgcacaaa taggcgctta ataa 5094

<210>5

<211>1529

<212>PRT

<213>Homo sapiens

<400>5

Met Arg Gly Val Gly Trp Gln Met Leu Ser Leu Ser Leu Gly Leu Val

1 5 10 15

Leu Ala Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys

20 25 30

Ser Cys Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser

35 40 45

Val Pro Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly

50 55 60

Asn Asn Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His

65 70 75 80

Leu Arg Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg

85 90 95

Gly Ala Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg

100 105 110

Asn His Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys

115 120 125

Leu Tyr Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg

130 135 140

Lys Ala Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr

145 150 155 160

Asn Gln Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp

165 170 175

Leu Glu Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val

180 185 190

Ala Ser Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser

195 200 205

Asn Asn Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu

210 215 220

Arg Gln Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser

225 230 235 240

His Leu Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val

245 250 255

Cys Ser Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His

260 265 270

Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly

275 280 285

Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu

290 295 300

Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe

305 310 315 320

Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile

325 330 335

Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser

340 345 350

Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe

355 360 365

Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile

370 375 380

Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu

385 390 395 400

Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe

405 410 415

Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe

420 425 430

Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn

435 440 445

Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala

450 455 460

Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala

465 470 475 480

Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu

485 490 495

Ser Gly Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys

500 505 510

Glu Gly Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro

515 520 525

Glu His Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu

530 535 540

Phe Thr Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu

545 550 555 560

Arg Lys Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly

565 570 575

Ala Phe Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn

580 585 590

Arg Leu Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu

595 600 605

Lys Thr Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp

610 615 620

Ser Phe Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn

625 630 635 640

Gln Ile Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu

645 650 655

Ser Thr Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu

660 665 670

Ala Trp Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn

675 680 685

Pro Arg Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp

690 695 700

Val Ala Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser

705 710 715 720

Cys Ser Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr

725 730 735

Val Val Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile

740 745 750

Pro Arg Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu

755 760 765

Val Pro Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu

770 775 780

Ser Asn Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met

785 790 795 800

Thr Gln Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile

805 810 815

Pro Pro Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu

820 825 830

His Gly Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu

835 840 845

Ser Ala Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp

850 855 860

Cys Asn Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu

865 870 875 880

Pro Gly Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu

885 890 895

Leu Leu Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp

900 905 910

Val Asn Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys

915 920 925

Asn Asp Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr

930 935 940

Cys Pro Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala

945 950 955 960

Cys Ile Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu

965 970 975

Gly Glu Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly

980 985 990

Glu Asn Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu

995 1000 1005

Asn Asn Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu

1010 1015 1020

Cys Pro Pro Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp

1025 1030 1035

Phe Cys Ala Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys

1040 1045 1050

Ile Leu Thr Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr

1055 1060 1065

Val Gly Glu His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn

1070 1075 1080

Lys Cys Lys Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr

1085 1090 1095

Thr Cys Ile Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe

1100 1105 1110

Ser Pro Pro Met Val Leu Pro Arg Thr Ser Pro Cys Asp Asn Phe

1115 1120 1125

Asp Cys Gln Asn Gly Ala Gln Cys Ile Val Arg Ile Asn Glu Pro

1130 1135 1140

Ile Cys Gln Cys Leu Pro Gly Tyr Gln Gly Glu Lys Cys Glu Lys

1145 1150 1155

Leu Val Ser Val Asn Phe Ile Asn Lys Glu Ser Tyr Leu Gln Ile

1160 1165 1170

Pro Ser Ala Lys Val Arg Pro Gln Thr Asn Ile Thr Leu Gln Ile

1175 1180 1185

Ala Thr Asp Glu Asp Ser Gly Ile Leu Leu Tyr Lys Gly Asp Lys

1190 1195 1200

Asp His Ile Ala Val Glu Leu Tyr Arg Gly Arg Val Arg Ala Ser

1205 1210 1215

Tyr Asp Thr Gly Ser His Pro Ala Ser Ala Ile Tyr Ser Val Glu

1220 1225 1230

Thr Ile Asn Asp Gly Asn Phe His Ile Val Glu Leu Leu Ala Leu

1235 1240 1245

Asp Gln Ser Leu Ser Leu Ser Val Asp Gly Gly Asn Pro Lys Ile

1250 1255 1260

Ile Thr Asn Leu Ser Lys Gln Ser Thr Leu Asn Phe Asp Ser Pro

1265 1270 1275

Leu Tyr Val Gly Gly Met Pro Gly Lys Ser Asn Val Ala Ser Leu

1280 1285 1290

Arg Gln Ala Pro Gly Gln Asn Gly Thr Ser Phe His Gly Cys Ile

1295 1300 1305

Arg Asn Leu Tyr Ile Asn Ser Glu Leu Gln Asp Phe Gln Lys Val

1310 1315 1320

Pro Met Gln Thr Gly Ile Leu Pro Gly Cys Glu Pro Cys His Lys

1325 1330 1335

Lys Val Cys Ala His Gly Thr Cys Gln Pro Ser Ser Gln Ala Gly

1340 1345 1350

Phe Thr Cys Glu Cys Gln Glu Gly Trp Met Gly Pro Leu Cys Asp

1355 1360 1365

Gln Arg Thr Asn Asp Pro Cys Leu Gly Asn Lys Cys Val His Gly

1370 1375 1380

Thr Cys Leu Pro Ile Asn Ala Phe Ser Tyr Ser Cys Lys Cys Leu

1385 1390 1395

Glu Gly His Gly Gly Val Leu Cys Asp Glu Glu Glu Asp Leu Phe

1400 1405 1410

Asn Pro Cys Gln Ala Ile Lys Cys Lys His Gly Lys Cys Arg Leu

1415 1420 1425

Ser Gly Leu Gly Gln Pro Tyr Cys Glu Cys Ser Ser Gly Tyr Thr

1430 1435 1440

Gly Asp Ser Cys Asp Arg Glu Ile Ser Cys Arg Gly Glu Arg Ile

1445 1450 1455

Arg Asp Tyr Tyr Gln Lys Gln Gln Gly Tyr Ala Ala Cys Gln Thr

1460 1465 1470

Thr Lys Lys Val Ser Arg Leu Glu Cys Arg Gly Gly Cys Ala Gly

1475 1480 1485

Gly Gln Cys Cys Gly Pro Leu Arg Ser Lys Arg Arg Lys Tyr Ser

1490 1495 1500

Phe Glu Cys Thr Asp Gly Ser Ser Phe Val Asp Glu Val Glu Lys

1505 1510 1515

Val Val Lys Cys Gly Cys Thr Arg Cys Val Ser

1520 1525

<210>6

<211>3872

<212>DNA

<213>Homo sapiens

<400>6

gcggccgcga attcggcacg agcagcagga caaagtgctc gggacaagga catagggctg 60

agagtagcca tgggctctgg aggagacagc ctcctggggg gcaggggttc cctgcctctg 120

ctgctcctgc tcatcatggg aggcatggct caggactccc cgccccagat cctagtccac 180

ccccaggacc agctgttcca gggccctggc cctgccagga tgagctgcca agcctcaggc 240

cagccacctc ccaccatccg ctggttgctg aatgggcagc ccctgagcat ggtgccccca 300

gacccacacc acctcctgcc tgatgggacc cttctgctgc tacagccccc tgcccgggga 360

catgcccacg atggccaggc cctgtccaca gacctgggtg tctacacatg tgaggccagc 420

aaccggcttg gcacggcagt cagcagaggc gctcggctgt ctgtggctgt cctccgggag 480

gatttccaga tccagcctcg ggacatggtg gctgtggtgg gtgagcagtt tactctggaa 540

tgtgggccgc cctggggcca cccagagccc acagtctcat ggtggaaaga tgggaaaccc 600

ctggccctcc agcccggaag gcacacagtg tccggggggt ccctgctgat ggcaagagca 660

gagaagagtg acgaagggac ctacatgtgt gtggccacca acagcgcagg acatagggag 720

agccgcgcag cccgggtttc catccaggag ccccaggact acacggagcc tgtggagctt 780

ctggctgtgc gaattcagct ggaaaatgtg acactgctga acccggatcc tgcagagggc 840

cccaagccta gaccggcggt gtggctcagc tggaaggtca gtggccctgc tgcgcctgcc 900

caatcttaca cggccttgtt caggacccag actgccccgg gaggccaggg agctccgtgg 960

gcagaggagc tgctggccgg ctggcagagc gcagagcttg gaggcctcca ctggggccaa 1020

gactacgagt tcaaagtgag accatcctct ggccgggctc gaggccctga cagcaacgtg 1080

ctgctcctga ggctgccgga aaaagtgccc agtgccccac ctcaggaagt gactctaaag 1140

cctggcaatg gcactgtctt tgtgagctgg gtcccaccac ctgctgaaaa ccacaatggc 1200

atcatccgtg gctaccaggt ctggagcctg ggcaacacat cactgccacc agccaactgg 1260

actgtagttg gtgagcagac ccagctggaa atcgccaccc atatgccagg ctcctactgc 1320

gtgcaagtgg ctgcagtcac tggtgctgga gctggggagc ccagtagacc tgtctgcctc 1380

cttttagagc aggccatgga gcgagccacc caagaaccca gtgagcatgg tccctggacc 1440

ctggagcagc tgagggctac cttgaagcgg cctgaggtca ttgccacctg cggtgttgca 1500

ctctggctgc tgcttctggg caccgccgtg tgtatccacc gccggcgccg agctagggtg 1560

cacctgggcc caggtctgta cagatatacc agtgaggatg ccatcctaaa acacaggatg 1620

gatcacagtg actcccagtg gttggcagac acttggcgtt ccacctctgg ctctcgggac 1680

ctgagcagca gcagcagcct cagcagtcgg ctgggggcgg atgcccggga cccactagac 1740

tgtcgtcgct ccttgctctc ctgggactcc cgaagccccg gcgtgcccct gcttccagac 1800

accagcactt tttatggctc cctcatcgct gagctgccct ccagtacccc agccaggcca 1860

agtccccagg tcccagctgt caggcgcctc ccaccccagc tggcccagct ctccagcccc 1920

tgttccagct cagacagcct ctgcagccgc aggggactct cttctccccg cttgtctctg 1980

gcccctgcag aggcttggaa ggccaaaaag aagcaggagc tgcagcatgc caacagttcc 2040

ccactgctcc ggggcagcca ctccttggag ctccgggcct gtgagttagg aaatagaggt 2100

tccaagaacc tttcccaaag cccaggagct gtgccccaag ctctggttgc ctggcgggcc 2160

ctgggaccga aactcctcag ctcctcaaat gagctggtta ctcgtcatct ccctccagca 2220

cccctctttc ctcatgaaac tcccccaact cagagtcaac agacccagcc tccggtggca 2280

ccacaggctc cctcctccat cctgctgcca gcagccccca tccccatcct tagcccctgc 2340

agtcccccta gcccccaggc ctcttccctc tctggcccca gcccagcttc cagtcgcctg 2400

tccagctcct cactgtcatc cctgggggag gatcaagaca gcgtgctgac ccctgaggag 2460

gtagccctgt gcttggaact cagtgagggt gaggagactc ccaggaacag cgtctctccc 2520

atgccaaggg ctccttcacc ccccaccacc tatgggtaca tcagcgtccc aacagcctca 2580

gagttcacgg acatgggcag gactggagga ggggtggggc ccaagggggg agtcttgctg 2640

tgcccacctc ggccctgcct cacccccacc cccagcgagg gctccttagc caatggttgg 2700

ggctcagcct ctgaggacaa tgccgccagc gccagagcca gccttgtcag ctcctccgat 2760

ggctccttcc tcgctgatgc tcactttgcc cgggccctgg cagtggctgt ggatagcttt 2820

ggtttcggtc tagagcccag ggaggcagac tgcgtcttca tagatgcctc atcacctccc 2880

tccccacggg atgagatctt cctgaccccc aacctctccc tgcccctgtg ggagtggagg 2940

ccagactggt tggaagacat ggaggtcagc cacacccagc ggctgggaag ggggatgcct 3000

ccctggcccc ctgactctca gatctcttcc cagagaagtc agctccactg tcgtatgccc 3060

aaggctggtg cttctcctgt agattactcc tgaaccgtgt ccctgagact tcccagacgg 3120

gaatcagaac cacttctcct gtccacccac aagacctggg ctgtggtgtg tgggtcttgg 3180

cctgtgtttc tctgcagctg gggtccacct tcccaagcct ccagagagtt ctccctccac 3240

gattgtgaaa acaaatgaaa acaaaattag agcaaagctg acctggagcc ctcagggagc 3300

aaaacatcat ctccacctga ctcctagcca ctgctttctc ctctgtgcca tccactccca 3360

ccaccaggtt gttttggcct gaggagcagc cctgcctgct gctcttcccc caccatttgg 3420

atcacaggaa gtggaggagc cagaggtgcc tttgtggagg acagcagtgg ctgctgggag 3480

agggctgtgg aggaaggagc ttctcggagc cccctctcag ccttacctgg gcccctcctc 3540

tagagaagag ctcaactctc tcccaacctc accatggaaa gaaaataatt atgaatgcca 3600

ctgaggcact gaggccctac ctcatgccaa acaaagggtt caaggctggg tctagcgagg 3660

atgctgaagg aagggaggta tgagaccgta ggtcaaaagc accatcctcg tactgttgtc 3720

actatgagct taagaaattt gataccataa aatggtaaag acttgaaaaa aaaaaaaaaa 3780

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 3872

<210>7

<211>1523

<212>PRT

<213>Homo sapiens

<400>7

Met Ala Pro Gly Trp Ala Gly Val Gly Ala Ala Val Arg Ala Arg Leu

1 5 10 15

Ala Leu Ala Leu Ala Leu Ala Ser Val Leu Ser Gly Pro Pro Ala Val

20 25 30

Ala Cys Pro Thr Lys Cys Thr Cys Ser Ala Ala Ser Val Asp Cys His

35 40 45

Gly Leu Gly Leu Arg Ala Val Pro Arg Gly Ile Pro Arg Asn Ala Glu

50 55 60

Arg Leu Asp Leu Asp Arg Asn Asn Ile Thr Arg Ile Thr Lys Met Asp

65 70 75 80

Phe Ala Gly Leu Lys Asn Leu Arg Val Leu His Leu Glu Asp Asn Gln

85 90 95

Val Ser Val Ile Glu Arg Gly Ala Phe Gln Asp Leu Lys Gln Leu Glu

100 105 110

Arg Leu Arg Leu Asn Lys Asn Lys Leu Gln Val Leu Pro Glu Leu Leu

115 120 125

Phe Gln Ser Thr Pro Lys Leu Thr Arg Leu Asp Leu Ser Glu Asn Gln

130 135 140

Ile Gln Gly Ile Pro Arg Lys Ala Phe Arg Gly Ile Thr Asp Val Lys

145 150 155 160

Asn Leu Gln Leu Asp Asn Asn His Ile Ser Cys Ile Glu Asp Gly Ala

165 170 175

Phe Arg Ala Leu Arg Asp Leu Glu Ile Leu Thr Leu Asn Asn Asn Asn

180 185 190

Ile Ser Arg Ile Leu Val Thr Ser Phe Asn His Met Pro Lys Ile Arg

195 200 205

Thr Leu Arg Leu His Ser Asn His Leu Tyr Cys Asp Cys His Leu Ala

210 215 220

Trp Leu Ser Asp Trp Leu Arg Gln Arg Arg Thr Val Gly Gln Phe Thr

225 230 235 240

Leu Cys Met Ala Pro Val His Leu Arg Gly Phe Asn Val Ala Asp Val

245 250 255

Gln Lys Lys Glu Tyr Val Cys Pro Ala Pro His Ser Glu Pro Pro Ser

260 265 270

Cys Asn Ala Asn Ser Ile Ser Cys Pro Ser Pro Cys Thr Cys Ser Asn

275 280 285

Asn Ile Val Asp Cys Arg Gly Lys Gly Leu Met Glu Ile Pro Ala Asn

290 295 300

Leu Pro Glu Gly Ile Val Glu Ile Arg Leu Glu Gln Asn Ser Ile Lys

305 310 315 320

Ala Ile Pro Ala Gly Ala Phe Thr Gln Tyr Lys Lys Leu Lys Arg Ile

325 330 335

Asp Ile Ser Lys Asn Gln Ile Ser Asp Ile Ala Pro Asp Ala Phe Gln

340 345 350

Gly Leu Lys Ser Leu Thr Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr

355 360 365

Glu Ile Ala Lys Gly Leu Phe Asp Gly Leu Val Ser Leu Gln Leu Leu

370 375 380

Leu Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asn Thr Phe Gln

385 390 395 400

Asp Leu Gln Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln

405 410 415

Thr Ile Ser Lys Gly Leu Phe Ala Pro Leu Gln Ser Ile Gln Thr Leu

420 425 430

His Leu Ala Gln Asn Pro Phe Val Cys Asp Cys His Leu Lys Trp Leu

435 440 445

Ala Asp Tyr Leu Gln Asp Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys

450 455 460

Ser Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Ser Gln Ile Lys Ser

465 470 475 480

Lys Lys Phe Arg Cys Ser Gly Ser Glu Asp Tyr Arg Ser Arg Phe Ser

485 490 495

Ser Glu Cys Phe Met Asp Leu Val Cys Pro Glu Lys Cys Arg Cys Glu

500 505 510

Gly Thr Ile Val Asp Cys Ser Asn Gln Lys Leu Val Arg Ile Pro Ser

515 520 525

His Leu Pro Glu Tyr Val Thr Asp Leu Arg Leu Asn Asp Asn Glu Val

530 535 540

Ser Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Asn Leu Arg

545 550 555 560

Lys Ile Asn Leu Ser Asn Asn Lys Ile Lys Glu Val Arg Glu Gly Ala

565 570 575

Phe Asp Gly Ala Ala Ser Val Gln Glu Leu Met Leu Thr Gly Asn Gln

580 585 590

Leu Glu Thr Val His Gly Arg Val Phe Arg Gly Leu Ser Gly Leu Lys

595 600 605

Thr Leu Met Leu Arg Ser Asn Leu Ile Gly Cys Val Ser Asn Asp Thr

610 615 620

Phe Ala Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Arg

625 630 635 640

Ile Thr Thr Ile Thr Pro Gly Ala Phe Thr Thr Leu Val Ser Leu Ser

645 650 655

Thr Ile Asn Leu Leu Ser Asn Pro Phe Asn Cys Asn Cys His Leu Ala

660 665 670

Trp Leu Gly Lys Trp Leu Arg Lys Arg Arg Ile Val Ser Gly Asn Pro

675 680 685

Arg Cys Gln Lys Pro Phe Phe Leu Lys Glu Ile Pro Ile Gln Asp Val

690 695 700

Ala Ile Gln Asp Phe Thr Cys Asp Gly Asn Glu Glu Ser Ser Cys Gln

705 710 715 720

Leu Ser Pro Arg Cys Pro Glu Gln Cys Thr Cys Met Glu Thr Val Val

725 730 735

Arg Cys Ser Asn Lys Gly Leu Arg Ala Leu Pro Arg Gly Met Pro Lys

740 745 750

Asp Val Thr Glu Leu Tyr Leu Glu Gly Asn His Leu Thr Ala Val Pro

755 760 765

Arg Glu Leu Ser Ala Leu Arg His Leu Thr Leu Ile Asp Leu Ser Asn

770 775 780

Asn Ser Ile Ser Met Leu Thr Asn Tyr Thr Phe Ser Asn Met Ser His

785 790 795 800

Leu Ser Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Val

805 810 815

His Ala Phe Asn Gly Leu Arg Ser Leu Arg Val Leu Thr Leu His Gly

820 825 830

Asn Asp Ile Ser Ser Val Pro Glu Gly Ser Phe Asn Asp Leu Thr Ser

835 840 845

Leu Ser His Leu Ala Leu Gly Thr Asn Pro Leu His Cys Asp Cys Ser

850 855 860

Leu Arg Trp Leu Ser Glu Trp Val Lys Ala Gly Tyr Lys Glu Pro Gly

865 870 875 880

Ile Ala Arg Cys Ser Ser Pro Glu Pro Met Ala Asp Arg Leu Leu Leu

885 890 895

Thr Thr Pro Thr His Arg Phe Gln Cys Lys Gly Pro Val Asp Ile Asn

900 905 910

Ile Val Ala Lys Cys Asn Ala Cys Leu Ser Ser Pro Cys Lys Asn Asn

915 920 925

Gly Thr Cys Thr Gln Asp Pro Val Glu Leu Tyr Arg Cys Ala Cys Pro

930 935 940

Tyr Ser Tyr Lys Gly Lys Asp Cys Thr Val Pro Ile Asn Thr Cys Ile

945 950 955 960

Gln Asn Pro Cys Gln His Gly Gly Thr Cys His Leu Ser Asp Ser His

965 970 975

Lys Asp Gly Phe Ser Cys Ser Cys Pro Leu Gly Phe Glu Gly Gln Arg

980 985 990

Cys Glu Ile Asn Pro Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn

995 1000 1005

Ala Thr Cys Val Asp Gly Ile Asn Asn Tyr Val Cys Ile Cys Pro

1010 1015 1020

Pro Asn Tyr Thr Gly Glu Leu Cys Asp Glu Val Ile Asp His Cys

1025 1030 1035

Val Pro Glu Leu Asn Leu Cys Gln His Glu Ala Lys Cys Ile Pro

1040 1045 1050

Leu Asp Lys Gly Phe Ser Cys Glu Cys Val Pro Gly Tyr Ser Gly

1055 1060 1065

Lys Leu Cys Glu Thr Asp Asn Asp Asp Cys Val Ala His Lys Cys

1070 1075 1080

Arg His Gly Ala Gln Cys Val Asp Thr Ile Asn Gly Tyr Thr Cys

1085 1090 1095

Thr Cys Pro Gln Gly Phe Ser Gly Pro Phe Cys Glu His Pro Pro

1100 1105 1110

Pro Met Val Leu Leu Gln Thr Ser Pro Cys Asp Gln Tyr Glu Cys

1115 1120 1125

Gln Asn Gly Ala Gln Cys Ile Val Val Gln Gln Glu Pro Thr Cys

1130 1135 1140

Arg Cys Pro Pro Gly Phe Ala Gly Pro Arg Cys Glu Lys LeuIle

1145 1150 1155

Thr Val Asn Phe Val Gly Lys Asp Ser Tyr Val Glu Leu Ala Ser

1160 1165 1170

Ala Lys Val Arg Pro Gln Ala Asn Ile Ser Leu Gln Val Ala Thr

1175 1180 1185

Asp Lys Asp Asn Gly Ile Leu Leu Tyr Lys Gly Asp Asn Asp Pro

1190 1195 1200

Leu Ala Leu Glu Leu Tyr Gln Gly His Val Arg Leu Val Tyr Asp

1205 1210 1215

Ser Leu Ser Ser Pro Pro Thr Thr Val Tyr Ser Val Glu Thr Val

1220 1225 1230

Asn Asp Gly Gln Phe His Ser Val Glu Leu Val Thr Leu Asn Gln

1235 1240 1245

Thr Leu Asn Leu Val Val Asp Lys Gly Thr Pro Lys Ser Leu Gly

1250 1255 1260

Lys Leu Gln Lys Gln Pro Ala Val Gly Ile Asn Ser Pro Leu Tyr

1265 1270 1275

Leu Gly Gly Ile Pro Thr Ser Thr Gly Leu Ser Ala Leu Arg Gln

1280 1285 1290

Gly Thr Asp Arg Pro Leu Gly Gly Phe His Gly Cys Ile His Glu

1295 1300 1305

Val Arg Ile Asn Asn Glu Leu Gln Asp Phe Lys Ala Leu Pro Pro

1310 1315 1320

Gln Ser Leu Gly Val Ser Pro Gly Cys Lys Ser Cys Thr Val Cys

1325 1330 1335

Lys His Gly Leu Cys Arg Ser Val Glu Lys Asp Ser Val Val Cys

1340 1345 1350

Glu Cys Arg Pro Gly Trp Thr Gly Pro Leu Cys Asp Gln Glu Ala

1355 1360 1365

Arg Asp Pro Cys Leu Gly His Arg Cys His His Gly Lys Cys Val

1370 1375 1380

Ala Thr Gly Thr Ser Tyr Met Cys Lys Cys Ala Glu Gly Tyr Gly

1385 1390 1395

Gly Asp Leu Cys Asp Asn Lys Asn Asp Ser Ala Asn Ala Cys Ser

1400 1405 1410

Ala Phe Lys Cys His His Gly Gln Cys His Ile Ser Asp Gln Gly

1415 1420 1425

Glu Pro Tyr Cys Leu Cys Gln Pro Gly Phe Ser Gly Glu His Cys

1430 1435 1440

Gln Gln Glu Asn Pro Cys Leu Gly Gln Val Val Arg Glu Val Ile

1445 1450 1455

Arg Arg Gln Lys Gly Tyr Ala Ser Cys Ala Thr Ala Ser Lys Val

1460 1465 1470

Pro Ile Met Glu Cys Arg Gly Gly Cys Gly Pro Gln Cys Cys Gln

1475 1480 1485

Pro Thr Arg Ser Lys Arg Arg Lys Tyr Val Phe Gln Cys Thr Asp

1490 1495 1500

Gly Ser Ser Phe Val Glu Glu Val Glu Arg His Leu Glu Cys Gly

1505 1510 1515

Cys Leu Ala Cys Ser

1520

<210>8

<211>1523

<212>PRT

<213>Homo sapiens

<400>8

Met Ala Pro Gly Trp Ala Gly Val Gly Ala Ala Val Arg Ala Arg Leu

1 5 10 15

Ala Leu Ala Leu Ala Leu Ala Ser Val Leu Ser Gly Pro Pro Ala Val

20 25 30

Ala Cys Pro Thr Lys Cys Thr Cys Ser Ala Ala Ser Val Asp Cys His

35 40 45

Gly Leu Gly Leu Arg Ala Val Pro Arg Gly Ile Pro Arg Asn Ala Glu

50 55 60

Arg Leu Asp Leu Asp Arg Asn Asn Ile Thr Arg Ile Thr Lys Met Asp

65 70 75 80

Phe Ala Gly Leu Lys Asn Leu Arg Val Leu His Leu Glu Asp Asn Gln

85 90 95

Val Ser Val Ile Glu Arg Gly Ala Phe Gln Asp Leu Lys Gln Leu Glu

100 105 110

Arg Leu Arg Leu Asn Lys Asn Lys Leu Gln Val Leu Pro Glu Leu Leu

115 120 125

Phe Gln Ser Thr Pro Lys Leu Thr Arg Leu Asp Leu Ser Glu Asn Gln

130 135 140

Ile Gln Gly Ile Pro Arg Lys Ala Phe Arg Gly Ile Thr Asp Val Lys

145 150 155 160

Asn Leu Gln Leu Asp Asn Asn His Ile Ser Cys Ile Glu Asp Gly Ala

165 170 175

Phe Arg Ala Leu Arg Asp Leu Glu Ile Leu Thr Leu Asn Asn Asn Asn

180 185 190

Ile Ser Arg Ile Leu Val Thr Ser Phe Asn His Met Pro Lys Ile Arg

195 200 205

Thr Leu Arg Leu His Ser Asn His Leu Tyr Cys Asp Cys His Leu Ala

210 215 220

Trp Leu Ser Asp Trp Leu Arg Gln Arg Arg Thr Val Gly Gln Phe Thr

225 230 235 240

Leu Cys Met Ala Pro Val His Leu Arg Gly Phe Asn Val Ala Asp Val

245 250 255

Gln Lys Lys Glu Tyr Val Cys Pro Ala Pro His Ser Glu Pro Pro Ser

260 265 270

Cys Asn Ala Asn Ser Ile Ser Cys Pro Ser Pro Cys Thr Cys Ser Asn

275 280 285

Asn Ile Val Asp Cys Arg Gly Lys Gly Leu Met Glu Ile Pro Ala Asn

290 295 300

Leu Pro Glu Gly Ile Val Glu Ile Arg Leu Glu Gln Asn Ser Ile Lys

305 310 315 320

Ala Ile Pro Ala Gly Ala Phe Thr Gln Tyr Lys Lys Leu Lys Arg Ile

325 330 335

Asp Ile Ser Lys Asn Gln Ile Ser Asp Ile Ala Pro Asp Ala Phe Gln

340 345 350

Gly Leu Lys Ser Leu Thr Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr

355 360 365

Glu Ile Ala Lys Gly Leu Phe Asp Gly Leu Val Ser Leu Gln Leu Leu

370 375 380

Leu Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asn Thr Phe Gln

385 390 395 400

Asp Leu Gln Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln

405 410 415

Thr Ile Ser Lys Gly Leu Phe Ala Pro Leu Gln Ser Ile Gln Thr Leu

420 425 430

His Leu Ala Gln Asn Pro Phe Val Cys Asp Cys His Leu Lys Trp Leu

435 440 445

Ala Asp Tyr Leu Gln Asp Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys

450 455 460

Ser Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Ser Gln Ile Lys Ser

465 470 475 480

Lys Lys Phe Arg Cys Ser Gly Ser Glu Asp Tyr Arg Ser Arg Phe Ser

485 490 495

Ser Glu Cys Phe Met Asp Leu Val Cys Pro Glu Lys Cys Arg Cys Glu

500 505 510

Gly Thr Ile Val Asp Cys Ser Asn Gln Lys Leu Val Arg Ile Pro Ser

515 520 525

His Leu Pro Glu Tyr Val Thr Asp Leu Arg Leu Asn Asp Asn Glu Val

530 535 540

Ser Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Asn Leu Arg

545 550 555 560

Lys Ile Asn Leu Ser Asn Asn Lys Ile Lys Glu Val Arg Glu Gly Ala

565 570 575

Phe Asp Gly Ala Ala Ser Val Gln Glu Leu Met Leu Thr Gly Asn Gln

580 585 590

Leu Glu Thr Val His Gly Arg Val Phe Arg Gly Leu Ser Gly Leu Lys

595 600 605

Thr Leu Met Leu Arg Ser Asn Leu Ile Gly Cys Val Ser Asn Asp Thr

610 615 620

Phe Ala Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Arg

625 630 635 640

Ile Thr Thr Ile Thr Pro Gly Ala Phe Thr Thr Leu Val Ser Leu Ser

645 650 655

Thr Ile Asn Leu Leu Ser Asn Pro Phe Asn Cys Asn Cys His Leu Ala

660 665 670

Trp Leu Gly Lys Trp Leu Arg Lys Arg Arg Ile Val Ser Gly Asn Pro

675 680 685

Arg Cys Gln Lys Pro Phe Phe Leu Lys Glu Ile Pro Ile Gln Asp Val

690 695 700

Ala Ile Gln Asp Phe Thr Cys Asp Gly Asn Glu Glu Ser Ser Cys Gln

705 710 715 720

Leu Ser Pro Arg Cys Pro Glu Gln Cys Thr Cys Met Glu Thr Val Val

725 730 735

Arg Cys Ser Asn Lys Gly Leu Arg Ala Leu Pro Arg Gly Met Pro Lys

740 745 750

Asp Val Thr Glu Leu Tyr Leu Glu Gly Asn His Leu Thr Ala Val Pro

755 760 765

Arg Glu Leu Ser Ala Leu Arg His Leu Thr Leu Ile Asp Leu Ser Asn

770 775 780

Asn Ser Ile Ser Met Leu Thr Asn Tyr Thr Phe Ser Asn Met Ser His

785 790 795 800

Leu Ser Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Val

805 810 815

His Ala Phe Asn Gly Leu Arg Ser Leu Arg Val Leu Thr Leu His Gly

820 825 830

Asn Asp Ile Ser Ser Val Pro Glu Gly Ser Phe Asn Asp Leu Thr Ser

835 840 845

Leu Ser His Leu Ala Leu Gly Thr Asn Pro Leu His Cys Asp Cys Ser

850 855 860

Leu Arg Trp Leu Ser Glu Trp Val Lys Ala Gly Tyr Lys Glu Pro Gly

865 870 875 880

Ile Ala Arg Cys Ser Ser Pro Glu Pro Met Ala Asp Arg Leu Leu Leu

885 890 895

Thr Thr Pro Thr His Arg Phe Gln Cys Lys Gly Pro Val Asp Ile Asn

900 905 910

Ile Val Ala Lys Cys Asn Ala Cys Leu Ser Ser Pro Cys Lys Asn Asn

915 920 925

Gly Thr Cys Thr Gln Asp Pro Val Glu Leu Tyr Arg Cys Ala Cys Pro

930 935 940

Tyr Ser Tyr Lys Gly Lys Asp Cys Thr Val Pro Ile Asn Thr Cys Ile

945 950 955 960

Gln Asn Pro Cys Gln His Gly Gly Thr Cys His Leu Ser Asp Ser His

965 970 975

Lys Asp Gly Phe Ser Cys Ser Cys Pro Leu Gly Phe Glu Gly Gln Arg

980 985 990

Cys Glu Ile Asn Pro Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn

995 1000 1005

Ala Thr Cys Val Asp Gly Ile Asn Asn Tyr Val Cys Ile Cys Pro

1010 1015 1020

Pro Asn Tyr Thr Gly Glu Leu Cys Asp Glu Val Ile Asp His Cys

1025 1030 1035

Val Pro Glu Leu Asn Leu Cys Gln His Glu Ala Lys Cys Ile Pro

1040 1045 1050

Leu Asp Lys Gly Phe Ser Cys Glu Cys Val Pro Gly Tyr Ser Gly

1055 1060 1065

Lys Leu Cys Glu Thr Asp Asn Asp Asp Cys Val Ala His Lys Cys

1070 1075 1080

Arg His Gly Ala Gln Cys Val Asp Thr Ile Asn Gly Tyr Thr Cys

1085 1090 1095

Thr Cys Pro Gln Gly Phe Ser Gly Pro Phe Cys Glu His Pro Pro

1100 1105 1110

Pro Met Val Leu Leu Gln Thr Ser Pro Cys Asp Gln Tyr Glu Cys

1115 1120 1125

Gln Asn Gly Ala Gln Cys Ile Val Val Gln Gln Glu Pro Thr Cys

1130 1135 1140

Arg Cys Pro Pro Gly Phe Ala Gly Pro Arg Cys Glu Lys Leu Ile

1145 1150 1155

Thr Val Asn Phe Val Gly Lys Asp Ser Tyr Val Glu Leu Ala Ser

1160 1165 1170

Ala Lys Val Arg Pro Gln Ala Asn Ile Ser Leu Gln Val Ala Thr

1175 1180 1185

Asp Lys Asp Asn Gly Ile Leu Leu Tyr Lys Gly Asp Asn Asp Pro

1190 1195 1200

Leu Ala Leu Glu Leu Tyr Gln Gly His Val Arg Leu Val Tyr Asp

1205 1210 1215

Ser Leu Ser Ser Pro Pro Thr Thr Val Tyr Ser Val Glu Thr Val

1220 1225 1230

Asn Asp Gly Gln Phe His Ser Val Glu Leu Val Thr Leu Asn Gln

1235 1240 1245

Thr Leu Asn Leu Val Val Asp Lys Gly Thr Pro Lys Ser Leu Gly

1250 1255 1260

Lys Leu Gln Lys Gln Pro Ala Val Gly Ile Asn Ser Pro Leu Tyr

1265 1270 1275

Leu Gly Gly Ile Pro Thr Ser Thr Gly Leu Ser Ala Leu Arg Gln

1280 1285 1290

Gly Thr Asp Arg Pro Leu Gly Gly Phe His Gly Cys Ile His Glu

1295 1300 1305

Val Arg Ile Asn Asn Glu Leu Gln Asp Phe Lys Ala Leu Pro Pro

1310 1315 1320

Gln Ser Leu Gly Val Ser Pro Gly Cys Lys Ser Cys Thr Val Cys

1325 1330 1335

Lys His Gly Leu Cys Arg Ser Val Glu Lys Asp Ser Val Val Cys

1340 1345 1350

Glu Cys Arg Pro Gly Trp Thr Gly Pro Leu Cys Asp Gln Glu Ala

1355 1360 1365

Arg Asp Pro Cys Leu Gly His Arg Cys His His Gly Lys Cys Val

1370 1375 1380

Ala Thr Gly Thr Ser Tyr Met Cys Lys Cys Ala Glu Gly Tyr Gly

1385 1390 1395

Gly Asp Leu Cys Asp Asn Lys Asn Asp Ser Ala Asn Ala Cys Ser

1400 1405 1410

Ala Phe Lys Cys His His Gly Gln Cys His Ile Ser Asp Gln Gly

1415 1420 1425

Glu Pro Tyr Cys Leu Cys Gln Pro Gly Phe Ser Gly Glu His Cys

1430 1435 1440

Gln Gln Glu Asn Pro Cys Leu Gly Gln Val Val Arg Glu Val Ile

1445 1450 1455

Arg Arg Gln Lys Gly Tyr Ala Ser Cys Ala Thr Ala Ser Lys Val

1460 1465 1470

Pro Ile Met Glu Cys Arg Gly Gly Cys Gly Pro Gln Cys Cys Gln

1475 1480 1485

Pro Thr Arg Ser Lys Arg Arg Lys Tyr Val Phe Gln Cys Thr Asp

1490 1495 1500

Gly Ser Ser Phe Val Glu Glu Val Glu Arg His Leu Glu Cys Gly

1505 1510 1515

Cys Leu Ala Cys Ser

1520

<210>9

<211>771

<212>PRT

<213>Homo sapiens

<400>9

Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly Val Leu Leu

1 5 10 15

Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu

20 25 30

Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr Phe

35 40 45

Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu

50 55 60

Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe

65 70 75 80

Asp Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser

85 90 95

Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys

100 105 110

Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His

115 120 125

Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile

130 135 140

Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser

145 150 155 160

His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu

165 170 175

Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala

180 185 190

Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His

195 200 205

His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro

210 215 220

Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp

225 230 235 240

Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His

245 250 255

Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp

260 265 270

Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys

275 280 285

Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His

290 295 300

Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys

305 310 315 320

Asn Pro Val Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys

325 330 335

Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe

340 345 350

Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro

355 360 365

Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys

370 375 380

Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile

385 390 395 400

Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met

405 410 415

Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr

420 425 430

Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val

435 440 445

Met Phe Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile

450 455 460

Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met

465 470 475 480

Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr

485 490 495

Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu

500 505 510

Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys

515 520 525

Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg

530 535 540

Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn

545 550 555 560

Gly Asp Pro Leu Thr His Cys Ser Asp Leu His His Asp Asn His His

565 570 575

Gly His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser

580 585 590

Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp

595 600 605

Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp

610 615 620

Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln

625 630 635 640

Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe

645 650 655

Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu His

660 665 670

Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr

675 680 685

Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg

690 695 700

Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu

705 710 715 720

Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg

725 730 735

Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu

740 745 750

Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro

755 760 765

Arg Ser Val

770

<210>10

<211>5672

<212>DNA

<213>Homo sapiens

<400>10

aagcaccact gcagcagacc ttgttaattt tttttttttt tctttccaca caacagttgt 60

gcctcattat ccggtgcctg gctcggaatt tttttttttt tttttctttt tggagggttt 120

gaagtttctg tgcttcagtg actgttacag aagaagaggt gttagtgttg ccatgaggtc 180

ttgattgtct gcatttatga atgaaactga cctaaatcac ctgttacctc cagtttccag 240

attgtttgaa cttctctggc cgcacaatac aggaaggaag actaaagcag caaagggacc 300

tacagcgtct gcagcatggg ctggttaact aggattgtct gtcttttctg gggagtatta 360

cttacagcaa gagcaaacta tcagaatggg aagaacaatg tgccaaggct gaaattatcc 420

tacaaagaaa tgttggaatc caacaatgtg atcactttca atggcttggc caacagctcc 480

agttatcata ccttcctttt ggatgaggaa cggagtaggc tgtatgttgg agcaaaggat 540

cacatatttt cattcgacct ggttaatatc aaggattttc aaaagattgt gtggccagta 600

tcttacacca gaagagatga atgcaagtgg gctggaaaag acatcctgaa agaatgtgct 660

aatttcatca aggtacttaa ggcatataat cagactcact tgtacgcctg tggaacgggg 720

gcttttcatc caatttgcac ctacattgaa attggacatc atcctgagga caatattttt 780

aagctggaga actcacattt tgaaaacggc cgtgggaaga gtccatatga ccctaagctg 840

ctgacagcat cccttttaat agatggagaa ttatactctg gaactgcagc tgattttatg 900

gggcgagact ttgctatctt ccgaactctt gggcaccacc acccaatcag gacagagcag 960

catgattcca ggtggctcaa tgatccaaag ttcattagtg cccacctcat ctcagagagt 1020

gacaatcctg aagatgacaa agtatacttt ttcttccgtg aaaatgcaat agatggagaa 1080

cactctggaa aagctactca cgctagaata ggtcagatat gcaagaatga ctttggaggg 1140

cacagaagtc tggtgaataa atggacaaca ttcctcaaag ctcgtctgat ttgctcagtg 1200

ccaggtccaa atggcattga cactcatttt gatgaactgc aggatgtatt cctaatgaac 1260

tttaaagatc ctaaaaatcc agttgtatat ggagtgttta cgacttccag taacattttc 1320

aagggatcag ccgtgtgtat gtatagcatg agtgatgtga gaagggtgtt ccttggtcca 1380

tatgcccaca gggatggacc caactatcaa tgggtgcctt atcaaggaag agtcccctat 1440

ccacggccag gaacttgtcc cagcaaaaca tttggtggtt ttgactctac aaaggacctt 1500

cctgatgatg ttataacctt tgcaagaagt catccagcca tgtacaatcc agtgtttcct 1560

atgaacaatc gcccaatagt gatcaaaacg gatgtaaatt atcaatttac acaaattgtc 1620

gtagaccgag tggatgcaga agatggacag tatgatgtta tgtttatcgg aacagatgtt 1680

gggaccgttc ttaaagtagt ttcaattcct aaggagactt ggtatgattt agaagaggtt 1740

ctgctggaag aaatgacagt ttttcgggaa ccgactgcta tttcagcaat ggagctttcc 1800

actaagcagc aacaactata tattggttca acggctgggg ttgcccagct ccctttacac 1860

cggtgtgata tttacgggaa agcgtgtgct gagtgttgcc tcgcccgaga cccttactgt 1920

gcttgggatg gttctgcatg ttctcgctat tttcccactg caaagagacg cacaagacga 1980

caagatataa gaaatggaga cccactgact cactgttcag acttacacca tgataatcac 2040

catggccaca gccctgaaga gagaatcatc tatggtgtag agaatagtag cacatttttg 2100

gaatgcagtc cgaagtcgca gagagcgctg gtctattggc aattccagag gcgaaatgaa 2160

gagcgaaaag aagagatcag agtggatgat catatcatca ggacagatca aggccttctg 2220

ctacgtagtc tacaacagaa ggattcaggc aattacctct gccatgcggt ggaacatggg 2280

ttcatacaaa ctcttcttaa ggtaaccctg gaagtcattg acacagagca tttggaagaa 2340

cttcttcata aagatgatga tggagatggc tctaagacca aagaaatgtc caatagcatg 2400

acacctagcc agaaggtctg gtacagagac ttcatgcagc tcatcaacca ccccaatctc 2460

aacacaatgg atgagttctg tgaacaagtt tggaaaaggg accgaaaaca acgtcggcaa 2520

aggccaggac ataccccagg gaacagtaac aaatggaagc acttacaaga aaataagaaa 2580

ggtagaaaca ggaggaccca cgaatttgag agggcaccca ggagtgtctg agctgcatta 2640

cctctagaaa cctcaaacaa gtagaaactt gcctagacaa taactggaaa aacaaatgca 2700

atatacatga acttttttca tggcattatg tggatgttta caatggtggg aaattcagct 2760

gagttccacc aattataaat taaatccatg agtaactttc ctaataggct ttttttccta 2820

ataccaccac ctaacagaga acacaggtga atgcagatgt tcactttagc agacttaatg 2880

tttcctatga gatttcactg tacaggtttg tctttcttct ttgcctgaga aataaaaatg 2940

tcatttgcca tattgccatc taaaggagaa aaactgcatc agcaaagcca ttgtattgaa 3000

ctaaaagttt aaaatgaact gcatggattt actaagctga tgaatattcc aaaacgtggt 3060

tggattcaag gatatatttt gtctaccggc cctcatgttt gtatgtactt gaggagtaaa 3120

atgagtaaaa tgatactgaa tgaaatgttc tgtggaaata ttaaaaaaaa aaaaaaacat 3180

aagccatcca tcatccagaa gaaaaatgga atacactgat ctactactga tgtcttcttt 3240

cagctttgat ctaaagatgt attttattaa aactataatt taaatgtacc atgaaaaata 3300

tgcagtaaaa attagttgtt ttctaagcta gagtaggatt tgtcttacaa ttattgtgct 3360

atgtagtttt tgttttaaaa attccaatgg tgtgctgctt tctttggaca ttttattttc 3420

aattctataa gagggataga tgacattgtt ctagaaacac atatacatca ttaagagtga 3480

atctctaaaa ccaggatata aattatgctt tatttctctg agaaaatcaa acaaatggaa 3540

gctgttcaca cctccccttc tttaagcatt atctaaatta atttttactt gcataatgtt 3600

cttagaaaaa aaaacagaac atttaagcag gaaaaaagga agaaacaagt tgatttttaa 3660

gtgcatttta ctataatgaa tcaatgaagg gaaaaggaac tgcatatttc atgaaaataa 3720

taagcattgt cttaatatac tgttaataga aaatgtgtct taattccgtg cttgaatccc 3780

tgcatgatat ttgagactaa gatctctctt atgattctac caagaattat atctgtgtca 3840

cttaattttt ttaaaagaga gagatcaata actattcaga gcaacatgtt aaaggcaaag 3900

tttccaatca tttacatctg tatcaggtgc ctcttacctt tccttattta agacaattat 3960

ttgtacaaga aacacatgac tcttttcata tcaatgggag ggacttttct acaaagtatt 4020

ttccaggatg caacccacat ttaaacaatg taaaattctt tgtttcctgc aacaacttac 4080

aaaataaggt aaaagactaa aattcaagat ttgcttcctt cattgtccta agacgattcg 4140

ttgagaatca ctgactttga gatatttaaa actttcagca ttatactgtg gtttcttttg 4200

cactgcactc acctattcag gactcctccc ccaggttcct catcatgcac aaaaatgcaa 4260

agaaaacatc ttattagtaa ttaatgaagc aacattgaaa ttctaactct agctgtcttt 4320

ggattctaat taactcagca tcaatttctc acctcagact acagtgaatt tttatttcct 4380

atcagctgaa atatttcaca gatggaagct catgtttcag ttttaatgac tgccttgaat 4440

aaacaagttg ttgccacttg tttcaaacaa aagcctaaaa ataatctaca ttcaatttta 4500

ggctccattg actaatatgg tgttgctttt ggaagtactg tatatcctca catggaagcc 4560

aaattgttaa attatttgaa ggacacacca ctgtacagaa agtagtgttt caaatataaa 4620

tcgaagaaca aagagtgctc caaaaaatag gtcattcttt tattttcata aagtatctaa 4680

actgtactaa cattcagtgt tgtgtttcat tctaaatttg cagctgaaat aaatttattt 4740

gcgatagcag aaatatctta ttattcatcc tcagaaataa aggatttgaa gggatagaga 4800

ttatatgata aatttataga agactttcag aatttgaatg cattttgttt agtgttatga 4860

aatgacaata gaaaaaagtc tcgacttcaa ttaaaagtta cacaaacaaa caaatctaca 4920

ggcatgtctt tatataccat caggtctaag ttttcaaaga aaattgtaga tataacttgc 4980

agataactca ttacagtcat aatctctgcc catgtgtatt gagagggggc agtttgcacg 5040

aaaaagaatt attggcccat ttaataattc agctttaaat agactttgtc atatgcatga 5100

atcatcagag atgaaactgt ttgagagact catgtgacct tacgaaaatt acaacagcag 5160

tcttaaagta tgaaaaagat gcatcacagc agagacatta tggcccagtt gatatcaaat 5220

gtaaaatgta aatgcatgta aatgcacact tcattttatg tattatttag taatttgcag 5280

tggtatgtgt ttaatatttt tgctacctac acattaggca aaaaaaagat gtaaataatt 5340

tgggagaaaa agaggaagaa cagtgtaaaa taaaactttc tataagtact ccatttcaat 5400

gtgttcaaca tcatcctaaa aggcaagatt ttcccacgca ggtgacaagg tggtttatgt 5460

actatttaag ggcggaaggt gcgtgcccgt tcaataagca tgttttttgc caggtaggaa 5520

atatgttcca tatctttact tatcattgca tttcagatgg gaactagaaa aactggagag 5580

aaaaatgtaa tgaaactgct gctgtaaatt attcctttta gcatgtattc acttgctaaa 5640

tacacatttc ttcaaaataa aaaaaaaaaa aa 5672

<210>11

<211>775

<212>PRT

<213>Homo sapiens

<400>11

Met Ala Ser Ala Gly His Ile Ile Thr Leu Leu Leu Trp Gly Tyr Leu

1 5 10 15

Leu Glu Leu Trp Thr Gly Gly His Thr Ala Asp Thr Thr His Pro Arg

20 25 30

Leu Arg Leu Ser His Lys Glu Leu Leu Asn Leu Asn Arg Thr Ser Ile

35 40 45

Phe His Ser Pro Phe Gly Phe Leu Asp Leu His Thr Met Leu Leu Asp

50 55 60

Glu Tyr Gln Glu Arg Leu Phe Val Gly Gly Arg Asp Leu Val Tyr Ser

65 70 75 80

Leu Ser Leu Glu Arg Ile Ser Asp Gly Tyr Lys Glu Ile His Trp Pro

85 90 95

Ser Thr Ala Leu Lys Met Glu Glu Cys Ile Met Lys Gly Lys Asp Ala

100 105 110

Gly Glu Cys Ala Asn Tyr Val Arg Val Leu His His Tyr Asn Arg Thr

115 120 125

His Leu Leu Thr Cys Gly Thr Gly Ala Phe Asp Pro Val Cys Ala Phe

130 135 140

Ile Arg Val Gly Tyr His Leu Glu Asp Pro Leu Phe His Leu Glu Ser

145 150 155 160

Pro Arg Ser Glu Arg Gly Arg Gly Arg Cys Pro Phe Asp Pro Ser Ser

165 170 175

Ser Phe Ile Ser Thr Leu Ile Gly Ser Glu Leu Phe Ala Gly Leu Tyr

180 185 190

Ser Asp Tyr Trp Ser Arg Asp Ala Ala Ile Phe Arg Ser Met Gly Arg

195 200 205

Leu Ala His Ile Arg Thr Glu His Asp Asp Glu Arg Leu Leu Lys Glu

210 215 220

Pro Lys Phe Val Gly Ser Tyr Met Ile Pro Asp Asn Glu Asp Arg Asp

225 230 235 240

Asp Asn Lys Val Tyr Phe Phe Phe Thr Glu Lys Ala Leu Glu Ala Glu

245 250 255

Asn Asn Ala His Ala Ile Tyr Thr Arg Val Gly Arg Leu Cys Val Asn

260 265 270

Asp Val Gly Gly Gln Arg Ile Leu Val Asn Lys Trp Ser Thr Phe Leu

275 280 285

Lys Ala Arg Leu Val Cys Ser Val Pro Gly Met Asn Gly Ile Asp Thr

290 295 300

Tyr Phe Asp Glu Leu Glu Asp Val Phe Leu Leu Pro Thr Arg Asp His

305 310 315 320

Lys Asn Pro Val Ile Phe Gly Leu Phe Asn Thr Thr Ser Asn Ile Phe

325 330 335

Arg Gly His Ala Ile Cys Val Tyr His Met Ser Ser Ile Arg Ala Ala

340 345 350

Phe Asn Gly Pro Tyr Ala His Lys Glu Gly Pro Glu Tyr His Trp Ser

355 360 365

Val Tyr Glu Gly Lys Val Pro Tyr Pro Arg Pro Gly Ser Cys Ala Ser

370 375 380

Lys Val Asn Gly Gly Arg Tyr Gly Thr Thr Lys Asp Tyr Pro Asp Asp

385 390 395 400

Ala Ile Arg Phe Ala Arg Ser His Pro Leu Met Tyr Gln Ala Ile Lys

405 410 415

Pro Ala His Lys Lys Pro Ile Leu Val Lys Thr Asp Gly Lys Tyr Asn

420 425 430

Leu Lys Gln Ile Ala Val Asp Arg Val Glu Ala Glu Asp Gly Gln Tyr

435 440 445

Asp Val Leu Phe Ile Gly Thr Asp Asn Gly Ile Val Leu Lys Val Ile

450 455 460

Thr Ile Tyr Asn Gln Glu Met Glu Ser Met Glu Glu Val Ile Leu Glu

465 470 475 480

Glu Leu Gln Ile Phe Lys Asp Pro Val Pro Ile Ile Ser Met Glu Ile

485 490 495

Ser Ser Lys Arg Gln Gln Leu Tyr Ile Gly Ser Ala Ser Ala Val Ala

500 505 510

Gln Val Arg Phe His His Cys Asp Met Tyr Gly Ser Ala Cys Ala Asp

515 520 525

Cys Cys Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ile Ser Cys

530 535 540

Ser Arg Tyr Tyr Pro Thr Gly Thr His Ala Lys Arg Arg Phe Arg Arg

545 550 555 560

Gln Asp Val Arg His Gly Asn Ala Ala Gln Gln Cys Phe Gly Gln Gln

565 570 575

Phe Val Gly Asp Ala Leu Asp Lys Thr Glu Glu His Leu Ala Tyr Gly

580 585 590

Ile Glu Asn Asn Ser Thr Leu Leu Glu Cys Thr Pro Arg Ser Leu Gln

595 600 605

Ala Lys Val Ile Trp Phe Val Gln Lys Gly Arg Glu Thr Arg Lys Glu

610 615 620

Glu Val Lys Thr Asp Asp Arg Val Val Lys Met Asp Leu Gly Leu Leu

625 630 635 640

Phe Leu Arg Leu His Lys Ser Asp Ala Gly Thr Tyr Phe Cys Gln Thr

645 650 655

Val Glu His Ser Phe Val His Thr Val Arg Lys Ile Thr Leu Glu Val

660 665 670

Val Glu Glu Glu Lys Val Glu Asp Met Phe Asn Lys Asp Asp Glu Glu

675 680 685

Asp Arg His His Arg Met Pro Cys Pro Ala Gln Ser Ser Ile Ser Gln

690 695 700

Gly Ala Lys Pro Trp Tyr Lys Glu Phe Leu Gln Leu Ile Gly Tyr Ser

705 710 715 720

Asn Phe Gln Arg Val Glu Glu Tyr Cys Glu Lys Val Trp Cys Thr Asp

725 730 735

Arg Lys Arg Lys Lys Leu Lys Met Ser Pro Ser Lys Trp Lys Tyr Ala

740 745 750

Asn Pro Gln Glu Lys Lys Leu Arg Ser Lys Pro Glu His Tyr Arg Leu

755 760 765

Pro Arg His Thr Leu Asp Ser

770 775

<210>12

<211>6474

<212>DNA

<213>Homo sapiens

<400>12

gtttggcaag tcagtgcaag aggctgactt ctgagaggct tccaggagcc cgaagagagg 60

acctccacgg gagaagggag tgcgtgtgct cggttttttt tttttctctc tttttttttt 120

ttttttctga atgaacagct ttgcccaagt gactgaaaaa tacagcttct tcctgaatct 180

accggcgtag ttgctgaaga gcgctctaga caggacatgg ctctgaagac tcactctttg 240

gaatgtcctc ttgctcccgg cttataaaca actgtcccga ggaaagaaag gttttacata 300

gccaaataca gcctgacaaa tggcacttcg gaactgtgct ttctgatgac aacgcgttcg 360

atttctgaca aagcctctcg cacgctgccc ctggagggaa gtcctaagta aaactcagac 420

cctccttaaa gtgaggagcg agggcttgga cggtgaacac ggcagcatgg catccgcggg 480

gcacattatc accttgctcc tgtggggtta cttactggag ctttggacag gaggtcatac 540

agctgatact acccaccccc ggttacgcct gtcacataaa gagctcttga atctgaacag 600

aacatcaata tttcatagcc cttttggatt tcttgatctc catacaatgc tgctggatga 660

atatcaagag aggctcttcg tgggaggcag ggaccttgta tattccctca gcttggagag 720

aatcagtgac ggctataaag agatacactg gccgagtaca gctctaaaaa tggaagaatg 780

cataatgaag ggaaaagatg cgggtgaatg tgcaaattat gttcgggttt tgcatcacta 840

taacaggaca caccttctga cctgtggtac tggagctttt gatccagttt gtgccttcat 900

cagagttgga tatcatttgg aggatcctct gtttcacctg gaatcaccca gatctgagag 960

aggaaggggc agatgtcctt ttgaccccag ctcctccttc atctccactt taattggtag 1020

tgaattgttt gctggactct acagtgacta ctggagcaga gacgctgcga tcttccgcag 1080

catggggcga ctggcccata tccgcactga gcatgacgat gagcgtctgt tgaaagaacc 1140

aaaatttgta ggttcataca tgattcctga caatgaagac agagatgaca acaaagtata 1200

tttctttttt actgagaagg cactggaggc agaaaacaat gctcacgcaa tttacaccag 1260

ggtcgggcga ctctgtgtga atgatgtagg agggcagaga atactggtga ataagtggag 1320

cactttccta aaagcgagac tcgtttgctc agtaccagga atgaatggaa ttgacacata 1380

ttttgatgaa ttagaggacg tttttttgct acctaccaga gatcataaga atccagtgat 1440

atttggactc tttaacacta ccagtaatat ttttcgaggg catgctatat gtgtctatca 1500

catgtctagc attcgggcag ccttcaacgg accatatgca cataaggaag gacctgaata 1560

ccactggtca gtctatgaag gaaaagtccc ttatccaagg cctggttctt gtgccagcaa 1620

agtaaatgga gggagatacg gaaccaccaa ggactatcct gatgatgcca tccgatttgc 1680

aagaagtcat ccactaatgt accaggccat aaaacctgcc cataaaaaac caatattggt 1740

aaaaacagat ggaaaatata acctgaaaca aatagcagta gategagtgg aagctgagga 1800

tggccaatat gacgtcttgt ttattgggac agataatgga attgtgctga aagtaatcac 1860

aatttacaac caagaaatgg aatcaatgga agaagtaatt ctagaagaac ttcagatatt 1920

caaggatcca gttcctatta tttctatgga gatttcttca aaacggcaac agctgtatat 1980

tggatctgct tctgctgtgg ctcaagtcag attccatcac tgtgacatgt atggaagtgc 2040

ttgtgctgac tgctgcctgg ctcgagaccc ttactgtgcc tgggatggca tatcctgctc 2100

ccggtattac ccaacaggca cacatgcaaa aaggcgtttc cggagacaag atgttcgaca 2160

tggaaatgca gctcagcagt gctttggaca acagtttgtt ggggatgctt tggataagac 2220

tgaagaacat ctggcttatg gcatagagaa caacagtact ttgctggaat gtaccccacg 2280

atctttacaa gcgaaagtta tctggtttgt acagaaagga cgtgagacaa gaaaagagga 2340

ggtgaagaca gatgacagag tggttaagat ggaccttggt ttactcttcc taaggttaca 2400

caaatcagat gctgggacct atttttgcca gacagtagag catagctttg tccatacggt 2460

ccgtaaaatc accttggagg tagtggaaga ggagaaagtc gaggatatgt ttaacaagga 2520

cgatgaggag gacaggcatc acaggatgcc ttgtcctgct cagagtagca tctcgcaggg 2580

agcaaaacca tggtacaagg aattcttgca gctgatcggt tatagcaact tccagagagt 2640

ggaagaatac tgcgagaaag tatggtgcac agatagaaag aggaaaaagc ttaaaatgtc 2700

accctccaag tggaagtatg ccaaccctca ggaaaagaag ctccgttcca aacctgagca 2760

ttaccgcctg cccaggcaca cgctggactc ctgatggggt gagactatct actgtctttt 2820

gaagaattta tatttggaaa gtaaaaaagt aaaaaaataa atcatccaac ttctttgcat 2880

tacttaaaag agatttctgt aatacaggaa tgactatgaa ggtgttataa taaattattc 2940

tacatactca tttgactgga taaactttac ataaaattaa ctaatttttt aaataaatgc 3000

attgcttaat ggtttctcat tatgtttatc aaaaaacaac tgtagctgtt attttcagta 3060

cttggctgct tttctgtgaa aattattatt ttacttttgg aagacaagat tattagaata 3120

ttgaagaaaa attggagact tataatcatg gtaaatataa aactaaatat gttttaatat 3180

ttctgaattt ttcttttcca tcacaatgta agatatgcag aatacaagat actttggcat 3240

tctcatgtga actttctgta ctctttaagg attattttat tagtgttgtt taagccatga 3300

gtgttaagta gcaggtgtgt tgtgagtgct gtaacccatg aaaggaaaaa tgtcattctg 3360

aggcttgtgc ccttcgtaaa atattcatta aagtacattc acactatttt tgctttataa 3420

cacagtcttt aattttcact cactgtggaa ataaaaacta aggtaacttc tcagaaagat 3480

atcaaatctc agaaagaatg tcaaatcaga tgaagttata gttaggattc taactactgt 3540

aaaagatttt tgcttccctc ttgtggtaaa aaaaattata ttctcacaca tttctttttt 3600

ctctacagac ggatatctgt ttaggaaaga tttgaaagca gattatcagt aggtacatgg 3660

atacatcaag ttcatttgca gaaacaaata actgaaataa aaaacatgtt aatccttgta 3720

tcatacttta atatgaaagt attgtttata gataatttat ctcacaagtc aaaaatgaag 3780

attttgcagc actgaaaatc tattaaagct ccaaatttta agtttctaaa taatcttcgc 3840

tgaaatctaa aatatactat aacaaccgtg ttttatttgt gaaaaaaata ttaaagtgat 3900

ttgctctcaa atatcaaatt ttcttctctc ttttatatta agagacagaa aattgtttca 3960

tgagttcact taactactga gatattcaga gcatttttac ctctctctta aatgttataa 4020

aaaacaattg tatttttaag aatgtttatt tatcaaagtc tttccttctt ctattaaata 4080

tttagcaatt acctttctaa aatatgaaat tttgtaagat gttttcacct aaataaaaat 4140

tgaaagcaag tggattacac aggagaacca ttatgaacat ttatttagat attaatctta 4200

aacagtgttt atttcagttt tcaaagttag cttataggtt atacatttaa gttaaagtgc 4260

tcataatcac ttgcaatttc attgtaaaat gaacaaatac ataaatattt taagaaaaat 4320

ttaagtttat tcagataagt caccatgctt caaaagatct aagaaatgca aatatactga 4380

aaattgacat cctctgaaaa ttccacttgc tatttaccca agaatccact ggaggtcatt 4440

actgccatta aataataact gaaaagacta tgtagtgaaa tgtattttta aaaactatat 4500

tcagtaaaag cctgctcaat ttggagaaat agaaccacaa acacagatca caggggcctt 4560

acaaagttta tgtctgaaca aataagtcaa ttaagtacac tttattgaaa attgccttcc 4620

attaacacac aagaaagaaa gcaggatttt ctcctgtatc tgaattttaa aattaaaaag 4680

gcagataaga cataaatagt tatcatttta attgcaataa cacagacaag tagttaatga 4740

tgataacaat ggtgtaactt gtaaactaaa tatttggtaa ctgaagcaat aggcagagga 4800

aaatagcttt tctatgacac aagtcataag aagtccatat actgaagagc gtttgattaa 4860

aataaagtga ctattaacca gaaaagaaac attttacata aaatgctaaa atttattata 4920

ggaaaataaa tcaaacccaa agaaagttta ttcaatgcta atttgaaaga aaattgataa 4980

gaaaactttg agggcccaag tccacaattt ggtgagacca ctaaatttta catataatta 5040

tacacacaca tatgtacata tatatgtata taatcttgct tcccgcctgt ttatggcagt 5100

actgaagaga aatgggaaag aagagggagg gagagagaaa gacgaaggga gagagaaagc 5160

agtttccaag gatatgtttc atgtcccacc attttctcag tttctccctc tctctcccaa 5220

cacacacaca cacacacccc tcacatacta taaaataaat cttcactgcc ctatcaaaat 5280

acaaataaat caatctatgc tgttctgtcc ttcttgagaa tctaaaacat accacaaaaa 5340

tacatcccca gtcttttgtt ctgtctgagg ttagaattaa ttcaaattca gaatctgttg 5400

tgagaaatgc ccaggcttta aaaattaaaa atggatggat cttctctgaa ctcagggagg 5460

gcacatactt agatacctac aagacttgga ggaattaaga gttcaccctt catctcacca 5520

aattttcccc atttttctct ttcttgtaga aggagagaaa ccatgctctc tagcaacatt 5580

gagcaaaaat cataaccact catctaattt ctaagaggca cctccatcga gggccggtct 5640

cctgcttctt tagacctctt ctatctttgt tacaggagag gacctgtgga tagacttagt 5700

tttgacataa aacaatgccc attcacctcc tccttcagca caacgtcacc cattgggcaa 5760

gagatccaga tttgttaaca aaaaagattt tacttcgtga ttccacgtct ataattctat 5820

attgctaatt ttttcttttg tgtgaattac tgaatatttc agagcaaagc tatcaacttg 5880

gagaaacagg gattaaaaat aaggataaac actaataaga gctctagaaa aaagggaaca 5940

gaaagtctgc ctgtttagta agtggcaatt ccatacatat tttagagttt tttctatcta 6000

aaattagtta aatacttaga atgtttgtaa tgagtgttcg atatttgcta taggttttag 6060

ggttttgtaa atcttcatag taattataaa catttgtaaa atttgtaaaa tactataagt 6120

cattttgagt gttggtgtta agcatgaaac aaacagcagc tgttgtcctt aaaaatgaat 6180

tgacctggcc gggcgcggtg gctcacgcct gtaatcccag cactttggga ggccgaggcg 6240

ggtggatcat gaggtcagga gatggagacc atcctggcta acaaggtgaa accccgtctc 6300

tactaaaaat acaaaaaatt agccgggcgc ggtggcgggc gcctgtagtc ccagctactt 6360

gggaggctga ggcaggagaa tggcgtgaac ccgggaagcg gagcttgcag tgagccgaga 6420

ttgcgccact gcagtccgca gtccggcctg ggcgacagag cgagactccg tctc 6474

<210>13

<211>785

<212>PRT

<213>Homo sapiens

<400>13

Met Leu Val Ala Gly Leu Leu Leu Trp Ala Ser Leu Leu Thr Gly Ala

1 5 10 15

Trp Pro Ser Phe Pro Thr Gln Asp His Leu Pro Ala Thr Pro Arg Val

20 25 30

Arg Leu Ser Phe Lys Glu Leu Lys Ala Thr Gly Thr Ala His Phe Phe

35 40 45

Asn Phe Leu Leu Asn Thr Thr Asp Tyr Arg Ile Leu Leu Lys Asp Glu

50 55 60

Asp His Asp Arg Met Tyr Val Gly Ser Lys Asp Tyr Val Leu Ser Leu

65 70 75 80

Asp Leu His Asp Ile Asn Arg Glu Pro Leu Ile Ile His Trp Ala Ala

85 90 95

Ser Pro Gln Arg Ile Glu Glu Cys Val Leu Ser Gly Lys Asp Val Asn

100 105 110

Gly Glu Cys Gly Asn Phe Val Arg Leu Ile Gln Pro Trp Asn Arg Thr

115 120 125

His Leu Tyr Val Cys Gly Thr Gly Ala Tyr Asn Pro Met Cys Thr Tyr

130 135 140

Val Asn Arg Gly Arg Arg Ala Gln Ala Thr Pro Trp Thr Gln Thr Gln

145 150 155 160

Ala Val Arg Gly Arg Gly Ser Arg Ala Thr Asp Gly Ala Leu Arg Pro

165 170 175

Met Pro Thr Ala Pro Arg Gln Asp Tyr Ile Phe Tyr Leu Glu Pro Glu

180 185 190

Arg Leu Glu Ser Gly Lys Gly Lys Cys Pro Tyr Asp Pro Lys Leu Asp

195 200 205

Thr Ala Ser Ala Leu Ile Asn Glu Glu Leu Tyr Ala Gly Val Tyr Ile

210 215 220

Asp Phe Met Gly Thr Asp Ala Ala Ile Phe Arg Thr Leu Gly Lys Gln

225 230 235 240

Thr Ala Met Arg Thr Asp Gln Tyr Asn Ser Arg Trp Leu Asn Asp Pro

245 250 255

Ser Phe Ile His Ala Glu Leu Ile Pro Asp Ser Ala Glu Arg Asn Asp

260 265 270

Asp Lys Leu Tyr Phe Phe Phe Arg Glu Arg Ser Ala Glu Ala Pro Gln

275 280 285

Ser Pro Ala Val Tyr Ala Arg Ile Gly Arg Ile Cys Leu Asn Asp Asp

290 295 300

Gly Gly His Cys Cys Leu Val Asn Lys Trp Ser Thr Phe Leu Lys Ala

305 310 315 320

Arg Leu Val Cys Ser Val Pro Gly Glu Asp Gly Ile Glu Thr His Phe

325 330 335

Asp Glu Leu Gln Asp Val Phe Val Gln Gln Thr Gln Asp Val Arg Asn

340 345 350

Pro Val Ile Tyr Ala Val Phe Thr Ser Ser Gly Ser Val Phe Arg Gly

355 360 365

Ser Ala Val Cys Val Tyr Ser Met Ala Asp Ile Arg Met Val Phe Asn

370 375 380

Gly Pro Phe Ala His Lys Glu Gly Pro Asn Tyr Gln Trp Met Pro Phe

385 390 395 400

Ser Gly Lys Met Pro Tyr Pro Arg Pro Gly Thr Cys Pro Gly Gly Thr

405 410 415

Phe Thr Pro Ser Met Lys Ser Thr Lys Asp Tyr Pro Asp Glu Val Ile

420 425 430

Asn Phe Met Arg Ser His Pro Leu Met Tyr Gln Ala Val Tyr Pro Leu

435 440 445

Gln Arg Arg Pro Leu Val Val Arg Thr Gly Ala Pro Tyr Arg Leu Thr

450 455 460

Thr Ile Ala Val Asp Gln Val Asp Ala Ala Asp Gly Arg Tyr Glu Val

465 470 475 480

Leu Phe Leu Gly Thr Asp Arg Gly Thr Val Gln Lys Val Ile Val Leu

485 490 495

Pro Lys Asp Asp Gln Glu Met Glu Glu Leu Met Leu Glu Glu Val Glu

500 505 510

Val Phe Lys Asp Pro Ala Pro Val Lys Thr Met Thr Ile Ser Ser Lys

515 520 525

Arg Gln Gln Leu Tyr Val Ala Ser Ala Val Gly Val Thr His Leu Ser

530 535 540

Leu His Arg Cys Gln Ala Tyr Gly Ala Ala Cys Ala Asp Cys Cys Leu

545 550 555 560

Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Gln Ala Cys Ser Arg Tyr

565 570 575

Thr Ala Ser Ser Lys Arg Arg Ser Arg Arg Gln Asp Val Arg His Gly

580 585 590

Asn Pro Ile Arg Gln Cys Arg Gly Phe Asn Ser Asn Ala Asn Lys Asn

595 600 605

Ala Val Glu Ser Val Gln Tyr Gly Val Ala Gly Ser Ala Ala Phe Leu

610 615 620

Glu Cys Gln Pro Arg Ser Pro Gln Ala Thr Val Lys Trp Leu Phe Gln

625 630 635 640

Arg Asp Pro Gly Asp Arg Arg Arg Glu Ile Arg Ala Glu Asp Arg Phe

645 650 655

Leu Arg Thr Glu Gln Gly Leu Leu Leu Arg Ala Leu Gln Leu Ser Asp

660 665 670

Arg Gly Leu Tyr Ser Cys Thr Ala Thr Glu Asn Asn Phe Lys His Val

675 680 685

Val Thr Arg Val Gln Leu His Val Leu Gly Arg Asp Ala Val His Ala

690 695 700

Ala Leu Phe Pro Pro Leu Ser Met Ser Ala Pro Pro Pro Pro Gly Ala

705 710 715 720

Gly Pro Pro Thr Pro Pro Tyr Gln Glu Leu Ala Gln Leu Leu Ala Gln

725 730 735

Pro Glu Val Gly Leu Ile His Gln Tyr Cys Gln Gly Tyr Trp Arg His

740 745 750

Val Pro Pro Ser Pro Arg Glu Ala Pro Gly Ala Pro Arg Ser Pro Glu

755 760 765

Pro Gln Asp Gln Lys Lys Pro Arg Asn Arg Arg His His Pro Pro Asp

770 775 780

Thr

785

<210>14

<211>3540

<212>DNA

<213>Homo sapiens

<400>14

ccgcggcgcc gatcccggct gaggcgcagc ggcgagaggt cgcgggcagg gccatggccc 60

cggggggccg ctagcgcgga ccggcccaac gggagccgct ccgtgccgcc gccgccgccc 120

gggcgcccag gccccgccgc tgcggaagag gtttctagag agtggagcct gcttcctggg 180

ccctaggccc ctcccacaat gcttgtcgcc ggtcttcttc tctgggcttc cctactgacc 240

ggggcctggc catccttccc cacccaggac cacctcccgg ccacgccccg ggtccggctc 300

tcattcaaag agctgaaggc cacaggcacc gcccacttct tcaacttcct gctcaacaca 360

accgactacc gaatcttgct caaggacgag gaccacgacc gcatgtacgt gggcagcaag 420

gactacgtgc tgtccctgga cctgcacgac atcaaccgcg agcccctcat tatacactgg 480

gcagcctccc cacagcgcat cgaggaatgc gtgctctcag gcaaggatgt caacggcgag 540

tgtgggaact tcgtcaggct catccagccc tggaaccgaa cacacctgta tgtgtgcggg 600

acaggtgcct acaaccccat gtgcacctat gtgaaccgcg gacgccgcgc ccaggccaca 660

ccatggaccc agactcaggc ggtcagaggc cgcggcagca gagccacgga tggtgccctc 720

cgcccgatgc ccacagcccc acgccaggat tacatcttct acctggagcc tgagcgactc 780

gagtcaggga agggcaagtg tccgtacgat cccaagctgg acacagcatc ggccctcatc 840

aatgaggagc tctatgctgg tgtgtacatc gattttatgg gcactgatgc agccatcttc 900

cgcacacttg gaaagcagac agccatgcgc acggatcagt acaactcccg gtggctgaac 960

gacccgtcgt tcatccatgc tgagctcatt cctgacagtg cggagcgcaa tgatgataag 1020

ctttacttct tcttccgtga gcggtcggca gaggcgccgc agagccccgc ggtgtacgcc 1080

cgcatcgggc gcatttgcct gaacgatgac ggtggtcact gttgcctggt caacaagtgg 1140

agcacattcc tgaaggcgcg gctcgtctgc tctgtcccgg gcgaggatgg cattgagact 1200

cactttgatg agctccagga cgtgtttgtc cagcagaccc aggacgtgag gaaccctgtc 1260

atttacgctg tctttacctc ctctggctcc gtgttccgag gctctgccgt gtgtgtctac 1320

tccatggctg atattcgcat ggtcttcaac gggccctttg cccacaaaga ggggcccaac 1380

taccagtgga tgcccttctc agggaagatg ccctacccac ggccgggcac gtgccctggt 1440

ggaaccttca cgccatctat gaagtccacc aaggattatc ctgatgaggt gatcaacttc 1500

atgcgcagcc acccactcat gtaccaggcc gtgtaccctc tgcagcggcg gcccctggta 1560

gtccgcacag gtgctcccta ccgccttacc actattgccg tggaccaggt ggatgcagcc 1620

gacgggcgct atgaggtgct tttcctgggc acagaccgcg ggacagtgca gaaggtcatt 1680

gtgctgccca aggatgacca ggagatggag gagctcatgc tggaggaggt ggaggtcttc 1740

aaggatccag cacccgtcaa gaccatgacc atctcttcta agaggcaaca actctacgtg 1800

gcgtcagccg tgggtgtcac acacctgagc ctgcaccgct gccaggcgta tggggctgcc 1860

tgtgctgact gctgccttgc ccgggaccct tactgtgcct gggatggcca ggcctgctcc 1920

cgctatacag catcctccaa gaggcggagc cgccggcagg acgtccggca cggaaacccc 1980

atcaggcagt gccgtgggtt caactccaat gccaacaaga atgccgtgga gtctgtgcag 2040

tatggcgtgg ccggcagcgc agccttcctt gagtgccagc cccgctcgcc ccaagccact 2100

gttaagtggc tgttccagcg agatcctggt gaccggcgcc gagagattcg tgcagaggac 2160

cgcttcctgc gcacagagca gggcttgttg ctccgtgcac tgcagctcag cgatcgtggc 2220

ctctactcct gcacagccac tgagaacaac tttaagcacg tcgtcacacg agtgcagctg 2280

catgtactgg gccgggacgc cgtccatgct gccctcttcc caccactgtc catgagcgcc 2340

ccgccacccc caggcgcagg ccccccaacg cctccttacc aggagttagc ccagctgctg 2400

gcccagccag aagtgggcct catccaccag tactgccagg gttactggcg ccatgtgccc 2460

cccagcccca gggaggctcc aggggcaccc cggtctcctg agccccagga ccagaaaaag 2520

ccccggaacc gccggcacca ccctccggac acatgaggcc agctgcctgt gcctgccatg 2580

ggccagccta gcccttgtcc cttttaatat aaaagatata tatatatata tatatatata 2640

tataaaatat ctatattcta tacacaccct gcccctgcaa agacagtatt tattggtggg 2700

ttgaatatag cctgcctcag tggcagcatc ctccaaaact tagacccatg ctggtcagag 2760

acggcagaaa acagagcctg cctaaccagg cccagccagt tggtggggcc aggccaggac 2820

cacacagtcc ccagactcag ctggaagtct acctgctgga cagcctccgc caagatctac 2880

aggacaaagg gagggagcaa gccctactcg gatggggcac ggactgtcca ccttttctga 2940

tgtgtgttgt cagcctgtgc tgtggcatag acatggatgc gaggaccact ttggagactg 3000

gggtggcctc aagagcacac agagaaggga agaaggggcc atcacaggat gccagcccct 3060

gcctgggttg ggggcactca gccacgacca gccccttcct gggtatttat tctctattta 3120

ttggggatag gagaagaggc atcctgcctg ggtgggacag ccccttcagc cccttctccc 3180

ctccccgcct ggccagggca gggccacccc actctacctc cttagctttc cctgtgccac 3240

tttgactcag aggctgggag catagcagag gggccaggcc caggcagagc tgacgggagg 3300

ccccagctct gaggggaggg ggtccgtggt agaggcctgg ggccggtaga ggctccccag 3360

ggctccctta tgtccaccac ttcaggggat gggtgtggat gtaattagct ctggggggca 3420

gttgggtaga tgggtggggg ctcctggtgg ccttctgctg cccaggccac agccgccttt 3480

gggttccatc ttgctaataa acactggctc tgggactaga aaaaaaaaaa aaaaaaaaaa 3540

<210>15

<211>231

<212>PRT

<213>Homo sapiens

<400>15

Ser Cys Pro Ala Met Cys Thr Cys Ser Asn Gly Ile Val Asp Cys Arg

1 5 10 15

Gly Lys Gly Leu Thr Ala Ile Pro Ala Asn Leu Pro Glu Thr Met Thr

20 25 30

Glu Ile Arg Leu Glu Leu Asn Gly Ile Lys Ser Ile Pro Pro Gly Ala

35 40 45

Phe Ser Pro Tyr Arg Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln

50 55 60

Ile Ala Glu Ile Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn

65 70 75 80

Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr Asp Leu Pro Arg Gly Val

85 90 95

Phe Gly Gly Leu Tyr Thr Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys

100 105 110

Ile Asn Cys Ile Arg Pro Asp Ala Phe Gln Asp Leu Gln Asn Leu Ser

115 120 125

Leu Leu Ser Leu Tyr Asp Asn Lys Ile Gln Ser Leu Ala Lys Gly Thr

130 135 140

Phe Thr Ser Leu Arg Ala Ile Gln Thr Leu His Leu Ala Gln Asn Pro

145 150 155 160

Phe Ile Cys Asp Cys Asn Leu Lys Trp Leu Ala Asp Phe Leu Arg Thr

165 170 175

Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Ala Ser Pro Arg Arg Leu

180 185 190

Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser

195 200 205

Ala Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Gln Leu Asn

210 215 220

Ser Glu Cys Asn Ser Asp Val

225 230

<210>16

<211>234

<212>PRT

<213>Homo sapiens

<400>16

Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys

1 5 10 15

Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile

20 25 30

Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly

35 40 45

Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn

50 55 60

Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu

65 70 75 80

Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser

85 90 95

Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn

100 105 110

Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu

115 120 125

Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly

130 135 140

Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn

145 150 155 160

Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His

165 170 175

Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg

180 185 190

Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys

195 200 205

Ser Ala Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser

210 215 220

Lys Leu Ser Gly Asp Cys Phe Ala Asp Leu

225 230

<210>17

<211>604

<212>PRT

<213>Homo sapiens

<400>17

Met Met Arg Ala Val Trp Glu Ala Leu Ala Ala Leu Ala Ala Val Ala

1 5 10 15

Cys Leu Val Gly Ala Val Arg Gly Gly Pro Gly Leu Ser Met Phe Ala

20 25 30

Gly Gln Ala Ala Gln Pro Asp Pro Cys Ser Asp Glu Asn Gly His Pro

35 40 45

Arg Arg Cys Ile Pro Asp Phe Val Asn Ala Ala Phe Gly Lys Asp Val

50 55 60

Arg Val Ser Ser Thr Cys Gly Arg Pro Pro Ala Arg Tyr Cys Val Val

65 70 75 80

Ser Glu Arg Gly Glu Glu Arg Leu Arg Ser Cys His Leu Cys Asn Ala

85 90 95

Ser Asp Pro Lys Lys Ala His Pro Pro Ala Phe Leu Thr Asp Leu Asn

100 105 110

Asn Pro His Asn Leu Thr Cys Trp Gln Ser Glu Asn Tyr Leu Gln Phe

115 120 125

Pro His Asn Val Thr Leu Thr Leu Ser Leu Gly Lys Lys Phe Glu Val

130 135 140

Thr Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro Glu Ser Met Ala

145 150 155 160

Ile Tyr Lys Ser Met Asp Tyr Gly Arg Thr Trp Val Pro Phe Gln Phe

165 170 175

Tyr Ser Thr Gln Cys Arg Lys Met Tyr Asn Arg Pro His Arg Ala Pro

180 185 190

Ile Thr Lys Gln Asn Glu Gln Glu Ala Val Cys Thr Asp Ser His Thr

195 200 205

Asp Met Arg Pro Leu Ser Gly Gly Leu Ile Ala Phe Ser Thr Leu Asp

210 215 220

Gly Arg Pro Ser Ala His Asp Phe Asp Asn Ser Pro Val Leu Gln Asp

225 230 235 240

Trp Val Thr Ala Thr Asp Ile Arg Val Ala Phe Ser Arg Leu His Thr

245 250 255

Phe Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala Arg Asp Ser Tyr

260 265 270

Phe Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg Cys Lys Cys Asn

275 280 285

Gly His Ala Ala Arg Cys Val Arg Asp Arg Thr Asp Ser Leu Val Cys

290 295 300

Asp Cys Arg His Asn Thr Ala Gly Pro Glu Cys Asp Arg Cys Lys Pro

305 310 315 320

Phe His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg Glu Ala Asn

325 330 335

Glu Cys Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg Cys Arg Phe

340 345 350

Asn Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly Gly Val Cys

355 360 365

Leu Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys Lys

370 375 380

Glu Gly Tyr Tyr Arg Asp Met Gly Lys Pro Ile Thr His Arg Lys Ala

385 390 395 400

Cys Lys Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys Thr Cys

405 410 415

Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Ile

420 425 430

Thr Cys Asn Arg Cys Ala Lys Gly Tyr Gln Gln Ser Arg Ser Pro Ile

435 440 445

Ala Pro Cys Ile Lys Ile Pro Val Ala Pro Pro Thr Thr Ala Ala Ser

450 455 460

Ser Val Glu Glu Pro Glu Asp Cys Asp Ser Tyr Cys Lys Ala Ser Lys

465 470 475 480

Gly Lys Leu Lys Ile Asn Met Lys Lys Tyr Cys Lys Lys Asp Tyr Ala

485 490 495

Val Gln Ile His Ile Leu Lys Ala Asp Lys Ala Gly Asp Trp Trp Lys

500 505 510

Phe Thr Val Asn Ile Ile Ser Val Tyr Lys Gln Gly Thr Ser Arg Ile

515 520 525

Arg Arg Gly Asp Gln Ser Leu Trp Ile Arg Ser Arg Asp Ile Ala Cys

530 535 540

Lys Cys Pro Lys Ile Lys Pro Leu Lys Lys Tyr Leu Leu Leu Gly Asn

545 550 555 560

Ala Glu Asp Ser Pro Asp Gln Ser Gly Ile Val Ala Asp Lys Ser Ser

565 570 575

Leu Val Ile Gln Trp Arg Asp Thr Trp Ala Arg Arg Leu Arg Lys Phe

580 585 590

Gln Gln Arg Glu Lys Lys Gly Lys Cys Lys Lys Ala

595 600

<210>18

<211>1989

<212>DNA

<213>Homo sapiens

<400>18

agcttcgggg gcgagcgctc gtgtgtgtga gtgcgcgccg gccagcgcgc cttctgcggc 60

aggcggacag atcctcggcg cggcagggcc ggggcaagct ggacgcagca tgatgcgcgc 120

agtgtgggag gcgctggcgg cgctggcggc ggtggcgtgc ctggtgggcg cggtgcgcgg 180

cgggcccggg ctcagcatgt tcgcgggcca ggcggcgcag cccgatccct gctcggacga 240

gaacggccac ccgcgccgct gcatcccgga ctttgtcaat gcggccttcg gcaaggacgt 300

gcgcgtgtcc agcacctgcg gccggccccc ggcgcgctac tgcgtggtga gcgagcgcgg 360

cgaggagcgg ctgcgctcgt gccacctctg caacgcgtcc gaccccaaga aggcgcaccc 420

gcccgccttc ctcaccgacc tcaacaaccc gcacaacctg acgtgctggc agtccgagaa 480

ctacctgcag ttcccgcaca acgtcacgct cacactgtcc ctcggcaaga agttcgaagt 540

gacctacgtg agcctgcagt tctgctcgcc gcggcccgag tccatggcca tctacaagtc 600

catggactac gggcgcacgt gggtgccctt ccagttctac tccacgcagt gccgcaagat 660

gtacaaccgg ccgcaccgcg cgcccatcac caagcagaac gagcaggagg ccgtgtgcac 720

cgactcgcac accgacatgc gcccgctctc gggcggcctc atcgccttca gcacgctgga 780

cgggcggccc tcggcgcacg acttcgacaa ctcgcccgtg ctgcaggact gggtcacggc 840

cacagacatc cgcgtggcct tcagccgcct gcacacgttc ggcgaegaga acgaggacga 900

ctcggagctg gcgcgcgact cgtacttcta cgcggtgtcc gacctgcagg tgggcggccg 960

gtgcaagtgc aacggccacg cggcccgctg cgtgcgcgac cgcaccgaca gcctggtgtg 1020

cgactgcagg cacaacacgg ccggcccgga gtgcgaccgc tgcaagccct tccactacga 1080

ccggccctgg cagcgcgcca cagcccgcga agccaacgag tgcgtggcct gtaactgcaa 1140

cctgcatgcc cggcgctgcc gcttcaacat ggagctctac aagctttcgg ggcgcaagag 1200

cggaggtgtc tgcctcaact gtcgccacaa caccgccggc cgccactgcc attactgcaa 1260

ggagggctac taccgcgaca tgggcaagcc catcacccac cggaaggcct gcaaagcctg 1320

tgattgccac cctgtgggtg ctgctggcaa aacctgcaac caaaccaccg gccagtgtcc 1380

ctgcaaggac ggcgtgacgg gtatcacctg caaccgctgc gccaaaggct accagcagag 1440

ccgctctccc atcgccccct gcataaagat ccctgtagcg ccgccgacga ctgcagccag 1500

cagcgtggag gagcctgaag actgcgattc ctactgcaag gcctccaagg ggaagctgaa 1560

gattaacatg aaaaagtact gcaagaagga ctatgccgtc cagatccaca tcctgaaggc 1620

ggacaaggcg ggggactggt ggaagttcac ggtgaacatc atctccgtgt ataagcaggg 1680

cacgagccgc atccgccgcg gtgaccagag cctgtggatc cgctcgcggg acatcgcctg 1740

caagtgtccc aaaatcaagc ccctcaagaa gtacctgctg ctgggcaacg cggaggactc 1800

tccggaccag agcggcatcg tggccgataa aagcagcctg gtgatccagt ggcgggacac 1860

gtgggcgcgg cggctgcgca agttccagca gcgtgagaag aagggcaagt gcaagaaggc 1920

ctagcgccga ggcagcgggc gggcgggccg ggcgggcccg agggcggggc gagcgagacg 1980

gcgcttggc 1989

<210>19

<211>580

<212>PRT

<213>Homo sapiens

<400>19

Met Pro Gly Trp Pro Trp Gly Leu Leu Leu Thr Ala Gly Thr Leu Phe

1 5 10 15

Ala Ala Leu Ser Pro Gly Pro Pro Ala Pro Ala Asp Pro Cys His Asp

20 25 30

Glu Gly Gly Ala Pro Arg Gly Cys Val Pro Gly Leu Val Asn Ala Ala

35 40 45

Leu Gly Arg Glu Val Leu Ala Ser Ser Thr Cys Gly Arg Pro Ala Thr

50 55 60

Arg Ala Cys Asp Ala Ser Asp Pro Arg Arg Ala His Ser Pro Ala Leu

65 70 75 80

Leu Thr Ser Pro Gly Gly Thr Ala Ser Pro Leu Cys Trp Arg Ser Glu

85 90 95

Ser Leu Pro Arg Ala Pro Leu Asn Val Thr Leu Thr Val Pro Leu Gly

100 105 110

Lys Ala Phe Glu Leu Val Phe Val Ser Leu Arg Phe Cys Ser Ala Pro

115 120 125

Pro Ala Ser Val Ala Leu Leu Lys Ser Gln Asp His Gly Arg Ser Trp

130 135 140

Ala Pro Leu Gly Phe Phe Ser Ser His Cys Asp Leu Asp Tyr Gly Arg

145 150 155 160

Leu Pro Ala Pro Ala Asn Gly Pro Ala Gly Pro Gly Pro Glu Ala Leu

165 170 175

Cys Phe Pro Ala Pro Leu Ala Gln Pro Asp Gly Ser Gly Leu Leu Ala

180 185 190

Phe Ser Met Gln Asp Ser Ser Pro Pro Gly Leu Asp Leu Asp Ser Ser

195 200 205

Pro Val Leu Gln Asp Trp Val Thr Ala Thr Asp Val Arg Val Val Leu

210 215 220

Thr Arg Pro Ser Thr Ala Gly Asp Pro Arg Asp Met Glu Ala Val Val

225 230 235 240

Pro Tyr Ser Tyr Ala Ala Thr Asp Leu Gln Val Gly Gly Arg Cys Lys

245 250 255

Cys Asn Gly His Ala Ser Arg Cys Leu Leu Asp Thr Gln Gly His Leu

260 265 270

Ile Cys Asp Cys Arg His Gly Thr Glu Gly Pro Asp Cys Gly Arg Cys

275 280 285

Lys Pro Phe Tyr Cys Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg Glu

290 295 300

Ser His Ala Cys Leu Ala Cys Ser Cys Asn Gly His Ala Arg Arg Cys

305 310 315 320

Arg Phe Asn Met Glu Leu Tyr Arg Leu Ser Gly Arg Arg Ser Gly Gly

325 330 335

Val Cys Leu Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr

340 345 350

Cys Arg Glu Gly Phe Tyr Arg Asp Pro Gly Arg Ala Leu Ser Asp Arg

355 360 365

Arg Ala Cys Arg Ala Cys Asp Cys His Pro Val Gly Ala Ala Gly Lys

370 375 380

Thr Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr

385 390 395 400

Gly Leu Thr Cys Asn Arg Cys Ala Pro Gly Phe Gln Gln Ser Arg Ser

405 410 415

Pro Val Ala Pro Cys Val Lys Thr Pro Ile Pro Gly Pro Thr Glu Asp

420 425 430

Ser Ser Pro Val Gln Pro Gln Asp Cys Asp Ser His Cys Lys Pro Ala

435 440 445

Arg Gly Ser Tyr Arg Ile Ser Leu Lys Lys Phe Cys Lys Lys Asp Tyr

450 455 460

Ala Val Gln Val Ala Val Gly Ala Arg Gly Glu Ala Arg Gly Ala Trp

465 470 475 480

Thr Arg Phe Pro Val Ala Val Leu Ala Val Phe Arg Ser Gly Glu Glu

485 490 495

Arg Ala Arg Arg Gly Ser Ser Ala Leu Trp Val Pro Ala Gly Asp Ala

500 505 510

Ala Cys Gly Cys Pro Arg Leu Leu Pro Gly Arg Arg Tyr Leu Leu Leu

515 520 525

Gly Gly Gly Pro Gly Ala Ala Ala Gly Gly Ala Gly Gly Arg Gly Pro

530 535 540

Gly Leu Ile Ala Ala Arg Gly Ser Leu Val Leu Pro Trp Arg Asp Ala

545 550 555 560

Trp Thr Arg Arg Leu Arg Arg Leu Gln Arg Arg Glu Arg Arg Gly Arg

565 570 575

Cys Ser Ala Ala

580

<210>20

<211>1986

<212>DNA

<213>Homo sapiens

<400>20

gaggacgcgc caacatcccc gctgctgtgc tgggcccggg gcgtgcccgc cgctgctccc 60

acctctgggc cgggctgggg ccgcccgggg gccctgttcc tcggcattgc gggcctggtg 120

ggcagaaccg cggagagggc ttcttttccc caagggcagc gtcttggggc ccggccactg 180

gctgacccgc agcggctccg gccatgcctg gctggccctg ggggctgctg ctgacggcag 240

gcacgctctt cgccgccctg agtcctgggc cgccggcgcc cgccgacccc tgccacgatg 300

aggggggtgc gccccgcggc tgcgtgccag gactggtgaa cgccgccctg ggccgcgagg 360

tgctggcttc cagcacgtgc gggcggccgg ccactcgggc ctgcgacgcc tccgacccgc 420

gacgggcaca ctcccccgcc ctccttactt ccccaggggg cacggccagc cctctgtgct 480

ggcgctcgga gtccctgcct cgggcgcccc tcaacgtgac tctcacggtg cccctgggca 540

aggcttttga gctggtcttc gtgagcctgc gcttctgctc agctccccca gcctccgtgg 600

ccctgctcaa gtctcaggac catggccgca gctgggcccc gctgggcttc ttctcctccc 660

actgtgacct ggactatggc cgtctgcctg cccctgccaa tggcccagct ggcccagggc 720

ctgaggccct gtgcttcccc gcacccctgg cccagcctga tggcagcggc cttctggcct 780

tcagcatgca ggacagcagc cccccaggcc tggacctgga cagcagccca gtgctccaag 840

actgggtgac cgccaccgac gtccgtgtag tgctcacaag gcctagcacg gcaggtgacc 900

ccagggacat ggaggccgtc gtcccttact cctacgcagc caccgacctc caggtgggcg 960

ggcgctgcaa gtgcaatgga catgcctcac ggtgcctgct ggacacacag ggccacctga 1020

tctgcgactg tcggcatggc accgagggcc ctgactgcgg ccgctgcaag cccttctact 1080

gcgacaggcc atggcagcgg gccactgccc gggaatccca cgcctgcctc gcttgctcct 1140

gcaacggcca tgcccgccgc tgccgcttca acatggagct gtaccgactg tccggccgcc 1200

gcagcggggg tgtctgtctc aactgccggc acaacaccgc cggccgccac tgccactact 1260

gccgggaggg cttctatcga gaccctggcc gtgccctgag tgaccgtcgg gcttgcaggg 1320

cctgcgactg tcacccggtt ggtgctgctg gcaagacctg caaccagacc acaggccagt 1380

gtccctgcaa ggatggcgtc actggcctca cctgcaaccg ctgcgcgcct ggcttccagc 1440

aaagccgctc cccagtggcg ccctgtgtta agacccctat ccctggaccc actgaggaca 1500

gcagccctgt gcagccccag gactgtgact cgcactgcaa acctgcccgt ggcagctacc 1560

gcatcagcct aaagaagttc tgcaagaagg actatgcggt gcaggtggcg gtgggtgcgc 1620

gcggcgaggc gcgcggcgcg tggacacgct tcccggtggc ggtgctcgcc gtgttccgga 1680

gcggagagga gcgcgcgcgg cgcgggagta gcgcgctgtg ggtgcccgcc ggggatgcgg 1740

cctgcggctg cccgcgcctg ctccccggcc gccgctacct cctgctgggg ggcgggcctg 1800

gagccgcggc tgggggcgcg gggggccggg ggcccgggct catcgccgcc cgcggaagcc 1860

tcgtgctacc ctggagggac gcgtggacgc ggcgcctgcg gaggctgcag cgacgcgaac 1920

ggcgggggcg ctgcagcgcc gcctgagccc gccggctggg cagggcggcc gctgctccca 1980

catcta 1986

<210>21

<211>628

<212>PRT

<213>Homo sapiens

<400>21

Met Gly Ser Cys Ala Arg Leu Leu Leu Leu Trp Gly Cys Thr Val Val

1 5 10 15

Ala Ala Gly Leu Ser Gly Val Ala Gly Val Ser Ser Arg Cys Glu Lys

20 25 30

Ala Cys Asn Pro Arg Met Gly Asn Leu Ala Leu Gly Arg Lys Leu Trp

35 40 45

Ala Asp Thr Thr Cys Gly Gln Asn Ala Thr Glu Leu Tyr Cys Phe Tyr

50 55 60

Ser Glu Asn Thr Asp Leu Thr Cys Arg Gln Pro Lys Cys Asp Lys Cys

65 70 75 80

Asn Ala Ala Tyr Pro His Leu Ala His Leu Pro Ser Ala Met Ala Asp

85 90 95

Ser Ser Phe Arg Phe Pro Arg Thr Trp Trp Gln Ser Ala Glu Asp Val

100 105 110

His Arg Glu Lys Ile Gln Leu Asp Leu Glu Ala Glu Phe Tyr Phe Thr

115 120 125

His Leu Ile Val Met Phe Lys Ser Pro Arg Pro Ala Ala Met Val Leu

130 135 140

Asp Arg Ser Gln Asp Phe Gly Lys Thr Trp Lys Pro Tyr Lys Tyr Phe

145 150 155 160

Ala Thr Asn Cys Ser Ala Thr Phe Gly Leu Glu Asp Asp Val Val Lys

165 170 175

Lys Gly Ala Ile Cys Thr Ser Lys Tyr Ser Ser Pro Phe Pro Cys Thr

180 185 190

Gly Gly Glu Val Ile Phe Lys Ala Leu Ser Pro Pro Tyr Asp Thr Glu

195 200 205

Asn Pro Tyr Ser Ala Lys Val Gln Glu Gln Leu Lys Ile Thr Asn Leu

210 215 220

Arg Val Gln Leu Leu Lys Arg Gln Ser Cys Pro Cys Gln Arg Asn Asp

225 230 235 240

Leu Asn Glu Glu Pro Gln His Phe Thr His Tyr Ala Ile Tyr Asp Phe

245 250 255

Ile Val Lys Gly Ser Cys Phe Cys Asn Gly His Ala Asp Gln Cys Ile

260 265 270

Pro Val His Gly Phe Arg Pro Val Lys Ala Pro Gly Thr Phe His Met

275 280 285

Val His Gly Lys Cys Met Cys Lys His Asn Thr Ala Gly Ser His Cys

290 295 300

Gln His Cys Ala Pro Leu Tyr Asn Asp Arg Pro Trp Glu Ala Ala Asp

305 310 315 320

Gly Lys Thr Gly Ala Pro Asn Glu Cys Arg Thr Cys Lys Cys Asn Gly

325 330 335

His Ala Asp Thr Cys His Phe Asp Val Asn Val Trp Glu Ala Ser Gly

340 345 350

Asn Arg Ser Gly Gly Val Cys Asp Asp Cys Gln His Asn Thr Glu Gly

355 360 365

Gln Tyr Cys Gln Arg Cys Lys Pro Gly Phe Tyr Arg Asp Leu Arg Arg

370 375 380

Pro Phe Ser Ala Pro Asp Ala Cys Lys Pro Cys Ser Cys His Pro Val

385 390 395 400

Gly Ser Ala Val Leu Pro Ala Asn Ser Val Thr Phe Cys Asp Pro Ser

405 410 415

Asn Gly Asp Cys Pro Cys Lys Pro Gly Val Ala Gly Arg Arg Cys Asp

420 425 430

Arg Cys Met Val Gly Tyr Trp Gly Phe Gly Asp Tyr Gly Cys Arg Pro

435 440 445

Cys Asp Cys Ala Gly Ser Cys Asp Pro Ile Thr Gly Asp Cys Ile Ser

450 455 460

Ser His Thr Asp Ile Asp Trp Tyr His Glu Val Pro Asp Phe Arg Pro

465 470 475 480

Val His Asn Lys Ser Glu Pro Ala Trp Glu Trp Glu Asp Ala Gln Gly

485 490 495

Phe Ser Ala Leu Leu His Ser Gly Lys Cys Glu Cys Lys Glu Gln Thr

500 505 510

Leu Gly Asn Ala Lys Ala Phe Cys Gly Met Lys Tyr Ser Tyr Val Leu

515 520 525

Lys Ile Lys Ile Leu Ser Ala His Asp Lys Gly Thr His Val Glu Val

530 535 540

Asn Val Lys Ile Lys Lys Val Leu Lys Ser Thr Lys Leu Lys Ile Phe

545 550 555 560

Arg Gly Lys Arg Thr Leu Tyr Pro Glu Ser Trp Thr Asp Arg Gly Cys

565 570 575

Thr Cys Pro Ile Leu Asn Pro Gly Leu Glu Tyr Leu Val Ala Gly His

580 585 590

Glu Asp Ile Arg Thr Gly Lys Leu Ile Val Asn Met Lys Ser Phe Val

595 600 605

Gln His Trp Lys Pro Ser Leu Gly Arg Lys Val Met Asp Ile Leu Lys

610 615 620

Arg Glu Cys Lys

625

<210>22

<211>3634

<212>DNA

<213>Homo sapiens

<400>22

ggacgggacg gagccggggc agccagaaga ggtgggaaaa gcggaggagg acgcccagga 60

ggaggcggcg gcggcggccg ggaagtgaaa ggtctcgcaa agttcagcgg cggctgcggg 120

cgccgagccc cgggctagcg gcagacgagc ccgcagggcc gctccgcggg gcagcgcagc 180

caggccggct atggtcccgg ggctcccgcc gccccccagg tgcccgggac ccgccaggcc 240

ggtgcgcgag ggtcacccca cctccccgcg cggtcccggc ccctggctcc cagctgccgg 300

cgaccgctga ccgagcccgg cgccccagga ggaggaagaa accagggccc cgttccctcc 360

cgaggacggc ggcgcttcat cccgcagccc agaggtctcg gctccctccg gcacccgccc 420

ggcccggctg ctcccggctc ctcccggcca tggggagctg cgcgcggctg ctgctgctct 480

ggggctgcac ggtggtggcc gcaggactga gtggagtagc tggagtgagt tcccgctgtg 540

aaaaagcctg caaccctcgg atgggaaatt tggctttggg gcgaaaactc tgggcagaca 600

ccacctgcgg tcagaatgct accgaactgt actgcttcta cagtgagaac acggatctga 660

cttgtcggca gcccaaatgt gacaagtgca atgctgccta tcctcacctg gctcacctgc 720

catctgccat ggcagactca tccttccggt ttcctcgcac atggtggcag tctgcggagg 780

atgtgcacag agaaaagatc cagttagacc tggaagctga attctacttc actcacctaa 840

ttgtgatgtt caagtccccc aggccggctg ccatggtgct ggaccgctcc caggactttg 900

ggaaaacatg gaagccttat aagtactttg cgactaactg ctccgctaca tttggcctgg 960

aagatgatgt tgtcaagaag ggcgctattt gtacttctaa atactccagt ccttttccat 1020

gcactggagg agaggttatt ttcaaagctt tgtcaccacc atacgataca gagaaccctt 1080

acagtgccaa agttcaggag cagctgaaga tcaccaacct tcgcgtgcag ctgctgaaac 1140

gacagtcttg tccctgtcag agaaatgacc tgaacgaaga gcctcaacat tttacacact 1200

atgcaatcta tgatttcatt gtcaagggca gctgcttctg caatggccac gctgatcaat 1260

gcatacctgt tcatggcttc agacctgtca aggccccagg aacattccac atggtccatg 1320

ggaagtgtat gtgtaagcac aacacagcag gcagccactg ccagcactgt gccccgttat 1380

acaatgaccg gccatgggag gcagctgatg gcaaaacggg ggctcccaac gagtgcagaa 1440

cctgcaagtg taatgggcat gctgatacct gtcacttcga cgttaatgtg tgggaggcat 1500

cagggaatcg tagtggtggt gtctgtgatg actgtcagca caacacagaa ggacagtatt 1560

gccagaggtg caagccaggc ttctatcgtg acctgcggag acccttctca gctccagatg 1620

cttgcaaacc gtgttcctgc catccagtag gatcagctgt ccttcctgcc aactcagtga 1680

ccttctgcga ccccagcaat ggtgactgcc cttgcaagcc tggggtggca gggcgacgtt 1740

gtgacaggtg catggtggga tactggggct tcggagacta tggctgtcga ccatgtgact 1800

gtgcggggag ctgtgaccct atcaccggag actgcatcag cagccacaca gacatagact 1860

ggtatcatga agttcctgac ttccgtcccg tgcacaataa gagcgaacca gcctgggagt 1920

gggaggatgc gcaggggttt tctgcacttc tacactcagg taaatgcgaa tgtaaggaac 1980

agacattagg aaatgccaag gcattctgtg gaatgaaata ttcatatgtg ctaaaaataa 2040

agattttatc agctcatgat aaaggtactc atgttgaggt caatgtgaag attaaaaagg 2100

tcttaaaatc taccaaactg aagattttcc gaggaaagcg aacattatat ccagaatcat 2160

ggacggacag aggatgcact tgtccaatcc tcaatcctgg tttggaatac cttgtagcag 2220

gacatgagga tataagaaca ggcaaactaa ttgtgaatat gaaaagcttt gtccagcact 2280

ggaaaccttc tcttggaaga aaagtcatgg atattttaaa aagagagtgc aagtagcatt 2340

aagatggata gcacataatg gcacttgtct atgtacaaaa cacaaacttt agagcaagaa 2400

gacctcagac aggaaactgg aattttttaa agtgccaaaa catatagaaa tgtttgaatg 2460

catgggtctt atctaactta tctcttctgg acccatgttt aaatacagtt ttatttcatg 2520

aagagaaatg aaaaccccta cactgatatc tgttttctat gggactgatt ctgaaattct 2580

taactattaa gaatatttta atagcagcat gacatttagc agtaatccat taagggcagt 2640

acctctaaca aggacgcctt ccagcttcag cgatgttact tacgtttgat gctacttaaa 2700

gtaatgaatg acgttttaag gaatccctaa ccctactatc agaaaaggtg tttgttaaag 2760

agccttctct tgtgtgttac gcatgaactt tggtctgtag gtgttaaatg gaacctctcc 2820

atgtgtatat agtatttcct tgtataaagc actttactac ctaccacttg tgttgtgaac 2880

gtttggtgac tgctgttgaa agaaggaaaa gggtgtgtga gaaagcctac tgaagcagca 2940

gcactgccac tacatgtgga caaaagtgac catataaaag aagttgtgct atttaactct 3000

gaatacttgg agaaactagg tgaagatgca accagaaagg agaatatgta tgcgtgaagt 3060

ctcagctttg agctggaggc tagattccaa gatgacagcc atgatgaaac tttttaaaaa 3120

actaaaccag aagagacttt aaaataagag aaagaaatca taaatgtaga catatgcttg 3180

gctaaagggg aaatggactt taaattttaa agagctcatt tgcaatgcac ttgtatacac 3240

ttcaaaaatt attgtagaca cagaatttgt tatatttttg tgcttagtat ttaaacctga 3300

acattgaaac agttttcctc cttgtctttc ttaacagtaa tagtcattat atttacctgt 3360

tttttaacac aatgtatgtg atagtcaaaa aatcacagtt tttcattatt attcatcttc 3420

tgtacccacg cataaccact atacatagtt tcttttgtac ttgaatatac aaaacatgaa 3480

cacagtgcca tatgaataat ttcacataca gaaccttttt ttctctgaag tcctgtggac 3540

ttgcaaatat atatatatat tgctttgtta atttgttttt atatttcata tatgtaataa 3600

aggaatatga tctgaaaaaa aaaaaaaaaa aaaa 3634

<210>23

<211>438

<212>PRT

<213>Homo sapiens

<400>23

Met Tyr Leu Ser Arg Phe Leu Ser Ile His Ala Leu Trp Val Thr Val

1 5 10 15

Ser Ser Val Met Gln Pro Tyr Pro Leu Val Trp Gly His Tyr Asp Leu

20 25 30

Cys Lys Thr Gln Ile Tyr Thr Glu Glu Gly Lys Val Trp Asp Tyr Met

35 40 45

Ala Cys Gln Pro Glu Ser Thr Asp Met Thr Lys Tyr Leu Lys Val Lys

50 55 60

Leu Asp Pro Pro Asp Ile Thr Cys Gly Asp Pro Pro Glu Thr Phe Cys

65 70 75 80

Ala Met Gly Asn Pro Tyr Met Cys Asn Asn Glu Cys Asp Ala Ser Thr

85 90 95

Pro Glu Leu Ala His Pro Pro Glu Leu Met Phe Asp Phe Glu Gly Arg

100 105 110

His Pro Ser Thr Phe Trp Gln Ser Ala Thr Trp Lys Glu Tyr Pro Lys

115 120 125

Pro Leu Gln Val Asn Ile Thr Leu Ser Trp Ser Lys Thr Ile Glu Leu

130 135 140

Thr Asp Asn Ile Val Ile Thr Phe Glu Ser Gly Arg Pro Asp Gln Met

145 150 155 160

Ile Leu Glu Lys Ser Leu Asp Tyr Gly Arg Thr Trp Gln Pro Tyr Gln

165 170 175

Tyr Tyr Ala Thr Asp Cys Leu Asp Ala Phe His Met Asp Pro Lys Ser

180 185 190

Val Lys Asp Leu Ser Gln His Thr Val Leu Glu Ile Ile Cys Thr Glu

195 200 205

Glu Tyr Ser Thr Gly Tyr Thr Thr Asn Ser Lys Ile Ile His Phe Glu

210 215 220

Ile Lys Asp Arg Phe Ala Phe Phe Ala Gly Pro Arg Leu Arg Asn Met

225 230 235 240

Ala Ser Leu Tyr Gly Gln Leu Asp Thr Thr Lys Lys Leu Arg Asp Phe

245 250 255

Phe Thr Val Thr Asp Leu Arg Ile Arg Leu Leu Arg Pro Ala Val Gly

260 265 270

Glu Ile Phe Val Asp Glu Leu His Leu Ala Arg Tyr Phe Tyr Ala Ile

275 280 285

Ser Asp Ile Lys Val Arg Gly Arg Cys Lys Cys Asn Leu His Ala Thr

290 295 300

Val Cys Val Tyr Asp Asn Ser Lys Leu Thr Cys Glu Cys Glu His Asn

305 310 315 320

Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys Lys Asn Tyr Gln Gly Arg

325 330 335

Pro Trp Ser Pro Gly Ser Tyr Leu Pro Ile Pro Lys Gly Thr Ala Asn

340 345 350

Thr Cys Ile Pro Ser Ile Ser Ser Ile Gly Thr Asn Val Cys Asp Asn

355 360 365

Glu Leu Leu His Cys Gln Asn Gly Gly Thr Cys His Asn Asn Val Arg

370 375 380

Cys Leu Cys Pro Ala Ala Tyr Thr Gly Ile Leu Cys Glu Lys Leu Arg

385 390 395 400

Cys Glu Glu Ala Gly Ser Cys Gly Ser Asp Ser Gly Gln Gly Ala Pro

405 410 415

Pro His Gly Ser Pro Ala Leu Leu Leu Leu Thr Thr Leu Leu Gly Thr

420 425 430

Ala Ser Pro Leu Val Phe

435

<210>24

<211>3015

<212>DNA

<213>Homo sapiens

<400>24

tgcagccgga gcagcaccag caacagcaac agcgagcggg acggagttag gaccgctcgg 60

agcgcacagg tctcgagggt gttggtgcca gaagaaaaga atgattgatg ggaaacagac 120

accgggctat agacactcat ccttttgctt cagatactga tatctcagcc tgcttgagca 180

tcccttgtga gctgtgaaca ttgaggatca ctcagggtta tcggatgtac aacgggagag 240

ccatcgcttt gctaaattat tatctgcaat tggacatctt ttacaaaaac caaactagac 300

ctgagtctaa tagatatgtt ctaagacaaa gaaaaagctg caagttgtta acgcctaaca 360

cacaagtatg ttaggcttcc accaaagtcc tcaatatacc tgaatacgca caatatctta 420

actcttcata tttggttttg ggatctgctt tgaggtccca tcttcattta aaaaaaaata 480

cagagaccta cctacccgta cgcatacata catatgtgta tatatatgta aactagacaa 540

agatcgcaga tcataaagca agctctgctt tagtttccaa gaagattaca aagaatttag 600

agatgtattt gtcaagattc ctgtcgattc atgccctttg ggttacggtg tcctcagtga 660

tgcagcccta ccctttggtt tggggacatt atgatttgtg taagactcag atttacacgg 720

aagaagggaa agtttgggat tacatggcct gccagccgga atccacggac atgacaaaat 780

atctgaaagt gaaactcgat cctccggata ttacctgtgg agaccctcct gagacgttct 840

gtgcaatggg caatccctac atgtgcaata atgagtgtga tgcgagtacc cctgagctgg 900

cacacccccc tgagctgatg tttgattttg aaggaagaca tccctccaca ttttggcagt 960

ctgccacttg gaaggagtat cccaagcctc tccaggttaa catcactctg tcttggagca 1020

aaaccattga gctaacagac aacatagtta ttacctttga atctgggcgt ccagaccaaa 1080

tgatcctgga gaagtctctc gattatggac gaacatggca gccctatcag tattatgcca 1140

cagactgctt agatgctttt cacatggatc ctaaatccgt gaaggattta tcacagcata 1200

cggtcttaga aatcatttgc acagaagagt actcaacagg gtatacaaca aatagcaaaa 1260

taatccactt tgaaatcaaa gacaggttcg cgttttttgc tggacctcgc ctacgcaata 1320

tggcttccct ctacggacag ctggatacaa ccaagaaact cagagatttc tttacagtca 1380

cagacctgag gataaggctg ttaagaccag ccgttgggga aatatttgta gatgagctac 1440

acttggcacg ctacttttac gcgatctcag acataaaggt gcgaggaagg tgcaagtgta 1500

atctccatgc cactgtatgt gtgtatgaca acagcaaatt gacatgcgaa tgtgagcaca 1560

acactacagg tccagactgt gggaaatgca agaagaatta tcagggccga ccttggagtc 1620

caggctccta tctccccatc cccaaaggca ctgcaaatac ctgtatcccc agtatttcca 1680

gtattggtac gaatgtctgc gacaacgagc tcctgcactg ccagaacgga gggacgtgcc 1740

acaacaacgt gcgctgcctg tgcccggccg catacacggg catcctctgc gagaagctgc 1800

ggtgcgagga ggctggcagc tgcggctccg actctggcca gggcgcgccc ccgcacggct 1860

ccccagcgct gctgctgctg accacgctgc tgggaaccgc cagccccctg gtgttctagg 1920

tgtcacctcc agccacaccg gacgggcctg tgccgtgggg aagcagacac aacccaaaca 1980

tttgctacta acataggaaa cacacacata cagacacccc cactcagaca gtgtacaaac 2040

taagaaggcc taactgaact aagccatatt tatcacccgt ggacagcaca tccgagtcaa 2100

gactgttaat ttctgactcc agaggagttg gcagctgttg atattatcac tgcaaatcac 2160

attgccagct gcagagcata ttgtggattg gaaaggctgc gacagccccc caaacaggaa 2220

agacaaaaaa caaacaaatc aaccgaccta aaaacattgg ctactctagc gtggtgcgcc 2280

ctagtacgac tccgcccagt gtgtggacca accaaatagc attctttgct gtcaggtgca 2340

ttgtgggcat aaggaaatct gttacaagct gccatattgg cctgcttccg tccctgaatc 2400

ccttccaacc tgtgctttag tgaacgttgc tctgtaaccc ttgttggttg aaagatttct 2460

ttgtctgatg ttagtgatgc acatgtgtaa cagccccctc taaaagcgca agccagtcat 2520

acccctgtat atcttagcag cactgagtcc agtgcgagca cacacccact atacaagagt 2580

ggctatagga aaaaagaaag tgtatctatc cttttgtatt caaatgaagt tatttttctt 2640

gaactactgt aatatgtaga ttttttgtat tattgccaat ttgtgttacc agacaatctg 2700

ttaatgtatc taattcgaat cagcaaagac tgacatttta ttttgtcctc tttcgttctg 2760

ttttgtttca ctgtgcagag atttctctgt aagggcaacg aacgtgctgg catcaaagaa 2820

tatcagttta catatataac aagtgtaata agattccacc aaaggacatt ctaaatgttt 2880

tcttgttgct ttaacactgg aagatttaaa gaataaaaac tcctgcataa acaaaaaaaa 2940

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3000

aaaaaaaaaa aaaaa 3015

<210>25

<211>530

<212>PRT

<213>Homo sapiens

<400>25

Met Leu His Leu Leu Ala Leu Phe Leu His Cys Leu Pro Leu Ala Ser

1 5 10 15

Gly Asp Tyr Asp Ile Cys Lys Ser Trp Val Thr Thr Asp Glu Gly Pro

20 25 30

Thr Trp Glu Phe Tyr Ala Cys Gln Pro Lys Val Met Arg Leu Lys Asp

35 40 45

Tyr Val Lys Val Lys Val Glu Pro Ser Gly Ile Thr Cys Gly Asp Pro

50 55 60

Pro Glu Arg Phe Cys Ser His Glu Asn Pro Tyr Leu Cys Ser Asn Glu

65 70 75 80

Cys Asp Ala Ser Asn Pro Asp Leu Ala His Pro Pro Arg Leu Met Phe

85 90 95

Asp Lys Glu Glu Glu Gly Leu Ala Thr Tyr Trp Gln Ser Ile Thr Trp

100 105 110

Ser Arg Tyr Pro Ser Pro Leu Glu Ala Asn Ile Thr Leu Ser Trp Asn

115 120 125

Lys Thr Val Glu Leu Thr Asp Asp Val Val Met Thr Phe Glu Tyr Gly

130 135 140

Arg Pro Thr Val Met Val Leu Glu Lys Ser Leu Asp Asn Gly Arg Thr

145 150 155 160

Trp Gln Pro Tyr Gln Phe Tyr Ala Glu Asp Cys Met Glu Ala Phe Gly

165 170 175

Met Ser Ala Arg Arg Ala Arg Asp Met Ser Ser Ser Ser Ala His Arg

180 185 190

Val Leu Cys Thr Glu Glu Tyr Ser Arg Trp Ala Gly Ser Lys Lys Glu

195 200 205

Lys His Val Arg Phe Glu Val Arg Asp Arg Phe Ala Ile Phe Ala Gly

210 215 220

Pro Asp Leu Arg Asn Met Asp Asn Leu Tyr Thr Arg Leu Glu Ser Ala

225 230 235 240

Lys Gly Leu Lys Glu Phe Phe Thr Leu Thr Asp Leu Arg Met Arg Leu

245 250 255

Leu Arg Pro Ala Leu Gly Gly Thr Tyr Val Gln Arg Glu Asn Leu Tyr

260 265 270

Lys Tyr Phe Tyr Ala Ile Ser Asn Ile Glu Val Ile Gly Arg Cys Lys

275 280 285

Cys Asn Leu His Ala Asn Leu Cys Ser Met Arg Glu Gly Ser Leu Gln

290 295 300

Cys Glu Cys Glu His Asn Thr Thr Gly Pro Asp Cys Gly Lys Cys Lys

305 310 315 320

Lys Asn Phe Arg Thr Arg Ser Trp Arg Ala Gly Ser Tyr Leu Pro Leu

325 330 335

Pro His Gly Ser Pro Asn Ala Cys Ala Ala Ala Gly Ser Phe Gly Asn

340 345 350

Cys Glu Cys Tyr Gly His Ser Asn Arg Cys Ser Tyr Ile Asp Phe Leu

355 360 365

Asn Val Val Thr Cys Val Ser Cys Lys His Asn Thr Arg Gly Gln His

370 375 380

Cys Gln His Cys Arg Leu Gly Tyr Tyr Arg Asn Gly Ser Ala Glu Leu

385 390 395 400

Asp Asp Glu Asn Val Cys Ile Glu Cys Asn Cys Asn Gln Ile Gly Ser

405 410 415

Val His Asp Arg Cys Asn Glu Thr Gly Phe Cys Glu Cys Arg Glu Gly

420 425 430

Ala Ala Gly Pro Lys Cys Asp Asp Cys Leu Pro Thr His Tyr Trp Arg

435 440 445

Gln Gly Cys Tyr Pro Asn Val Cys Asp Asp Asp Gln Leu Leu Cys Gln

450 455 460

Asn Gly Gly Thr Cys Leu Gln Asn Gln Arg Cys Ala Cys Pro Arg Gly

465 470 475 480

Tyr Thr Gly Val Arg Cys Glu Gln Pro Arg Cys Asp Pro Ala Asp Asp

485 490 495

Asp Gly Gly Leu Asp Cys Asp Arg Ala Pro Gly Ala Ala Pro Arg Pro

500 505 510

Ala Thr Leu Leu Gly Cys Leu Leu Leu Leu Gly Leu Ala Ala Arg Leu

515 520 525

Gly Arg

530

<210>26

<211>2428

<212>DNA

<213>Homo sapiens

<400>26

ccatgctgag gccgcgagtc ccgcctgacc ccgtcgctgc ctctccaggg cttctctggg 60

ccgcgcctct gcagactgcg cagccatgct gcatctgctg gcgctcttcc tgcactgcct 120

ccctctggcc tctggggact atgacatctg caaatcctgg gtgaccacag atgagggccc 180

cacctgggag ttctacgcct gccagcccaa ggtgatgcgc ctgaaggact acgtcaaggt 240

gaaggtggag ccctcaggca tcacatgtgg agacccccct gagaggttct gctcccatga 300

gaatccctac ctatgcagca acgagtgtga cgcctccaac ccggacctgg cccacccgcc 360

caggctcatg ttcgacaagg aggaggaggg cctggccacc tactggcaga gcatcacctg 420

gagccgctac cccagcccgc tggaagccaa catcaccctt tcgtggaaca agaccgtgga 480

gctgaccgac gacgtggtga tgaccttcga gtacggccgg cccacggtca tggtcctgga 540

gaagtccctg gacaacgggc gcacctggca gccctaccag ttctacgccg aggactgcat 600

ggaggccttc ggtatgtccg cccgccgggc ccgcgacatg tcatcctcca gcgcgcaccg 660

cgtgctctgc accgaggagt actcgcgctg ggcaggctcc gagaaggaga agcacgtgcg 720

cttcgaggtg cgggaccgct tcgccatctt tgccggcccc gacctgcgca acatggacaa 780

cctctacacg cggctggaga gcgccaaggg cctcaaggag ttcttcaccc tcaccgacct 840

gcgcatgcgg ctgctgcgcc cggcgctggg cggcacctat gtgcagcggg agaacctcta 900

caagtacttc tacgccatct ccaacatcga ggtcatcggc aggtgcaagt gcaacctgca 960

tgccaacctg tgctccatgc gcgagggcag cctgcagtgc gagtgcgagc acaacaccac 1020

cggccccgac tgcggcaagt gcaagaagaa tttccgcacc cggtcctggc gggccggctc 1080

ctacctgccg ctgccccatg gctctcccaa cgcctgtgcc gctgcaggtt cctttggcaa 1140

ctgcgaatgc tacggtcact ccaaccgctg cagctacatt gacttcctga atgtggtgac 1200

ctgcgtcagc tgcaagcaca acacgcgagg tcagcactgc cagcactgcc ggctgggcta 1260

ctaccgcaac ggctcggcag agctggatga tgagaacgtc tgcattgagt gtaactgcaa 1320

ccagataggc tccgtgcacg accggtgcaa cgagaccggc ttctgcgagt gccgcgaggg 1380

cgcggcgggc cccaagtgcg acgactgcct ccccacgcac tactggcgcc agggctgcta 1440

ccccaacgtg tgcgacgacg accagctgct gtgccagaac ggaggcacct gcctgcagaa 1500

ccagcgctgc gcctgcccgc gcggctacac cggcgtgcgc tgcgagcagc cccgctgcga 1560

ccccgccgac gatgacggcg gtctggactg cgaccgcgcg cccggggccg ccccgcgccc 1620

cgccaccctg ctcggctgcc tgctgctgct ggggctggcc gcccgcctgg gccgctgagc 1680

cccgcccgga ggacgctccc cgcacccgga ggccgggggt cccggggtcc cggggcgggg 1740

ccggcgtccg aggccgggcg gtgagaaggg tgcggcccga ggtgctccca ggtgctactc 1800

agcagggccc cccgcccggc ccgcgctccc gcccgcactg ccctcccccc gcagcagggg 1860

cgccttggga ctccggtccc cgcgcctgcg atttggtttc gtttttcttt tgtattatcc 1920

gccgcccagt tccttttttg tctttctctc tctctctttt tttttttttt ttctggcggt 1980

gagccagagg gtcgggagaa acgctgctcg ccccacaccc cgtcctgcct cccaccacac 2040

ttacacacac gggactgtgg ccgacacccc ctggcctgtg ccaggctcac gggcggcggc 2100

ggaccccgac ctccagttgc ctacaattcc agtcgctgac ttggtcctgt tttctattct 2160

ttatttttcc tgcaacccac cagaccccag gcctcaccgg aggcccggtg accacggaac 2220

tcaccgtctg ggggaggagg agagaaggaa ggggtggggg gcctggaaac ttcgttctgt 2280

agagaactat ttttgtttgt attcactgtc ccctgcaagg gggacggggc gggagcactg 2340

gtcaccgcgg gggccgatgg tggagaatcc gaggagtaaa gagtttgctc actgctgcaa 2400

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 2428

<210>27

<211>36

<212>PRT

<213>Homo sapiens

<400>27

Val Arg Arg Leu Pro Pro Gln Leu Ala Gln Leu Ser Ser Pro Cys Ser

1 5 10 15

Ser Ser Asp Ser Leu Cys Ser Arg Arg Gly Leu Ser Ser Pro Arg Leu

20 25 30

Ser Leu Ala Pro

35

<210>28

<211>24

<212>DNA

<213>Homo sapiens

<400>28

tttttagcgt acctatgagc agtt 24

<210>29

<211>24

<212>DNA

<213>Homo sapiens

<400>29

caacaccaga cacttacgag tgcc 24

<210>30

<211>24

<212>DNA

<213>Homo sapiens

<400>30

ttcgaaggcc agaattctcc tggc 24

<210>31

<211>20

<212>DNA

<213>Homo sapiens

<400>31

cccaaggcca acagggaaaa 20

<210>32

<211>20

<212>DNA

<213>Homo sapiens

<400>32

ggtgcccatc tcctgctcaa 20

<210>33

<211>23

<212>DNA

<213>Homo sapiens

<400>33

cccugacgaa agagaagccu auu 23

<210>34

<211>23

<212>DNA

<213>Homo sapiens

<400>34

uaggcuucuc uuucgucagg guu 23

<210>35

<211>21

<212>DNA

<213>Homo sapiens

<400>35

cccctacaaa agaaaaacca a 21

<210>36

<211>1132

<212>PRT

<213>Homo sapiens

<400>36

Met Ala Leu Thr Pro Gly Trp Gly Ser Ser Ala Gly Pro Val Arg Pro

1 5 10 15

Glu Leu Trp Leu Leu Leu Trp Ala Ala Ala Trp Arg Leu Gly Ala Ser

20 25 30

Ala Cys Pro Ala Leu Cys Thr Cys Thr Gly Thr Thr Val Asp Cys His

35 40 45

Gly Thr Gly Leu Gln Ala Ile Pro Lys Asn Ile Pro Arg Asn Thr Glu

50 55 60

Arg Leu Glu Leu Asn Gly Asn Asn Ile Thr Arg Ile His Lys Asn Asp

65 70 75 80

Phe Ala Gly Leu Lys Gln Leu Arg Val Leu Gln Leu Met Glu Asn Gln

85 90 95

Ile Gly Ala Val Glu Arg Gly Ala Phe Asp Asp Met Lys Glu Leu Glu

100 105 110

Arg Leu Arg Leu Asn Arg Asn Gln Leu His Met Leu Pro Glu Leu Leu

115 120 125

Phe Gln Asn Asn Gln Ala Leu Ser Arg Leu Asp Leu Ser Glu Asn Ala

130 135 140

Ile Gln Ala Ile Pro Arg Lys Ala Phe Arg Gly Ala Thr Asp Leu Lys

145 150 155 160

Asn Leu Arg Leu Asp Lys Asn Gln Ile Ser Cys Ile Glu Glu Gly Ala

165 170 175

Phe Arg Ala Leu Arg Gly Leu Glu Val Leu Thr Leu Asn Asn Asn Asn

180 185 190

Ile Thr Thr Ile Pro Val Ser Ser Phe Asn His Met Pro Lys Leu Arg

195 200 205

Thr Phe Arg Leu His Ser Asn His Leu Phe Cys Asp Cys His Leu Ala

210 215 220

Trp Leu Ser Gln Trp Leu Arg Gln Arg Pro Thr Ile Gly Leu Phe Thr

225 230 235 240

Gln Cys Ser Gly Pro Ala Ser Leu Arg Gly Leu Asn Val Ala Glu Val

245 250 255

Gln Lys Ser Glu Phe Ser Cys Ser Gly Gln Gly Glu Ala Gly Arg Val

260 265 270

Pro Thr Cys Thr Leu Ser Ser Gly Ser Cys Pro Ala Met Cys Thr Cys

275 280 285

Ser Asn Gly Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Ala Ile Pro

290 295 300

Ala Asn Leu Pro Glu Thr Met Thr Glu Ile Arg Leu Glu Leu Asn Gly

305 310 315 320

Ile Lys Ser Ile Pro Pro Gly Ala Phe Ser Pro Tyr Arg Lys Leu Arg

325 330 335

Arg Ile Asp Leu Ser Asn Asn Gln Ile Ala Glu Ile Ala Pro Asp Ala

340 345 350

Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys

355 360 365

Ile Thr Asp Leu Pro Arg Gly Val Phe Gly Gly Leu Tyr Thr Leu Gln

370 375 380

Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys Ile Arg Pro Asp Ala

385 390 395 400

Phe Gln Asp Leu Gln Asn Leu Ser Leu Leu Ser Leu Tyr Asp Asn Lys

405 410 415

Ile Gln Ser Leu Ala Lys Gly Thr Phe Thr Ser Leu Arg Ala Ile Gln

420 425 430

Thr Leu His Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys Asn Leu Lys

435 440 445

Trp Leu Ala Asp Phe Leu Arg Thr Asn Pro Ile Glu Thr Ser Gly Ala

450 455 460

Arg Cys Ala Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile

465 470 475 480

Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu Gln Tyr Phe Ile Pro

485 490 495

Gly Thr Glu Asp Tyr Gln Leu Asn Ser Glu Cys Asn Ser Asp Val Val

500 505 510

Cys Pro His Lys Cys Arg Cys Glu Ala Asn Val Val Glu Cys Ser Ser

515 520 525

Leu Lys Leu Thr Lys Ile Pro Glu Arg Ile Pro Gln Ser Thr Ala Glu

530 535 540

Leu Arg Leu Asn Asn Asn Glu Ile Ser Ile Leu Glu Ala Thr Gly Met

545 550 555 560

Phe Lys Lys Leu Thr His Leu Lys Lys Ile Asn Leu Ser Asn Asn Lys

565 570 575

Val Ser Glu Ile Glu Asp Gly Ala Phe Glu Gly Ala Ala Ser Val Ser

580 585 590

Glu Leu His Leu Thr Ala Asn Gln Leu Glu Ser Ile Arg Ser Gly Met

595 600 605

Phe Arg Gly Leu Asp Gly Leu Arg Thr Leu Met Leu Arg Asn Asn Arg

610 615 620

Ile Ser Cys Ile His Asn Asp Ser Phe Thr Gly Leu Arg Asn Val Arg

625 630 635 640

Leu Leu Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ser Pro Gly Ala

645 650 655

Phe Asp Thr Leu Gln Ser Leu Ser Thr Leu Asn Leu Leu Ala Asn Pro

660 665 670

Phe Asn Cys Asn Cys Gln Leu Ala Trp Leu Gly Gly Trp Leu Arg Lys

675 680 685

Arg Lys Ile Val Thr Gly Asn Pro Arg Cys Gln Asn Pro Asp Phe Leu

690 695 700

Arg Gln Ile Pro Leu Gln Asp Val Ala Phe Pro Asp Phe Arg Cys Glu

705 710 715 720

Glu Gly Gln Glu Glu Gly Gly Cys Leu Pro Arg Pro Gln Cys Pro Gln

725 730 735

Glu Cys Ala Cys Leu Asp Thr Val Val Arg Cys Ser Asn Lys His Leu

740 745 750

Arg Ala Leu Pro Lys Gly Ile Pro Lys Asn Val Thr Glu Leu Tyr Leu

755 760 765

Asp Gly Asn Gln Phe Thr Leu Val Pro Gly Gln Leu Ser Thr Phe Lys

770 775 780

Tyr Leu Gln Leu Val Asp Leu Ser Asn Asn Lys Ile Ser Ser Leu Ser

785 790 795 800

Asn Ser Ser Phe Thr Asn Met Ser Gln Leu Thr Thr Leu Ile Leu Ser

805 810 815

Tyr Asn Ala Leu Gln Cys Ile Pro Pro Leu Ala Phe Gln Gly Leu Arg

820 825 830

Ser Leu Arg Leu Leu Ser Leu His Gly Asn Asp Ile Ser Thr Leu Gln

835 840 845

Glu Gly Ile Phe Ala Asp Val Thr Ser Leu Ser His Leu Ala Ile Gly

850 855 860

Ala Asn Pro Leu Tyr Cys Asp Cys His Leu Arg Trp Leu Ser Ser Trp

865 870 875 880

Val Lys Thr Gly Tyr Lys Glu Pro Gly Ile Ala Arg Cys Ala Gly Pro

885 890 895

Gln Asp Met Glu Gly Lys Leu Leu Leu Thr Thr Pro Ala Lys Lys Phe

900 905 910

Glu Cys Gln Gly Pro Pro Thr Leu Ala Val Gln Ala Lys Cys Asp Leu

915 920 925

Cys Leu Ser Ser Pro Cys Gln Asn Gln Gly Thr Cys His Asn Asp Pro

930 935 940

Leu Glu Val Tyr Arg Cys Ala Cys Pro Ser Gly Tyr Lys Gly Arg Asp

945 950 955 960

Cys Glu Val Ser Leu Asn Ser Cys Ser Ser Gly Pro Cys Glu Asn Gly

965 970 975

Gly Thr Cys His Ala Gln Glu Gly Glu Asp Ala Pro Phe Thr Cys Ser

980 985 990

Cys Pro Thr Gly Phe Glu Gly Pro Thr Cys Gly Val Asn Thr Asp Asp

995 1000 1005

Cys Val Asp His Ala Cys Ala Asn Gly Gly Val Cys Val Asp Gly

1010 1015 1020

Val Gly Asn Tyr Thr Cys Gln Cys Pro Leu Gln Tyr Glu Gly Lys

1025 1030 1035

Ala Cys Glu Gln Leu Val Asp Leu Cys Ser Pro Asp Leu Asn Pro

1040 1045 1050

Cys Gln His Glu Ala Gln Cys Val Gly Thr Pro Asp Gly Pro Arg

1055 1060 1065

Cys Glu Cys Met Pro Gly Tyr Ala Gly Asp Asn Cys Ser Glu Asn

1070 1075 1080

Gln Asp Asp Cys Arg Asp His Arg Cys Gln Asn Gly Ala Gln Cys

1085 1090 1095

Met Asp Glu Val Asn Ser Tyr Ser Cys Leu Cys Ala Glu Gly Tyr

1100 1105 1110

Ser Gly Gln Leu Cys Glu Ile Pro Pro His Leu Pro Ala Pro Lys

1115 1120 1125

Ser Pro Cys Glu

1130

<210>37

<211>1119

<212>PRT

<213>Homo sapiens

<400>37

Met Arg Gly Val Gly Trp Gln Met Leu Ser Leu Ser Leu Gly Leu Val

1 5 10 15

Leu Ala Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys

20 25 30

Ser Cys Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser

35 40 45

Val Pro Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly

50 55 60

Asn Asn Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His

65 70 75 80

Leu Arg Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg

85 90 95

Gly Ala Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg

100 105 110

Asn His Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys

115 120 125

Leu Tyr Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg

130 135 140

Lys Ala Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr

145 150 155 160

Asn Gln Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp

165 170 175

Leu Glu Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val

180 185 190

Ala Ser Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser

195 200 205

Asn Asn Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu

210 215 220

Arg Gln Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser

225 230 235 240

His Leu Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val

245 250 255

Cys Ser Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His

260 265 270

Cys Pro Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly

275 280 285

Lys Gly Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu

290 295 300

Ile Arg Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe

305 310 315 320

Ser Pro Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile

325 330 335

Ser Glu Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser

340 345 350

Leu Val Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe

355 360 365

Glu Gly Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile

370 375 380

Asn Cys Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu

385 390 395 400

Leu Ser Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe

405 410 415

Ser Pro Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe

420 425 430

Ile Cys Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn

435 440 445

Pro Ile Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala

450 455 460

Asn Lys Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala

465 470 475 480

Lys Glu Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu

485 490 495

Ser Gly Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys

500 505 510

Glu Gly Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro

515 520 525

Glu His Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu

530 535 540

Phe Thr Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu

545 550 555 560

Arg Lys Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly

565 570 575

Ala Phe Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn

580 585 590

Arg Leu Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu

595 600 605

Lys Thr Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp

610 615 620

Ser Phe Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn

625 630 635 640

Gln Ile Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu

645 650 655

Ser Thr Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu

660 665 670

Ala Trp Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn

675 680 685

Pro Arg Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp

690 695 700

Val Ala Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser

705 710 715 720

Cys Ser Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr

725 730 735

Val Val Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile

740 745 750

Pro Arg Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu

755 760 765

Val Pro Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu

770 775 780

Ser Asn Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Asn Met Thr

785 790 795 800

Gln Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro

805 810 815

Pro Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu His

820 825 830

Gly Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu Ser

835 840 845

Ala Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys

850 855 860

Asn Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro

865 870 875 880

Gly Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu

885 890 895

Leu Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val

900 905 910

Asn Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys Asn

915 920 925

Asp Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr Cys

930 935 940

Pro Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala Cys

945 950 955 960

Ile Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu Gly

965 970 975

Glu Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu

980 985 990

Asn Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn

995 1000 1005

Asn Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys

1010 1015 1020

Pro Pro Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp Phe

1025 1030 1035

Cys Ala Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys Ile

1040 1045 1050

Leu Thr Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr Val

1055 1060 1065

Gly Glu His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn Lys

1070 1075 1080

Cys Lys Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr Thr

1085 1090 1095

Cys Ile Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe Ser

1100 1105 1110

Pro Pro Met Val Leu Pro

1115

<210>38

<211>1118

<212>PRT

<213>Homo sapiens

<400>38

Met Ala Pro Gly Trp Ala Gly Val Gly Ala Ala Val Arg Ala Arg Leu

1 5 10 15

Ala Leu Ala Leu Ala Leu Ala Ser Val Leu Ser Gly Pro Pro Ala Val

20 25 30

Ala Cys Pro Thr Lys Cys Thr Cys Ser Ala Ala Ser Val Asp Cys His

35 40 45

Gly Leu Gly Leu Arg Ala Val Pro Arg Gly Ile Pro Arg Asn Ala Glu

50 55 60

Arg Leu Asp Leu Asp Arg Asn Asn Ile Thr Arg Ile Thr Lys Met Asp

65 70 75 80

Phe Ala Gly Leu Lys Asn Leu Arg Val Leu His Leu Glu Asp Asn Gln

85 90 95

Val Ser Val Ile Glu Arg Gly Ala Phe Gln Asp Leu Lys Gln Leu Glu

100 105 110

Arg Leu Arg Leu Asn Lys Asn Lys Leu Gln Val Leu Pro Glu Leu Leu

115 120 125

Phe Gln Ser Thr Pro Lys Leu Thr Arg Leu Asp Leu Ser Glu Asn Gln

130 135 140

Ile Gln Gly Ile Pro Arg Lys Ala Phe Arg Gly Ile Thr Asp Val Lys

145 150 155 160

Asn Leu Gln Leu Asp Asn Asn His Ile Ser Cys Ile Glu Asp Gly Ala

165 170 175

Phe Arg Ala Leu Arg Asp Leu Glu Ile Leu Thr Leu Asn Asn Asn Asn

180 185 190

Ile Ser Arg Ile Leu Val Thr Ser Phe Asn His Met Pro Lys Ile Arg

195 200 205

Thr Leu Arg Leu His Ser Asn His Leu Tyr Cys Asp Cys His Leu Ala

210 215 220

Trp Leu Ser Asp Trp Leu Arg Gln Arg Arg Thr Val Gly Gln Phe Thr

225 230 235 240

Leu Cys Met Ala Pro Val His Leu Arg Gly Phe Asn Val Ala Asp Val

245 250 255

Gln Lys Lys Glu Tyr Val Cys Pro Ala Pro His Ser Glu Pro Pro Ser

260 265 270

Cys Asn Ala Asn Ser Ile Ser Cys Pro Ser Pro Cys Thr Cys Ser Asn

275 280 285

Asn Ile Val Asp Cys Arg Gly Lys Gly Leu Met Glu Ile Pro Ala Asn

290 295 300

Leu Pro Glu Gly Ile Val Glu Ile Arg Leu Glu Gln Asn Ser Ile Lys

305 310 315 320

Ala Ile Pro Ala Gly Ala Phe Thr Gln Tyr Lys Lys Leu Lys Arg Ile

325 330 335

Asp Ile Ser Lys Asn Gln Ile Ser Asp Ile Ala Pro Asp Ala Phe Gln

340 345 350

Gly Leu Lys Ser Leu Thr Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr

355 360 365

Glu Ile Ala Lys Gly Leu Phe Asp Gly Leu Val Ser Leu Gln Leu Leu

370 375 380

Leu Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asn Thr Phe Gln

385 390 395 400

Asp Leu Gln Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln

405 410 415

Thr Ile Ser Lys Gly Leu Phe Ala Pro Leu Gln Ser Ile Gln Thr Leu

420 425 430

His Leu Ala Gln Asn Pro Phe Val Cys Asp Cys His Leu Lys Trp Leu

435 440 445

Ala Asp Tyr Leu Gln Asp Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys

450 455 460

Ser Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Ser Gln Ile Lys Ser

465 470 475 480

Lys Lys Phe Arg Cys Ser Gly Ser Glu Asp Tyr Arg Ser Arg Phe Ser

485 490 495

Ser Glu Cys Phe Met Asp Leu Val Cys Pro Glu Lys Cys Arg Cys Glu

500 505 510

Gly Thr Ile Val Asp Cys Ser Asn Gln Lys Leu Val Arg Ile Pro Ser

515 520 525

His Leu Pro Glu Tyr Val Thr Asp Leu Arg Leu Asn Asp Asn Glu Val

530 535 540

Ser Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Asn Leu Arg

545 550 555 560

Lys Ile Asn Leu Ser Asn Asn Lys Ile Lys Glu Val Arg Glu Gly Ala

565 570 575

Phe Asp Gly Ala Ala Ser Val Gln Glu Leu Met Leu Thr Gly Asn Gln

580 585 590

Leu Glu Thr Val His Gly Arg Val Phe Arg Gly Leu Ser Gly Leu Lys

595 600 605

Thr Leu Met Leu Arg Ser Asn Leu Ile Gly Cys Val Ser Asn Asp Thr

610 615 620

Phe Ala Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Arg

625 630 635 640

Ile Thr Thr Ile Thr Pro Gly Ala Phe Thr Thr Leu Val Ser Leu Ser

645 650 655

Thr Ile Asn Leu Leu Ser Asn Pro Phe Asn Cys Asn Cys His Leu Ala

660 665 670

Trp Leu Gly Lys Trp Leu Arg Lys Arg Arg Ile Val Ser Gly Asn Pro

675 680 685

Arg Cys Gln Lys Pro Phe Phe Leu Lys Glu Ile Pro Ile Gln Asp Val

690 695 700

Ala Ile Gln Asp Phe Thr Cys Asp Gly Asn Glu Glu Ser Ser Cys Gln

705 710 715 720

Leu Ser Pro Arg Cys Pro Glu Gln Cys Thr Cys Met Glu Thr Val Val

725 730 735

Arg Cys Ser Asn Lys Gly Leu Arg Ala Leu Pro Arg Gly Met Pro Lys

740 745 750

Asp Val Thr Glu Leu Tyr Leu Glu Gly Asn His Leu Thr Ala Val Pro

755 760 765

Arg Glu Leu Ser Ala Leu Arg His Leu Thr Leu Ile Asp Leu Ser Asn

770 775 780

Asn Ser Ile Ser Met Leu Thr Asn Tyr Thr Phe Ser Asn Met Ser His

785 790 795 800

Leu Ser Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Val

805 810 815

His Ala Phe Asn Gly Leu Arg Ser Leu Arg Val Leu Thr Leu His Gly

820 825 830

Asn Asp Ile Ser Ser Val Pro Glu Gly Ser Phe Asn Asp Leu Thr Ser

835 840 845

Leu Ser His Leu Ala Leu Gly Thr Asn Pro Leu His Cys Asp Cys Ser

850 855 860

Leu Arg Trp Leu Ser Glu Trp Val Lys Ala Gly Tyr Lys Glu Pro Gly

865 870 875 880

Ile Ala Arg Cys Ser Ser Pro Glu Pro Met Ala Asp Arg Leu Leu Leu

885 890 895

Thr Thr Pro Thr His Arg Phe Gln Cys Lys Gly Pro Val Asp Ile Asn

900 905 910

Ile Val Ala Lys Cys Asn Ala Cys Leu Ser Ser Pro Cys Lys Asn Asn

915 920 925

Gly Thr Cys Thr Gln Asp Pro Val Glu Leu Tyr Arg Cys Ala Cys Pro

930 935 940

Tyr Ser Tyr Lys Gly Lys Asp Cys Thr Val Pro Ile Asn Thr Cys Ile

945 950 955 960

Gln Asn Pro Cys Gln His Gly Gly Thr Cys His Leu Ser Asp Ser His

965 970 975

Lys Asp Gly Phe Ser Cys Ser Cys Pro Leu Gly Phe Glu Gly Gln Arg

980 985 990

Cys Glu Ile Asn Pro Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn

995 1000 1005

Ala Thr Cys Val Asp Gly Ile Asn Asn Tyr Val Cys Ile Cys Pro

1010 1015 1020

Pro Asn Tyr Thr Gly Glu Leu Cys Asp Glu Val Ile Asp His Cys

1025 1030 1035

Val Pro Glu Leu Asn Leu Cys Gln His Glu Ala Lys Cys Ile Pro

1040 1045 1050

Leu Asp Lys Gly Phe Ser Cys Glu Cys Val Pro Gly Tyr Ser Gly

1055 1060 1065

Lys Leu Cys Glu Thr Asp Asn Asp Asp Cys Val Ala His Lys Cys

1070 1075 1080

Arg His Gly Ala Gln Cys Val Asp Thr Ile Asn Gly Tyr Thr Cys

1085 1090 1095

Thr Cys Pro Gln Gly Phe Ser Gly Pro Phe Cys Glu His Pro Pro

1100 1105 1110

Pro Met Val Leu Leu

1115

<210>39

<211>1091

<212>PRT

<213>Homo sapiens

<400>39

Gln Ala Cys Pro Ala Gln Cys Ser Cys Ser Gly Ser Thr Val Asp Cys

1 5 10 15

His Gly Leu Ala Leu Arg Ser Val Pro Arg Asn Ile Pro Arg Asn Thr

20 25 30

Glu Arg Leu Asp Leu Asn Gly Asn Asn Ile Thr Arg Ile Thr Lys Thr

35 40 45

Asp Phe Ala Gly Leu Arg His Leu Arg Val Leu Gln Leu Met Glu Asn

50 55 60

Lys Ile Ser Thr Ile Glu Arg Gly Ala Phe Gln Asp Leu Lys Glu Leu

65 70 75 80

Glu Arg Leu Arg Leu Asn Arg Asn His Leu Gln Leu Phe Pro Glu Leu

85 90 95

Leu Phe Leu Gly Thr Ala Lys Leu Tyr Arg Leu Asp Leu Ser Glu Asn

100 105 110

Gln Ile Gln Ala Ile Pro Arg Lys Ala Phe Arg Gly Ala Val Asp Ile

115 120 125

Lys Asn Leu Gln Leu Asp Tyr Asn Gln Ile Ser Cys Ile Glu Asp Gly

130 135 140

Ala Phe Arg Ala Leu Arg Asp Leu Glu Val Leu Thr Leu Asn Asn Asn

145 150 155 160

Asn Ile Thr Arg Leu Ser Val Ala Ser Phe Asn His Met Pro Lys Leu

165 170 175

Arg Thr Phe Arg Leu His Ser Asn Asn Leu Tyr Cys Asp Cys His Leu

180 185 190

Ala Trp Leu Ser Asp Trp Leu Arg Gln Arg Pro Arg Val Gly Leu Tyr

195 200 205

Thr Gln Cys Met Gly Pro Ser His Leu Arg Gly His Asn Val Ala Glu

210 215 220

Val Gln Lys Arg Glu Phe Val Cys Ser Gly His Gln Ser Phe Met Ala

225 230 235 240

Pro Ser Cys Ser Val Leu His Cys Pro Ala Ala Cys Thr Cys Ser Asn

245 250 255

Asn Ile Val Asp Cys Arg Gly Lys Gly Leu Thr Glu Ile Pro Thr Asn

260 265 270

Leu Pro Glu Thr Ile Thr Glu Ile Arg Leu Glu Gln Asn Thr Ile Lys

275 280 285

Val Ile Pro Pro Gly Ala Phe Ser Pro Tyr Lys Lys Leu Arg Arg Ile

290 295 300

Asp Leu Ser Asn Asn Gln Ile Ser Glu Leu Ala Pro Asp Ala Phe Gln

305 310 315 320

Gly Leu Arg Ser Leu Asn Ser Leu Val Leu Tyr Gly Asn Lys Ile Thr

325 330 335

Glu Leu Pro Lys Ser Leu Phe Glu Gly Leu Phe Ser Leu Gln Leu Leu

340 345 350

Leu Leu Asn Ala Asn Lys Ile Asn Cys Leu Arg Val Asp Ala Phe Gln

355 360 365

Asp Leu His Asn Leu Asn Leu Leu Ser Leu Tyr Asp Asn Lys Leu Gln

370 375 380

Thr Ile Ala Lys Gly Thr Phe Ser Pro Leu Arg Ala Ile Gln Thr Met

385 390 395 400

His Leu Ala Gln Asn Pro Phe Ile Cys Asp Cys His Leu Lys Trp Leu

405 410 415

Ala Asp Tyr Leu His Thr Asn Pro Ile Glu Thr Ser Gly Ala Arg Cys

420 425 430

Thr Ser Pro Arg Arg Leu Ala Asn Lys Arg Ile Gly Gln Ile Lys Ser

435 440 445

Lys Lys Phe Arg Cys Ser Ala Lys Glu Gln Tyr Phe Ile Pro Gly Thr

450 455 460

Glu Asp Tyr Arg Ser Lys Leu Ser Gly Asp Cys Phe Ala Asp Leu Ala

465 470 475 480

Cys Pro Glu Lys Cys Arg Cys Glu Gly Thr Thr Val Asp Cys Ser Asn

485 490 495

Gln Lys Leu Asn Lys Ile Pro Glu His Ile Pro Gln Tyr Thr Ala Glu

500 505 510

Leu Arg Leu Asn Asn Asn Glu Phe Thr Val Leu Glu Ala Thr Gly Ile

515 520 525

Phe Lys Lys Leu Pro Gln Leu Arg Lys Ile Asn Phe Ser Asn Asn Lys

530 535 540

Ile Thr Asp Ile Glu Glu Gly Ala Phe Glu Gly Ala Ser Gly Val Asn

545 550 555 560

Glu Ile Leu Leu Thr Ser Asn Arg Leu Glu Asn Val Gln His Lys Met

565 570 575

Phe Lys Gly Leu Glu Ser Leu Lys Thr Leu Met Leu Arg Ser Asn Arg

580 585 590

Ile Thr Cys Val Gly Asn Asp Ser Phe Ile Gly Leu Ser Ser Val Arg

595 600 605

Leu Leu Ser Leu Tyr Asp Asn Gln Ile Thr Thr Val Ala Pro Gly Ala

610 615 620

Phe Asp Thr Leu His Ser Leu Ser Thr Leu Asn Leu Leu Ala Asn Pro

625 630 635 640

Phe Asn Cys Asn Cys Tyr Leu Ala Trp Leu Gly Glu Trp Leu Arg Lys

645 650 655

Lys Arg Ile Val Thr Gly Asn Pro Arg Cys Gln Lys Pro Tyr Phe Leu

660 665 670

Lys Glu Ile Pro Ile Gln Asp Val Ala Ile Gln Asp Phe Thr Cys Asp

675 680 685

Asp Gly Asn Asp Asp Asn Ser Cys Ser Pro Leu Ser Arg Cys Pro Thr

690 695 700

Glu Cys Thr Cys Leu Asp Thr Val Val Arg Cys Ser Asn Lys Gly Leu

705 710 715 720

Lys Val Leu Pro Lys Gly Ile Pro Arg Asp Val Thr Glu Leu Tyr Leu

725 730 735

Asp Gly Asn Gln Phe Thr Leu Val Pro Lys Glu Leu Ser Asn Tyr Lys

740 745 750

His Leu Thr Leu Ile Asp Leu Ser Asn Asn Arg Ile Ser Thr Leu Ser

755 760 765

Asn Gln Ser Phe Ser Asn Met Thr Gln Leu Leu Thr Leu Ile Leu Ser

770 775 780

Tyr Asn Arg Leu Arg Cys Ile Pro Pro Arg Thr Phe Asp Gly Leu Lys

785 790 795 800

Ser Leu Arg Leu Leu Ser Leu His Gly Asn Asp Ile Ser Val Val Pro

805 810 815

Glu Gly Ala Phe Asn Asp Leu Ser Ala Leu Ser His Leu Ala Ile Gly

820 825 830

Ala Asn Pro Leu Tyr Cys Asp Cys Asn Met Gln Trp Leu Ser Asp Trp

835 840 845

Val Lys Ser Glu Tyr Lys Glu Pro Gly Ile Ala Arg Cys Ala Gly Pro

850 855 860

Gly Glu Met Ala Asp Lys Leu Leu Leu Thr Thr Pro Ser Lys Lys Phe

865 870 875 880

Thr Cys Gln Gly Pro Val Asp Val Asn Ile Leu Ala Lys Cys Asn Pro

885 890 895

Cys Leu Ser Asn Pro Cys Lys Asn Asp Gly Thr Cys Asn Ser Asp Pro

900 905 910

Val Asp Phe Tyr Arg Cys Thr Cys Pro Tyr Gly Phe Lys Gly Gln Asp

915 920 925

Cys Asp Val Pro Ile His Ala Cys Ile Ser Asn Pro Cys Lys His Gly

930 935 940

Gly Thr Cys His Leu Lys Glu Gly Glu Glu Asp Gly Phe Trp Cys Ile

945 950 955 960

Cys Ala Asp Gly Phe Glu Gly Glu Asn Cys Glu Val Asn Val Asp Asp

965 970 975

Cys Glu Asp Asn Asp Cys Glu Asn Asn Ser Thr Cys Val Asp Gly Ile

980 985 990

Asn Asn Tyr Thr Cys Leu Cys Pro Pro Glu Tyr Thr Gly Glu Leu Cys

995 1000 1005

Glu Glu Lys Leu Asp Phe Cys Ala Gln Asp Leu Asn Pro Cys Gln

1010 1015 1020

His Asp Ser Lys Cys Ile Leu Thr Pro Lys Gly Phe Lys Cys Asp

1025 1030 1035

Cys Thr Pro Gly Tyr Val Gly Glu His Cys Asp Ile Asp Phe Asp

1040 1045 1050

Asp Cys Gln Asp Asn Lys Cys Lys Asn Gly Ala His Cys Thr Asp

1055 1060 1065

Ala Val Asn Gly Tyr Thr Cys Ile Cys Pro Glu Gly Tyr Ser Gly

1070 1075 1080

Leu Phe Cys Glu Phe Ser Pro Pro

1085 1090

<210>40

<211>1506

<212>PRT

<213>Homo sapiens

<400>40

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro

245 250 255

Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly

260 265 270

Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu Ile Arg

275 280 285

Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe Ser Pro

290 295 300

Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile Ser Glu

305 310 315 320

Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val

325 330 335

Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly

340 345 350

Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys

355 360 365

Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser

370 375 380

Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser Pro

385 390 395 400

Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe Ile Cys

405 410 415

Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn Pro Ile

420 425 430

Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala Asn Lys

435 440 445

Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu

450 455 460

Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu Ser Gly

465 470 475 480

Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly

485 490 495

Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His

500 505 510

Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr

515 520 525

Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys

530 535 540

Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe

545 550 555 560

Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg Leu

565 570 575

Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu Lys Thr

580 585 590

Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe

595 600 605

Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile

610 615 620

Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr

625 630 635 640

Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp

645 650 655

Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg

660 665 670

Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val Ala

675 680 685

Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser Cys Ser

690 695 700

Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr Val Val

705 710 715 720

Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg

725 730 735

Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro

740 745 750

Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn

755 760 765

Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met Thr Gln

770 775 780

Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Pro

785 790 795 800

Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu His Gly

805 810 815

Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu Ser Ala

820 825 830

Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn

835 840 845

Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly

850 855 860

Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu

865 870 875 880

Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val Asn

885 890 895

Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys Asn Asp

900 905 910

Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr Cys Pro

915 920 925

Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala Cys Ile

930 935 940

Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu Gly Glu

945 950 955 960

Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu Asn

965 970 975

Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn

980 985 990

Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys Pro Pro

995 1000 1005

Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp Phe Cys Ala

1010 1015 1020

Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys Ile Leu Thr

1025 1030 1035

Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr Val Gly Glu

1040 1045 1050

His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn Lys Cys Lys

1055 1060 1065

Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr Thr Cys Ile

1070 1075 1080

Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe Ser Pro Pro

1085 1090 1095

Met Val Leu Pro Arg Thr Ser Pro Cys Asp Asn Phe Asp Cys Gln

1100 1105 1110

Asn Gly Ala Gln Cys Ile Val Arg Ile Asn Glu Pro Ile Cys Gln

1115 1120 1125

Cys Leu Pro Gly Tyr Gln Gly Glu Lys Cys Glu Lys Leu Val Ser

1130 1135 1140

Val Asn Phe Ile Asn Lys Glu Ser Tyr Leu Gln Ile Pro Ser Ala

1145 1150 1155

Lys Val Arg Pro Gln Thr Asn Ile Thr Leu Gln Ile Ala Thr Asp

1160 1165 1170

Glu Asp Ser Gly Ile Leu Leu Tyr Lys Gly Asp Lys Asp His Ile

1175 1180 1185

Ala Val Glu Leu Tyr Arg Gly Arg Val Arg Ala Ser Tyr Asp Thr

1190 1195 1200

Gly Ser His Pro Ala Ser Ala Ile Tyr Ser Val Glu ThrIle Asn

1205 1210 1215

Asp Gly Asn Phe His Ile Val Glu Leu Leu Ala Leu Asp Gln Ser

1220 1225 1230

Leu Ser Leu Ser Val Asp Gly Gly Asn Pro Lys Ile Ile Thr Asn

1235 1240 1245

Leu Ser Lys Gln Ser Thr Leu Asn Phe Asp Ser Pro Leu Tyr Val

1250 1255 1260

Gly Gly Met Pro Gly Lys Ser Asn Val Ala Ser Leu Arg Gln Ala

1265 1270 1275

Pro Gly Gln Asn Gly Thr Ser Phe His Gly Cys Ile Arg Asn Leu

1280 1285 1290

Tyr Ile Asn Ser Glu Leu Gln Asp Phe Gln Lys Val Pro Met Gln

1295 1300 1305

Thr Gly Ile Leu Pro Gly Cys Glu Pro Cys His Lys Lys Val Cys

1310 1315 1320

Ala His Gly Thr Cys Gln Pro Ser Ser Gln Ala Gly Phe Thr Cys

1325 1330 1335

Glu Cys Gln Glu Gly Trp Met Gly Pro Leu Cys Asp Gln Arg Thr

1340 1345 1350

Asn Asp Pro Cys Leu Gly Asn Lys Cys Val His Gly Thr Cys Leu

1355 1360 1365

Pro Ile Asn Ala Phe Ser Tyr Ser Cys Lys Cys Leu Glu Gly His

1370 1375 1380

Gly Gly Val Leu Cys Asp Glu Glu Glu Asp Leu Phe Asn Pro Cys

1385 1390 1395

Gln Ala Ile Lys Cys Lys His Gly Lys Cys Arg Leu Ser Gly Leu

1400 1405 1410

Gly Gln Pro Tyr Cys Glu Cys Ser Ser Gly Tyr Thr Gly Asp Ser

1415 1420 1425

Cys Asp Arg Glu Ile Ser Cys Arg Gly Glu Arg Ile Arg Asp Tyr

1430 1435 1440

Tyr Gln Lys Gln Gln Gly Tyr Ala Ala Cys Gln Thr Thr Lys Lys

1445 1450 1455

Val Ser Arg Leu Glu Cys Arg Gly Gly Cys Ala Gly Gly Gln Cys

1460 1465 1470

Cys Gly Pro Leu Arg Ser Lys Arg Arg Lys Tyr Ser Phe Glu Cys

1475 1480 1485

Thr Asp Gly Ser Ser Phe Val Asp Glu Val Glu Lys Val Val Lys

1490 1495 1500

Cys Gly Cys

1505

<210>41

<211>1498

<212>PRT

<213>Homo sapiens

<400>41

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro

245 250 255

Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly

260 265 270

Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu Ile Arg

275 280 285

Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe Ser Pro

290 295 300

Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile Ser Glu

305 310 315 320

Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val

325 330 335

Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly

340 345 350

Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys

355 360 365

Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser

370 375 380

Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser Pro

385 390 395 400

Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe Ile Cys

405 410 415

Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn Pro Ile

420 425 430

Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala Asn Lys

435 440 445

Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu

450 455 460

Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu Ser Gly

465 470 475 480

Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly

485 490 495

Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His

500 505 510

Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr

515 520 525

Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys

530 535 540

Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe

545 550 555 560

Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg Leu

565 570 575

Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu Lys Thr

580 585 590

Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe

595 600 605

Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile

610 615 620

Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr

625 630 635 640

Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp

645 650 655

Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg

660 665 670

Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val Ala

675 680 685

Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser Cys Ser

690 695 700

Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr Val Val

705 710 715 720

Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg

725 730 735

Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro

740 745 750

Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn

755 760 765

Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met Thr Gln

770 775 780

Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Pro

785 790 795 800

Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu His Gly

805 810 815

Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu Ser Ala

820 825 830

Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn

835 840 845

Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly

850 855 860

Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu

865 870 875 880

Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val Asn

885 890 895

Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys Asn Asp

900 905 910

Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr Cys Pro

915 920 925

Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala Cys Ile

930 935 940

Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu Gly Glu

945 950 955 960

Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu Asn

965 970 975

Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn

980 985 990

Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys Pro Pro

995 1000 1005

Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp Phe Cys Ala

1010 1015 1020

Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys Ile Leu Thr

1025 1030 1035

Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr Val Gly Glu

1040 1045 1050

His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn Lys Cys Lys

1055 1060 1065

Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr Thr Cys Ile

1070 1075 1080

Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe Ser Pro Pro

1085 1090 1095

Met Val Leu Pro Arg Cys Gln Asn Gly Ala Gln Cys Ile Val Arg

1100 1105 1110

Ile Asn Glu Pro Ile Cys Gln Cys Leu Pro Gly Tyr Gln Gly Glu

1115 1120 1125

Lys Cys Glu Lys Leu Val Ser Val Asn Phe Ile Asn Lys Glu Ser

1130 1135 1140

Tyr Leu Gln Ile Pro Ser Ala Lys Val Arg Pro Gln Thr Asn Ile

1145 1150 1155

Thr Leu Gln Ile Ala Thr Asp Glu Asp Ser Gly Ile Leu Leu Tyr

1160 1165 1170

Lys Gly Asp Lys Asp His Ile Ala Val Glu Leu Tyr Arg Gly Arg

1175 1180 1185

Val Arg Ala Ser Tyr Asp Thr Gly Ser His Pro Ala Ser Ala Ile

1190 1195 1200

Tyr Ser Val Glu Thr Ile Asn Asp Gly Asn Phe His Ile Val Glu

1205 1210 1215

Leu Leu Ala Leu Asp Gln Ser Leu Ser Leu Ser Val Asp Gly Gly

1220 1225 1230

Asn Pro Lys Ile Ile Thr Asn Leu Ser Lys Gln Ser Thr Leu Asn

1235 1240 1245

Phe Asp Ser Pro Leu Tyr Val Gly Gly Met Pro Gly Lys Ser Asn

1250 1255 1260

Val Ala Ser Leu Arg Gln Ala Pro Gly Gln Asn Gly Thr Ser Phe

1265 1270 1275

His Gly Cys Ile Arg Asn Leu Tyr Ile Asn Ser Glu Leu Gln Asp

1280 1285 1290

Phe Gln Lys Val Pro Met Gln Thr Gly Ile Leu Pro Gly Cys Glu

1295 1300 1305

Pro Cys His Lys Lys Val Cys Ala His Gly Thr Cys Gln Pro Ser

1310 1315 1320

Ser Gln Ala Gly Phe Thr Cys Glu Cys Gln Glu Gly Trp Met Gly

1325 1330 1335

Pro Leu Cys Asp Gln Arg Thr Asn Asp Pro Cys Leu Gly Asn Lys

1340 1345 1350

Cys Val His Gly Thr Cys Leu Pro Ile Asn Ala Phe Ser Tyr Ser

1355 1360 1365

Cys Lys Cys Leu Glu Gly His Gly Gly Val Leu Cys Asp Glu Glu

1370 1375 1380

Glu Asp Leu Phe Asn Pro Cys Gln Ala Ile Lys Cys Lys His Gly

1385 1390 1395

Lys Cys Arg Leu Ser Gly Leu Gly Gln Pro Tyr Cys Glu Cys Ser

1400 1405 1410

Ser Gly Tyr Thr Gly Asp Ser Cys Asp Arg Glu Ile Ser Cys Arg

1415 1420 1425

Gly Glu Arg Ile Arg Asp Tyr Tyr Gln Lys Gln Gln Gly Tyr Ala

1430 1435 1440

Ala Cys Gln Thr Thr Lys Lys Val Ser Arg Leu Glu Cys Arg Gly

1445 1450 1455

Gly Cys Ala Gly Gly Gln Cys Cys Gly Pro Leu Arg Ser Lys Arg

1460 1465 1470

Arg Lys Tyr Ser Phe Glu Cys Thr Asp Gly Ser Ser Phe Val Asp

1475 1480 1485

Glu Val Glu Lys Val Val Lys Cys Gly Cys

1490 1495

<210>42

<211>244

<212>PRT

<213>Homo sapiens

<400>42

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser

<210>43

<211>464

<212>PRT

<213>Homo sapiens

<400>43

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro

245 250 255

Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly

260 265 270

Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu Ile Arg

275 280 285

Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe Ser Pro

290 295 300

Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile Ser Glu

305 310 315 320

Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val

325 330 335

Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly

340 345 350

Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys

355 360 365

Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser

370 375 380

Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser Pro

385 390 395 400

Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe Ile Cys

405 410 415

Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn Pro Ile

420 425 430

Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala Asn Lys

435 440 445

Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu

450 455 460

<210>44

<211>687

<212>PRT

<213>Homo sapiens

<400>44

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro

245 250 255

Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly

260 265 270

Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu Ile Arg

275 280 285

Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe Ser Pro

290 295 300

Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile Ser Glu

305 310 315 320

Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val

325 330 335

Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly

340 345 350

Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys

355 360 365

Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser

370 375 380

Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser Pro

385 390 395 400

Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe Ile Cys

405 410 415

Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn Pro Ile

420 425 430

Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala Asn Lys

435 440 445

Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu

450 455 460

Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu Ser Gly

465 470 475 480

Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly

485 490 495

Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His

500 505 510

Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr

515 520 525

Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys

530 535 540

Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe

545 550 555 560

Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg Leu

565 570 575

Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu Lys Thr

580 585 590

Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe

595 600 605

Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile

610 615 620

Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr

625 630 635 640

Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp

645 650 655

Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg

660 665 670

Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val

675 680 685

<210>45

<211>885

<212>PRT

<213>Homo sapiens

<400>45

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro

245 250 255

Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly

260 265 270

Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu Ile Arg

275 280 285

Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe Ser Pro

290 295 300

Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile Ser Glu

305 310 315 320

Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val

325 330 335

Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly

340 345 350

Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys

355 360 365

Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser

370 375 380

Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser Pro

385 390 395 400

Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe Ile Cys

405 410 415

Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn Pro Ile

420 425 430

Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala Asn Lys

435 440 445

Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu

450 455 460

Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu Ser Gly

465 470 475 480

Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly

485 490 495

Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His

500 505 510

Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr

515 520 525

Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys

530 535 540

Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe

545 550 555 560

Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg Leu

565 570 575

Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu Lys Thr

580 585 590

Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe

595 600 605

Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile

610 615 620

Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr

625 630 635 640

Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp

645 650 655

Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg

660 665 670

Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val Ala

675 680 685

Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser Cys Ser

690 695 700

Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr Val Val

705 710 715 720

Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg

725 730 735

Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro

740 745 750

Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn

755 760 765

Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met Thr Gln

770 775 780

Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Pro

785 790 795 800

Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu His Gly

805 810 815

Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu Ser Ala

820 825 830

Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn

835 840 845

Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly

850 855 860

Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu

865 870 875 880

Thr Thr Pro Ser Lys

885

<210>46

<211>1089

<212>PRT

<213>Homo sapiens

<400>46

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro

245 250 255

Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly

260 265 270

Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu Ile Arg

275 280 285

Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe Ser Pro

290 295 300

Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile Ser Glu

305 310 315 320

Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val

325 330 335

Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly

340 345 350

Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys

355 360 365

Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser

370 375 380

Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser Pro

385 390 395 400

Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe Ile Cys

405 410 415

Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn Pro Ile

420 425 430

Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala Asn Lys

435 440 445

Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu

450 455 460

Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu Ser Gly

465 470 475 480

Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly

485 490 495

Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His

500 505 510

Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr

515 520 525

Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys

530 535 540

Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe

545 550 555 560

Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg Leu

565 570 575

Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu Lys Thr

580 585 590

Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe

595 600 605

Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile

610 615 620

Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr

625 630 635 640

Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp

645 650 655

Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg

660 665 670

Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val Ala

675 680 685

Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser Cys Ser

690 695 700

Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr Val Val

705 710 715 720

Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg

725 730 735

Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro

740 745 750

Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn

755 760 765

Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met Thr Gln

770 775 780

Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Pro

785 790 795 800

Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu His Gly

805 810 815

Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu Ser Ala

820 825 830

Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn

835 840 845

Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly

850 855 860

Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu

865 870 875 880

Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val Asn

885 890 895

Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys Asn Asp

900 905 910

Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr Cys Pro

915 920 925

Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala Cys Ile

930 935 940

Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu Gly Glu

945 950 955 960

Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu Asn

965 970 975

Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn

980 985 990

Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys Pro Pro

995 1000 1005

Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp Phe Cys Ala

1010 1015 1020

Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys Ile Leu Thr

1025 1030 1035

Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr Val Gly Glu

1040 1045 1050

His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn Lys Cys Lys

1055 1060 1065

Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr Thr Cys Ile

1070 1075 1080

Cys Pro Glu Gly Tyr Ser

1085

<210>47

<211>1163

<212>PRT

<213>Homo sapiens

<400>47

Ile Leu Asn Lys Val Ala Pro Gln Ala Cys Pro Ala Gln Cys Ser Cys

1 5 10 15

Ser Gly Ser Thr Val Asp Cys His Gly Leu Ala Leu Arg Ser Val Pro

20 25 30

Arg Asn Ile Pro Arg Asn Thr Glu Arg Leu Asp Leu Asn Gly Asn Asn

35 40 45

Ile Thr Arg Ile Thr Lys Thr Asp Phe Ala Gly Leu Arg His Leu Arg

50 55 60

Val Leu Gln Leu Met Glu Asn Lys Ile Ser Thr Ile Glu Arg Gly Ala

65 70 75 80

Phe Gln Asp Leu Lys Glu Leu Glu Arg Leu Arg Leu Asn Arg Asn His

85 90 95

Leu Gln Leu Phe Pro Glu Leu Leu Phe Leu Gly Thr Ala Lys Leu Tyr

100 105 110

Arg Leu Asp Leu Ser Glu Asn Gln Ile Gln Ala Ile Pro Arg Lys Ala

115 120 125

Phe Arg Gly Ala Val Asp Ile Lys Asn Leu Gln Leu Asp Tyr Asn Gln

130 135 140

Ile Ser Cys Ile Glu Asp Gly Ala Phe Arg Ala Leu Arg Asp Leu Glu

145 150 155 160

Val Leu Thr Leu Asn Asn Asn Asn Ile Thr Arg Leu Ser Val Ala Ser

165 170 175

Phe Asn His Met Pro Lys Leu Arg Thr Phe Arg Leu His Ser Asn Asn

180 185 190

Leu Tyr Cys Asp Cys His Leu Ala Trp Leu Ser Asp Trp Leu Arg Gln

195 200 205

Arg Pro Arg Val Gly Leu Tyr Thr Gln Cys Met Gly Pro Ser His Leu

210 215 220

Arg Gly His Asn Val Ala Glu Val Gln Lys Arg Glu Phe Val Cys Ser

225 230 235 240

Gly His Gln Ser Phe Met Ala Pro Ser Cys Ser Val Leu His Cys Pro

245 250 255

Ala Ala Cys Thr Cys Ser Asn Asn Ile Val Asp Cys Arg Gly Lys Gly

260 265 270

Leu Thr Glu Ile Pro Thr Asn Leu Pro Glu Thr Ile Thr Glu Ile Arg

275 280 285

Leu Glu Gln Asn Thr Ile Lys Val Ile Pro Pro Gly Ala Phe Ser Pro

290 295 300

Tyr Lys Lys Leu Arg Arg Ile Asp Leu Ser Asn Asn Gln Ile Ser Glu

305 310 315 320

Leu Ala Pro Asp Ala Phe Gln Gly Leu Arg Ser Leu Asn Ser Leu Val

325 330 335

Leu Tyr Gly Asn Lys Ile Thr Glu Leu Pro Lys Ser Leu Phe Glu Gly

340 345 350

Leu Phe Ser Leu Gln Leu Leu Leu Leu Asn Ala Asn Lys Ile Asn Cys

355 360 365

Leu Arg Val Asp Ala Phe Gln Asp Leu His Asn Leu Asn Leu Leu Ser

370 375 380

Leu Tyr Asp Asn Lys Leu Gln Thr Ile Ala Lys Gly Thr Phe Ser Pro

385 390 395 400

Leu Arg Ala Ile Gln Thr Met His Leu Ala Gln Asn Pro Phe Ile Cys

405 410 415

Asp Cys His Leu Lys Trp Leu Ala Asp Tyr Leu His Thr Asn Pro Ile

420 425 430

Glu Thr Ser Gly Ala Arg Cys Thr Ser Pro Arg Arg Leu Ala Asn Lys

435 440 445

Arg Ile Gly Gln Ile Lys Ser Lys Lys Phe Arg Cys Ser Ala Lys Glu

450 455 460

Gln Tyr Phe Ile Pro Gly Thr Glu Asp Tyr Arg Ser Lys Leu Ser Gly

465 470 475 480

Asp Cys Phe Ala Asp Leu Ala Cys Pro Glu Lys Cys Arg Cys Glu Gly

485 490 495

Thr Thr Val Asp Cys Ser Asn Gln Lys Leu Asn Lys Ile Pro Glu His

500 505 510

Ile Pro Gln Tyr Thr Ala Glu Leu Arg Leu Asn Asn Asn Glu Phe Thr

515 520 525

Val Leu Glu Ala Thr Gly Ile Phe Lys Lys Leu Pro Gln Leu Arg Lys

530 535 540

Ile Asn Phe Ser Asn Asn Lys Ile Thr Asp Ile Glu Glu Gly Ala Phe

545 550 555 560

Glu Gly Ala Ser Gly Val Asn Glu Ile Leu Leu Thr Ser Asn Arg Leu

565 570 575

Glu Asn Val Gln His Lys Met Phe Lys Gly Leu Glu Ser Leu Lys Thr

580 585 590

Leu Met Leu Arg Ser Asn Arg Ile Thr Cys Val Gly Asn Asp Ser Phe

595 600 605

Ile Gly Leu Ser Ser Val Arg Leu Leu Ser Leu Tyr Asp Asn Gln Ile

610 615 620

Thr Thr Val Ala Pro Gly Ala Phe Asp Thr Leu His Ser Leu Ser Thr

625 630 635 640

Leu Asn Leu Leu Ala Asn Pro Phe Asn Cys Asn Cys Tyr Leu Ala Trp

645 650 655

Leu Gly Glu Trp Leu Arg Lys Lys Arg Ile Val Thr Gly Asn Pro Arg

660 665 670

Cys Gln Lys Pro Tyr Phe Leu Lys Glu Ile Pro Ile Gln Asp Val Ala

675 680 685

Ile Gln Asp Phe Thr Cys Asp Asp Gly Asn Asp Asp Asn Ser Cys Ser

690 695 700

Pro Leu Ser Arg Cys Pro Thr Glu Cys Thr Cys Leu Asp Thr Val Val

705 710 715 720

Arg Cys Ser Asn Lys Gly Leu Lys Val Leu Pro Lys Gly Ile Pro Arg

725 730 735

Asp Val Thr Glu Leu Tyr Leu Asp Gly Asn Gln Phe Thr Leu Val Pro

740 745 750

Lys Glu Leu Ser Asn Tyr Lys His Leu Thr Leu Ile Asp Leu Ser Asn

755 760 765

Asn Arg Ile Ser Thr Leu Ser Asn Gln Ser Phe Ser Asn Met Thr Gln

770 775 780

Leu Leu Thr Leu Ile Leu Ser Tyr Asn Arg Leu Arg Cys Ile Pro Pro

785 790 795 800

Arg Thr Phe Asp Gly Leu Lys Ser Leu Arg Leu Leu Ser Leu His Gly

805 810 815

Asn Asp Ile Ser Val Val Pro Glu Gly Ala Phe Asn Asp Leu Ser Ala

820 825 830

Leu Ser His Leu Ala Ile Gly Ala Asn Pro Leu Tyr Cys Asp Cys Asn

835 840 845

Met Gln Trp Leu Ser Asp Trp Val Lys Ser Glu Tyr Lys Glu Pro Gly

850 855 860

Ile Ala Arg Cys Ala Gly Pro Gly Glu Met Ala Asp Lys Leu Leu Leu

865 870 875 880

Thr Thr Pro Ser Lys Lys Phe Thr Cys Gln Gly Pro Val Asp Val Asn

885 890 895

Ile Leu Ala Lys Cys Asn Pro Cys Leu Ser Asn Pro Cys Lys Asn Asp

900 905 910

Gly Thr Cys Asn Ser Asp Pro Val Asp Phe Tyr Arg Cys Thr Cys Pro

915 920 925

Tyr Gly Phe Lys Gly Gln Asp Cys Asp Val Pro Ile His Ala Cys Ile

930 935 940

Ser Asn Pro Cys Lys His Gly Gly Thr Cys His Leu Lys Glu Gly Glu

945 950 955 960

Glu Asp Gly Phe Trp Cys Ile Cys Ala Asp Gly Phe Glu Gly Glu Asn

965 970 975

Cys Glu Val Asn Val Asp Asp Cys Glu Asp Asn Asp Cys Glu Asn Asn

980 985 990

Ser Thr Cys Val Asp Gly Ile Asn Asn Tyr Thr Cys Leu Cys Pro Pro

995 1000 1005

Glu Tyr Thr Gly Glu Leu Cys Glu Glu Lys Leu Asp Phe Cys Ala

1010 1015 1020

Gln Asp Leu Asn Pro Cys Gln His Asp Ser Lys Cys Ile Leu Thr

1025 1030 1035

Pro Lys Gly Phe Lys Cys Asp Cys Thr Pro Gly Tyr Val Gly Glu

1040 1045 1050

His Cys Asp Ile Asp Phe Asp Asp Cys Gln Asp Asn Lys Cys Lys

1055 1060 1065

Asn Gly Ala His Cys Thr Asp Ala Val Asn Gly Tyr Thr Cys Ile

1070 1075 1080

Cys Pro Glu Gly Tyr Ser Gly Leu Phe Cys Glu Phe Ser Pro Pro

1085 1090 1095

Met Val Leu Pro Arg Thr Ser Pro Cys Asp Asn Phe Asp Cys Gln

1100 1105 1110

Asn Gly Ala Gln Cys Ile Val Arg Ile Asn Glu Pro Ile Cys Gln

1115 1120 1125

Cys Leu Pro Gly Tyr Gln Gly Glu Lys Cys Glu Lys Leu Val Ser

1130 1135 1140

Val Asn Phe Ile Asn Lys Glu Ser Tyr Leu Gln Ile Pro Ser Ala

1145 1150 1155

Lys Val Arg Pro Gln

1160

Claims (35)

1. A method of inhibiting vascular permeability in a tissue comprising administering to the tissue a repulsive guidance cue of axons, blood vessels, or a combination thereof.

2. The method of claim 1, wherein the repulsive guidance cue is a ligand of roundabout receptor or Unc5 receptor.

3. The method of claim 2, wherein the repulsive guidance cue is a ligand of roundabout-4(Robo4) receptor.

4. The method of claim 3, wherein the repulsive guidance cue is slit2 or a fragment thereof that binds to Robo 4.

5. The method of claim 2, wherein the repulsive guidance cue is a ligand of the Unc5b receptor.

6. The method of claim 5, wherein the repulsive guidance cue is guidance factor-1 or a fragment thereof that binds to the Unc5b receptor.

7. A method of screening or evaluating an agent that inhibits vascular permeability comprising determining the ability of the agent to affect Robo-4 mediated activation of Git 1.

8. The method of claim 7, wherein Robo4 mediated activation of Git1 is determined by steps comprising:

(a) contacting a first cell expressing Robo4 with a candidate agent,

(b) contacting a second cell substantially identical to the first cell but substantially lacking Robo4 with a candidate agent

(c) Git1 activation was assayed in the first and second cells,

(d) Wherein a higher activation of Git1 is detectable in the first cell compared to the second cell, indicating that the agent causes Robo 4-mediated activation of Git 1.

9. The method of claim 8, wherein Git1 glossization is analyzed by detecting ARF6 inactivation.

10. The method of claim 9, wherein ARF6 inactivation is assayed by detecting Rac inactivation.

11. A method of screening for or evaluating an agent that inhibits vascular permeability comprising determining the ability of the agent to inhibit ARF6, Rac, Pak, Mek, or Erk.

12. The method of claim 11, wherein Robo 4-mediated inhibition of ARF6, Rac, Pak, Mek, or Erk is determined by the steps comprising:

(a) contacting a first cell expressing Robo4 with a candidate agent,

(b) contacting a second cell substantially identical to the first cell but substantially lacking Robo4 with a candidate agent

(c) Analyzing the inhibition of ARF6, Rac, Pak, Mek, Erk, or a combination thereof in the first and second cells,

(d) wherein a lower activation of ARF6, Rac, Pak, Mek, or Erk is detectable in the first cell compared to the second cell, indicating that the agent causes Robo 4-mediated inhibition of ARF6, Rac, Pak, Mek, or Erk.

13. The method of claim 7 or 11, wherein said method is performed in the substantial absence of VEGF, TNF, thrombin or histamine.

14. The method of claim 7 or 11, wherein said analysis is performed in the presence of a biologically active amount of VEGF, TNF, thrombin or histamine.

15. A method for treating or preventing Respiratory Distress Syndrome (RDS) in a subject comprising

(a) Identifying a subject having said RDS or at risk thereof, and

(b) administering to the lungs of the subject a repulsive guidance cue that binds to neuronal receptors and endothelial cells.

16. A method of treating or preventing retinopathy of prematurity (ROP) in a subject, comprising:

(a) identifying a subject having or at risk of having said ROP, and

(b) administering to the retina of the subject a repulsive guidance cue that binds to neuronal receptors and endothelial cells.

17. A method of treating or preventing diabetic retinopathy in a subject, comprising

(a) Identifying a subject suffering from or at risk of said diabetic retinopathy, and

(b) administering to the retina of the subject a repulsive guidance cue that binds to neuronal receptors and endothelial cells.

18. A method of treating or preventing wet macular degeneration in a subject, comprising

(a) Identifying a subject having or at risk of developing said wet macular degeneration, and

(b) administering to the retina of the subject a repulsive guidance cue that binds to neuronal receptors and endothelial cells.

19. A method of treating a subject with a repulsive signal or mimetic in a subject, comprising

(a) Identifying a subject having a indication of treatment with a VEGF blocker, TNF blocker, histamine blocker, or thrombin blocker, and

(b) administering to the subject a repulsive guidance cue that binds to neuronal receptors and endothelial cells.

20. The method of claim 15, 16, 17, 18 or 19, wherein the repulsive guidance cue is a ligand of roundabout receptor, Unc5 receptor, DCC receptor, neogenin receptor, DSCAM receptor or ICAM-2 receptor.

21. The method of claim 20, wherein the ligand is Slit 2.

22. An isolated polypeptide comprising the paxillin binding sequence of roundabout-4(Robo4), wherein the polypeptide does not comprise full-length Robo 4.

23. The isolated polypeptide of claim 22, wherein the paxillin binding sequence consists of SEQ id no: 27 or a fragment thereof of at least 10 residues in length.

An isolated polypeptide of from 24.10 to 400 amino acids comprising SEQ ID NO: 27 or a fragment thereof of at least 10 residues in length.

An isolated polypeptide of 25.10 to 400 amino acids comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ id no: 27 or a fragment thereof of at least 10 residues in length, has at least 80% sequence homology.

26. An isolated polypeptide comprising the Paxillin Binding Sequence (PBS) of roundabout-4(Robo4), wherein the polypeptide consists of the formula:

R¹-PBS-R²

wherein R is¹And R²Independently is H, acyl, NH₂Amino acid or peptide, wherein the polypeptide does not comprise full length Robo 4.

27. The isolated polypeptide of claim 26, wherein the PBS consists of a sequence identical to SEQ ID NO: 27 or a fragment thereof of at least 10 residues in length, having at least 80% sequence homology.

28. An isolated polypeptide consisting essentially of a polypeptide sequence identical to SEQ ID NO: 27 or a fragment thereof of at least 10 residues in length, having at least 80% sequence homology.

29. An isolated nucleic acid encoding the polypeptide of claim 22, 24, 26 or 28.

30. An isolated nucleic acid encoding a polypeptide comprising a paxillin binding sequence of roundabout-4(Robo4), wherein the polypeptide does not comprise full-length Robo 4.

31. An isolated nucleic acid comprising SEQ ID NO: 2 or a fragment thereof of at least 30 residues in length, wherein the nucleic acid does not encode full-length roundabout-4(Robo 4).

32. A vector comprising the isolated nucleic acid of claim 29, 30, or 31.

33. A method of promoting angiogenesis in a tissue, comprising administering a composition comprising the polypeptide of claim 22, 24, 26, or 28 to endothelial cells of the tissue.

34. A method of promoting angiogenesis in a tissue, comprising administering a composition comprising the nucleic acid of claim 29, 30, or 31 to endothelial cells of the tissue.

35. A method of promoting angiogenesis in a tissue, comprising administering to the tissue a composition comprising the vector of claim 32, wherein the vector transduces endothelial cells.