CN1993048A - Follistatin domain containing proteins - Google Patents

Abstract

The present invention relates to the use of proteins comprising at least one follistatin domain to modulate the level or activity of growth and differentiation factor-8 (GDF-8). More particularly, the invention relates to the use of proteins comprising at least one follistatin domain, excluding follistatin itself, for treating disorders that are related to modulation of the level or activity of GDF-8. The invention is useful for treating muscular diseases and disorders, particularly those in which an increase in muscle tissue would be therapeutically beneficial. The invention is also useful for treating diseases and disorders related to metabolism, adipose tissue, and bone degeneration.

Description

Follistatin Domain Containing Proteins

This application claims the benefit of US Provisional Application No. 60/357,846, filed February 21, 2002, and US Provisional Application No. 60/434,645, filed December 20, 2002.

field of invention

The present invention relates to the use of proteins comprising at least one follistatin domain to modulate the level or activity of growth and differentiation factor-8 (GDF-8). More particularly, the present invention relates to the use of proteins comprising at least one follistatin domain, excluding follistatin itself, for the treatment of disorders associated with modulation of GDF-8 levels or activity. The present invention is useful in the treatment of muscle diseases and conditions, particularly those in which muscle tissue increases. The invention is also useful in the treatment of diseases and conditions associated with metabolism, adipose tissue and bone degeneration.

Background of the invention

Growth and differentiation factor-8 (GDF-8), also known as myostatin, is a member of the transforming growth factor-β (TGF-β) superfamily of structurally related growth factors, all growth factors Has important physiological growth regulation and morphogenetic properties (Kingsley et al., (1994) Genes Dev., 8: 133-46; Hoodless et al. (1998) Curr. Topics Microbiol. Immunol., 228: 235-72). GDF-8 is a negative regulator of skeletal muscle mass and there is considerable interest in identifying factors that regulate its biological activity. For example GDF-8 is highly expressed in developing and adult skeletal muscle. Null mutations of GDF-8 in transgenic mice are characterized by marked hypertrophy and hyperplasia of skeletal muscle (McPherron et al., (1997) Nature, 387:83-90). Similar increases in skeletal muscle mass are evident in naturally occurring mutations of bovine GDF-8 (Ashmore et al. (1974) Growth, 38:501-507; Swatland and Kieffer (1994) J. Anim. Sci., 38: 752-757; McPherron and Lee (1997) Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461; and Kambadur et al. (1997) Genome Res., 7:910-915). Recent studies have shown that muscle wasting associated with HIV infection in humans is accompanied by increased GDF-8 protein expression (Gonzalez-Cadavid et al. (1998) Proc. Natl. Acad. Sci. U.S.A., 95:14938-43). In addition, GDF-8 can regulate the production of muscle-specific enzymes (eg, creatine kinase) as well as regulate the proliferation of myoblasts (WO 00/43781).

Many human and animal conditions are associated with loss or functional impairment of muscle tissue. To date, few reliable or effective treatments for these conditions exist. However, the dreadful symptoms associated with these conditions can be substantially alleviated in patients suffering from these conditions by treatments that increase muscle tissue mass. Although not a cure for the condition, this treatment can significantly improve the quality of life of these patients and can improve some of the effects of these diseases. Therefore, there is a need in the art to identify new therapies that can promote an overall increase in muscle tissue in patients suffering from these conditions.

In addition to the growth-regulatory and morphogenetic properties of GDF-8 in skeletal muscle, GDF-8 may also be involved in many other physiological processes (e.g. glucose homeostasis) and in adipose tissue disorders such as type 2 diabetes and obesity Anomalies in development. For example, GDF-8 regulates the differentiation of preadipocytes into adipocytes (Kim et al. (2001) B.B.R.C. 281:902-906). Therefore, modulation of GDF-8 may also be useful in the treatment of these diseases.

The GDF-8 protein is synthesized as a precursor protein consisting of an amino-terminal propeptide and a carboxy-terminal mature domain (McPherron and Lee, (1997) Proc. Nat. Acad. Sci. U.S.A., 94:12457-12461 ). Prior to cleavage, the precursor GDF-8 protein forms homodimers. The amino-terminal propeptide is then cleaved from the mature domain. The cleaved propeptide remains non-covalently associated with the mature domain dimer, inactivating its biological activity (Miyazono et al., (1988) J. Biol. Chem., 263:6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263:7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Two GDF-8 propeptides are believed to bind to the mature GDF-8 dimer (Thies et al. (2001) Growth Factors, 18:251-259). Because of this inactive property, the propeptide is called a "latently associated peptide" (LAP), and the complex of the mature domain and propeptide is often called a "small latent complex" (Gentry and Nash (1990 ) Biochemistry, 29:6851-6857; Derynck et al. (1995) Nature, 316:701-705; and Massague (1990) Ann. Rev. Cell Biol., 12:597-641). Other proteins are known to bind GDF-8 or structurally related proteins and inhibit its biological activity. Proteins with this inhibitory effect include follistatin (Gamer et al. (1999) Dev. Biol., 208:222-232). It is believed that the mature domain of GDF-8 is active as a homodimer when the propeptide is removed.

It is evident that GDF-8 is involved in the regulation of many crucial biological processes. Due to its critical function in these processes, GDF-8 may be a desirable target for therapeutic intervention. In particular, therapeutic agents that inhibit GDF-8 activity may be used to treat human or animal conditions in which an increase in muscle tissue is therapeutically effective.

Known proteins comprising at least one follistatin domain play a role in many biological processes, particularly in the regulation of TGF-β superfamily signaling and regulation of extracellular matrix-mediated processes such as cell adhesion. Follistatin, follistatin-related genes (FLRG, FSRP) and follistatin-related protein (FRP), through transcriptional regulation via TGF-β (Bartholin et al. 2001) Oncogene, 20:5409-5419; Shibanuma et al. (1993) Eur.J.Biochem.217:13-19) or by their ability to antagonize the TGF-β signaling pathway (Phillips and de Kretser (1998) Front. Neuroendocrin., 19:287-322; Tsuchida et al. (2000) J.Biol.Chem., 275:40788-40796; Patel et al. (1996) Dev.Biol., 178:327-342; Amthor et al. (1996) ) Dev. Biol., 178:343-362) and are all associated with TGF-β signaling. The names of proteins in parentheses are alternative names.

Insulin growth factor binding protein 7 (IGFBP7, mac25), comprising at least one follistatin domain, binds insulin and subsequently blocks the interaction with the insulin receptor. In addition, IGFBP7 has been shown to bind activin, a member of the TGF-beta family (Kato (2000) MoI. Med., 6: 126-135).

Agrin and agrin-related proteins contain more than nine follistatin domains and are secreted from nerve cells to facilitate the aggregation of acetylcholine receptors and other molecules involved in forming synapses. It has been shown that follistatin domains may help localize growth factors to synapses (Patthy et al. (1993) Trends Neurosci., 16:76-81).

Osteonectin (SPARC, BM40) and hevin (SC1, mast9, QR1) are closely related proteins that interact with proteins of the extracellular matrix and regulate cell growth and adhesion (Motamed (1999) Int. J. Biochem. Cell Biol., 31:1363-1366; Girard and Springer (1996) J. Biol. Chem., 271:4511-4517). These proteins contain at least one follistatin domain.

Other follistatin domain proteins have been described or published in the NCBI database (National Center for Biotechnology Information, Bethesda, MD, USA), however their function is currently unknown. These proteins include U19878 (G01639, very similar to fomoregulin-1), T46914, human GASP1 (GDF-associated serum protein 1; described here; Figure 7), human GASP2 (WFIKKN; Trexler et al. (2001) Proc.Natl.Acad.Sci.U.S.A., 98:3705-3709; Figure 9), and the proteoglycan family of testican (SPOCK) proteins (Alliel et al. (1993) Eur.J.Biochem., 214:347-350) . The amino acid and nucleotide sequences of mouse GASP1 (FIG. 6) and mouse GASP2 (FIG. 8) were also determined from the Celera database (Rockville, MD). As described here, the nucleotide sequence of cloned mouse GASP1 matched the predicted Celera sequence except for some base substitutions in the wobble codon that did not alter the predicted amino acid sequence (see Figure 13).

Summary of the invention

Accordingly, the present invention relates to proteins other than follistatin which comprise the unique structural feature of the presence of at least one follistatin domain. Follistatin itself is not included in the present invention. Proteins comprising at least one follistatin domain specifically react with mature GDF-8 protein or fragments thereof, whether the GDF-8 protein is in monomeric form, a dimeric active form, or a GDF- 8 Composite forms of potential complexes. A protein comprising at least one follistatin domain may bind an epitope on the mature GDF-8 protein, resulting in a reduction in one or more biological activities associated with GDF-8, relative to a protein that does not bind the same protein Mature GDF-8 protein.

The present invention provides methods for modulating the effects of GDF-8 on cells. This method comprises administering an effective amount of a protein comprising at least one follistatin domain. The invention also includes methods of expressing proteins in cells by administering a DNA molecule encoding a protein comprising at least one follistatin domain.

According to the present invention, a protein comprising at least one follistatin domain may be administered to a patient in a therapeutically effective dose for the treatment or prevention of medical conditions in which an increase in muscle tissue is therapeutically effective. Embodiments include the treatment of diseases, disorders, and cellular and tissue trauma associated with the production, metabolism, or activity of GDF-8.

A protein comprising at least one follistatin domain can be prepared in the form of a pharmaceutical formulation. The pharmaceutical formulation may contain additional components that assist in the binding of mature GDF-8 protein or fragments thereof, whether the GDF-8 is in monomeric form, a dimeric active form, or a complex form of a GDF-8 latent complex.

Furthermore, proteins comprising at least one follistatin domain can be used as diagnostic tools for the quantitative or qualitative detection of mature GDF-8 protein or fragments thereof, whether in monomeric form, the active form of a dimer, or Complex form of the GDF-8 latent complex. For example, a protein comprising at least one follistatin domain can be used to detect the presence, absence or amount of GDF-8 protein in a cell, body fluid, tissue or organism. The presence or amount of detected mature GDF-8 protein may correlate with one or more of the medical conditions listed herein.

Proteins comprising at least one follistatin domain may be provided in diagnostic kits to detect mature GDF-8 protein or fragments thereof, whether in monomeric form, dimeric active form, or GDF-8 Complex forms of potential complexes, and help correlate the results obtained with one or more of the medical conditions described herein. Such a kit may comprise at least one protein, whether labeled or unlabeled, comprising at least one follistatin domain, and at least one reagent, such as a labeled antibody, that binds the protein. The kit may also include appropriate biological standards and control samples, which can be used to compare the results of experimental assays. It may also include buffers or washes and instructions for using the kit. Structural components can be included for performing experiments, such as sticks, beads, paper, columns, vials, or gels.

Brief description of the drawings

Figure 1 shows antibody purification of GDF-8 complexes from wild-type mouse serum. Silver-stained reducing gel showing protein purified from wild-type mouse serum using JA16 monoclonal antibody covalently coupled to agarose beads. A control purification using mock-coupled beads was performed in parallel (0). Subsequent elution with buffer (mock elution), competing peptide and SDS sample buffer showed 2 visible protein bands (indicated by arrows) with specific elution of peptide from JA16-bound beads.

Figure 2 shows the identification of mature and unprocessed GDF-8 in affinity purified samples from normal mouse serum. Figure 2A shows a representative MS/MS spectrum of a GDF-8-derived peptide (SEQ ID NO: 19) identified from a visible 12 kDa band in an affinity purified sample. Both N-terminal fragment ions (b ions) and C-terminal fragment ions (y ions) are visible. Notably, the strongest y-fragment ions result from cleavage in front of proline residues, a usual feature of proline-containing peptides. Figure 2B shows a western blot probed with a polyclonal antibody recognizing the mature region of GDF-8, confirming the presence of GDF-8 in the affinity purified samples. Both mature and unprocessed forms of GDF-8 are visible.

Figure 3 shows the binding of GDF-8 propeptide and follistatin-like related gene (FLRG) to circulating GDF-8 isolated from normal mouse serum. Representative MS/MS spectra of derived peptides identified from GDF-8 propeptide (SEQ ID NO: 23) (Figure 3A) and FLRG (SEQ ID NO: 30) (Figure 3C) identified in the 36 kDa band are shown. Figure 3B shows a western blot of affinity purified GDF-8 complex probed with a polyclonal antibody that specifically recognizes the GDF-8 propeptide region, confirming the mass spectrometric identification of the protein in the GDF-8 complex. The cleaved propeptide and unprocessed GDF-8 are visible - under longer exposure times, unprocessed GDF-8 is also seen in the SDS eluted samples. Figure 3D shows a western blot of affinity purified GDF-8 complex probed with a monoclonal antibody to FLRG.

Figure 4 shows the results of a complete analysis of the large-scale GDF-8 purification of the identified GDF-8 propeptide, FLRG and the novel protein as the major GDF-8 binding protein in serum. Silver stained gels of the negative control and JA16 immunoprecipitated peptide eluted samples were cut into 13 fractions. Proteins in each fraction were trypsinized and identified using nanoflow-LC-MS/MS and database searches. Unique proteins of the JA16 sample included only unprocessed and mature GDF-8, GDF-8 propeptide, FLRG, and a novel multidomain protease inhibitor (GDF-associated serum protein 1, GASP1). These proteins were identified from labeled regions of the gel.

Figure 5 shows the novel multi-domain protease inhibitor, GASP1, which binds to GDF-8 in serum. Figure 5A (peptide designated as SEQ ID NO: 31) and 5B (peptide designated as SEQ ID NO: 33) show representative MS/MS values for two GASP1 peptides identified from band 3 of the silver-stained gel of Figure 4. MS spectrum.

Figure 6A shows the predicted nucleotide sequence of mouse GASP1. Figure 6B shows the predicted alternative nucleotide sequence of mouse GASP1. Figure 6C shows the predicted amino acid sequence encoded by the nucleotide sequences shown in Figures 6A and 6B. The protein sequences encoded by these two nucleotide sequences are identical because the nucleotide differences are at the positions of the wobble codons. The follistatin domain is shown in bold and underlined.

Figure 7A shows the predicted nucleotide sequence of human GASP1. Figure 7B shows the corresponding predicted amino acid sequence. The follistatin domain is shown in bold and underlined. Figure 7C shows the predicted nucleotide sequence of human GASP1 using alternative start sites. Figure 7D shows the corresponding predicted amino acid sequence. The domain of follistatin is shown in bold and underlined. The end of the sequence is indicated by an asterisk.

Figure 8A shows the predicted nucleotide sequence of mouse GASP2, while Figure 8B shows the corresponding predicted amino acid sequence. The domain of follistatin is shown in bold and underlined.

Figure 9A shows the predicted nucleotide sequence of human GASP2, while Figure 9B shows the corresponding predicted amino acid sequence. The domain of follistatin is shown in bold and underlined.

Figure 10 shows the expression of mouse GASP1 in a number of adult tissues and during development. The figure shows the tissue expression profile of mouse GASP1. A 551 bp fragment of GASP1 was amplified from a normalized first-strand cDNA set by Clontech (Palo Alto, CA). A portion of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was amplified as a control. G3PDH expression is known to be high in skeletal muscle and low in testis. The template normalized the cDNA set against β-actin, phospholipase A2 and ribosomal protein S29 in addition to G3PDH.

Figure 11 shows proteins isolated from human serum. Proteins from JA16 immunoprecipitated or control samples (0) were eluted in mock PBS elution, competitive peptide elution or SDS elution. Proteins in the indicated regions of the gel were trypsinized and analyzed by LS-MS/MS and database searches. The proteins present in the JA16 samples but not in the control samples were mature GDF-8 (lane 16), GDF-8 propeptide and FLRG (lane 11 ), and human GASP1 (lane 4). Figure 1 IB shows a western blot of the same JA16 immunoprecipitation probed with an antibody recognizing mature GDF-8. Bands corresponding to mature and unprocessed GDF-8 isolated from human serum are visible.

Figure 12 shows typical mass spectra of peptides and associated proteins derived from GDF-8 isolated from lanes 4, 11 and 16 (Figure 11). The peptide sequence and the N-terminus (b ion) and C-terminus (y ion) are shown. A list of identified peptides is provided in Table 1. The spectra shown are from GASP1 peptide (SEQ ID NO: 44) (FIG. 12A), FLRG peptide (SEQ ID NO: 41) (FIG. 12B), GDF-8 propeptide (SEQ ID NO: 24) (FIG. 12C) and mature GDF-8 peptide (SEQ ID NO: 13) (FIG. 12D).

Figure 13 shows the nucleotide (SEQ ID NO:48) and amino acid (SEQ ID NO:49) sequence of cloned mouse GASP1. Peptides identified in JA16 affinity purified samples by mass spectrometry are underlined. The end of the sequence is indicated by an asterisk.

Figure 14A shows the domain structure of GASP1. GASP1 has a signal sequence/cleavage site after amino acid 29. In addition, GASP1 contains 2 Kunitz/BPTI serine protease inhibitor domains that may inhibit metalloproteases, a follistatin domain (including the Kazal serine protease inhibitor motif) and a netrin domain. Figure 14B shows the phylogenetic dendrogram of GASP1 and GASP2 predicted from the mouse and human genome sequences. Mouse and human GASP1 are 90% identical. GASP1 and GASP2 are 54% identical.

Figure 15 shows that recombinantly produced GASP1 binds GDF-8 and GDF-8 propeptide, respectively. (A) GDF-8 was immunoprecipitated using JA16 from mock or GASP1-V5-His transfected COS cell conditioned media supplemented with recombinant purified GDF-8 and/or propeptide. The presence of these proteins in the immunoprecipitates was detected by Western blot using anti-V5 (upper panel), anti-GDF-8 (middle panel), or anti-propeptide polyclonal antibodies. (B) Recombinantly produced GASP1 protein was immunoprecipitated with anti-V5 tagged antibody from mock or GASP1-V5-His conditioned media supplemented with recombinant purified GDF-8 and/or propeptide. Immunoprecipitates were analyzed by western blot as described in (A).

Figure 16 shows that GASP1 inhibits the biological activity of GDF-8 and the highly related BMP-11, but not activin or TGF-[beta]. Multiple dilutions of conditioned medium from mock (open circles) or GASP1-V5-His (closed squares) transfectants were mixed with (A) 10 ng/ml GDF-8, (B) 10 ng/ml BMP-11 , (C) 10ng/ml activin, or (D) 0.5ng/ml TGF-β incubation. These samples were then assayed for luciferase reporter activity in A204 (A-C) or RD (D) cells to determine the activity of added growth factors. Luciferase activity is shown in relative luciferase units. Activity resulting from each growth factor alone is shown by solid diamonds and short dashed lines. Without the addition of any growth factor, the background activity in the assay was very low, as indicated by the long dashed line without a symbol.

Figure 17 shows the potency of GASPl inhibition of GDF-8. Purified GASPl was tested for its ability to inhibit 20 ng/ml myostatin in a (CAGA) ₁₂ (SEQ ID NO: 53) luciferase reporter assay of RD cells (filled squares). Activity produced by GDF-8 alone is shown by solid diamonds and short dashed lines. The activity present in the absence of added growth factors is shown with long dashed lines.

definition

The term "follistatin domain" refers to an amino acid domain characterized by a cysteine-rich repeat unit or a nucleotide domain encoding the amino acid domain. The follistatin domain generally comprises a span of 65-90 amino acids and contains 10 conserved cysteine residues and a region similar to the Kazal serine protease inhibitor domain. In general, the loop regions between cysteine residues show sequence variability in the follistatin domain, but some conservation is still evident. The loop between the 4th and 5th cysteines is usually small, containing only 1 or 2 amino acids. Amino acids in the loop between the 7th and 8th cysteines are generally the most highly conserved, containing (G,A)-(S,N)-(S,N,T)-(D,N) - Consensus sequence of (G,N) followed by (T,S)-Y motif. The region between the 9th and 10th cysteine generally includes a motif comprising 2 hydrophobic residues (specifically V, I or L) separated by another amino acid.

The term "protein comprising at least one follistatin domain" refers to a protein comprising at least one, but possibly more than one follistatin domain. The term also refers to any variant of this protein (including fragments; proteins with substitution, addition, or deletion mutations; and fusion proteins) that retain known biological activities associated with the native protein, particularly GDF-8 Known biological activity associated with binding activity includes sequences that have been modified with conservative or non-conservative amino acid sequence changes. These proteins may be derived from any source, natural or synthetic. The protein may be derived from human or animal sources including bovine, chicken, murine, rat, pig, sheep, turkey, baboons and fish. Follistatin itself is not included in the present invention.

The term "GDF-8" or "GDF-8 protein" refers to a specific growth and differentiation factor. The term includes the full-length unprocessed precursor form of the protein, as well as the mature and propeptide forms resulting from post-translational cleavage. The term also refers to any fragment of GDF-8 that retains the known biological activity associated with the protein, including sequences that have been modified with conservative or non-conservative amino acid sequence changes. These GDF-8 molecules may be derived from any source, natural or synthetic. The protein may be of human or animal origin, including bovine, chicken, murine, rat, pig, sheep, turkey, baboons, and fish. Various GDF-8 molecules have been described in McPherron et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 12457-12461.

"Mature GDF-8" refers to the protein cleaved from the carboxy-terminal domain of the GDF-8 precursor protein. Mature GDF-8 can exist as a monomer, a homodimer, or a GDF-8 latent complex. Depending on in vivo or in vitro conditions, mature GDF-8 can establish a balance between any or all of these different forms. Homodimers are believed to be biologically active. In its biologically active form, the mature GDF-8 is also referred to as "activated GDF-8".

"GDF-8 propeptide" refers to a polypeptide cleaved from the amino-terminal domain of the GDF-8 precursor protein. GDF-8 propeptide is capable of binding the propeptide binding domain on mature GDF-8.

"GDF-8 latent complex" refers to the protein complex formed between the homodimer of mature GDF-8 and the GDF-8 propeptide. The two GDF-8 propeptides are thought to bind two molecules of the homodimeric form of mature GDF-8 to form an inactive tetrameric complex. The potential complex may include other GDF inhibitors in place of or in addition to one or more GDF-8 propeptides.

The phrase "GDF-8 activity" refers to one or more physiological growth regulatory or morphogenetic activities associated with activated GDF-8 protein. For example, activated GDF-8 is a negative regulator of skeletal muscle. Activated GDF-8 can also regulate the production of muscle-specific enzymes (eg, creatine kinase), stimulate cell proliferation of myoblasts and regulate differentiation of preadipocytes into adipocytes. GDF-8 is also thought to increase insulin sensitivity and glucose uptake in peripheral tissues, especially in skeletal muscle or adipocytes. Thus, GDF-8 biological activities include, but are not limited to, inhibition of muscle formation, inhibition of myocyte growth, inhibition of muscle development, reduction of muscle mass, modulation of muscle-specific enzymes, inhibition of cell proliferation of myoblasts, modulation of preadipocyte differentiation into adipose Cells, increase sensitivity to insulin, regulate glucose uptake, glucose hemostasis and regulate the development and maintenance of neuronal cells.

The term "isolated" or "purified" refers to a molecule that is substantially free from its natural environment. For example, an isolated protein refers to a protein derived from a cell or tissue source that is substantially free of cellular material or other contaminating proteins derived from the cell or tissue. The phrase "substantially free of cellular material" means wherein the isolated protein is at least 70%-80% (w/w) pure, at least 80%-89% (w/w) pure, at least 90-95% pure , or a preparation that is at least 96%, 97%, 98%, 99% or 100% (w/w) pure.

The term "LC-MS/MS" refers to liquid chromatography arranged in tandem with a mass spectrometer to separate molecular ions of a specific mass/charge ratio, fragment the ions and record the mass/charge ratios of the fragment ions. When analyzing peptide samples, this technique enables upstream separation of complex samples by liquid chromatography, followed by recording of the masses of the fragment ions and subsequent determination of the sequence of the peptides.

The term "MS/MS" refers to a method of using a mass spectrometer to separate molecular ions of a specific mass/charge ratio, fragment the ions, and record the mass/charge ratios of the resulting fragment ions. This fragment ion provides information about the sequence of the peptide.

The term "treatment" or "therapy" refers to both treatment and prophylaxis or preventive measures. Those in need of treatment may include those already with the particular medical condition as well as those who may eventually develop the condition (ie, those in need of prophylactic measures). The term treatment includes measures to identify the underlying cause of a condition as well as measures to reduce the symptoms of a medical condition without necessarily affecting its cause. Therefore, improvement of quality of life and improvement of symptoms, as well as measures to counteract the etiology, are considered treatment.

The term "medical disorder" refers to a disturbance of muscle, bone, or glucose homeostasis, and includes disorders associated with GDF-8 and/or other members of the TGF-beta superfamily (eg, BMP-11). Examples of such conditions include, but are not limited to, metabolic diseases and disorders such as insulin-dependent (type 1) diabetes, non-insulin-dependent (type 2) diabetes, hyperglycemia, impaired glucose tolerance, metabolic syndrome (e.g., combined syndrome X), and insulin resistance induced by trauma (such as burns or nitrogen imbalance), and adipose tissue disorders (such as obesity); muscle and neuromuscular disorders such as muscular dystrophy (including but not limited to, severe or Benign X-linked muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral dystrophy, myotonic muscular dystrophy, distal muscular dystrophy, progressive dystrophic ophthalmoplegia, oculopharyngeal muscular dystrophy, Du muscular dystrophy and Fakuyama congenital muscular dystrophy); amyotrophic lateral sclerosis (ALS); muscular atrophy; organ atrophy; fragility; carpal tunnel syndrome; congestive obstructive lung disease; congenital myopathy; congenital Myotonia; familial periodic paralysis; sudden myoglobinuria; myasthenia gravis; Iran-Long syndrome; secondary myasthenia; denervation atrophy; sudden muscle atrophy; and sarcopenia ), cachexia, and other muscle-wasting syndromes. Other examples include osteoporosis, especially in elderly and/or postmenopausal women; glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; fractures associated with osteoporosis; And trauma or chronic damage to muscle tissue. Further examples include low bone mass resulting from chronic glucocorticoid therapy, precocious gonadal disorders, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa.

The term "mass gain" refers to the appearance of a greater amount of muscle after treatment with a protein comprising at least one follistatin domain relative to the muscle mass present before treatment.

The term "therapeutic benefit" refers to amelioration of symptoms of a disorder, slowing of progression of a disorder, or cessation of progression of a disorder. Therapeutic benefit is determined by comparing various aspects of the disorder, such as intramuscular amounts before and after administration of at least one protein comprising at least one follistatin domain.

The term "modulate" refers to changing the properties of a protein by increasing, decreasing or inhibiting its activity, performance or quantity. For example, proteins comprising at least one follistatin domain can modulate GDF-8 by inhibiting its activity.

The term "stabilizing modification" is any modification known in the art or proposed herein that is capable of stabilizing a protein, increasing its in vitro half-life, increasing its circulating half-life and/or reducing its proteolytic degradation. Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins of a protein comprising at least one follistatin domain and a second protein), modifications of glycosylation sites (including, for example, , adding glycosylation sites to proteins comprising at least one follistatin domain), and modifications of carbohydrate moieties (including, for example, removal of carbohydrates from proteins comprising at least one follistatin domain compound section). In the case of a stabilizing modification involving a fusion protein (for example, a fusion of a second protein to a protein comprising at least one follistatin domain), the second protein may be referred to as a "stabilizer moiety" or a "stabilizing moiety". agent protein". For example, a protein human protein comprising at least one follistatin domain can be fused to an IgG molecule, wherein the IgG functions as a stabilizer protein or part of a stabilizer. As used herein, "stabilizer moiety" also includes non-proteinaceous modifications, such as carbohydrate moieties or non-proteinaceous polymers, in addition to referring to the second protein of the fusion protein.

The term "Fc region of an IgG molecule" refers to the Fc domain of an IgG immunoglobulin of the isotype as known to those skilled in the art. The Fc region of an IgG molecule is the part of the IgG molecule (IgGl, IgG2, IgG3 and IgG4) responsible for increasing the in vivo serum half-life of the IgG molecule.

"In vitro half-life" refers to the stability of a protein measured in vitro in a living organism. Assays for measuring half-life in vitro are well known in the art and include, but are not limited to, SDS-PAGE, ELISA, cell-based assays, pulse-chase, western blotting, northern blotting, and the like. These and other useful assays are well known in the art.

"In vivo half-life" refers to the stability of a protein in an organism. In vivo half-life can be measured by a number of methods known in the art, including, but not limited to, in vivo serum half-life, circulating half-life and the assays illustrated in the Examples herein.

"In vivo serum half-life" refers to the half-life of a protein circulating in the blood of an organism. Methods known in the art can be used to measure serum half-life in vivo. For example, a radioactive protein can be administered to an animal and the amount of the labeled protein in the serum can be monitored over time.

To aid in the identification of the sequences listed in this specification and figures, the following tables are provided, listing the SEQ ID NO, position of the figure and description of the sequence.

SEQ ID NO: refer to illustrate 1 Figure 6A Predicted mouse GASP1 nucleotide sequence 2 Figure 6B Alternative Nucleotide Sequences of Predicted Mouse GASP1 3 Figure 6C Predicted Amino Acid Sequence of Mouse GASP1 Encoded by SEQ ID NO: 1 and 2 4 Figure 7A Predicted Human GASP1 Nucleotide Sequence 5 Figure 7B Predicted human GASP1 amino acid sequence encoded by SEQ ID NO: 4 6 Figure 7C Predicted Human GASP1 Nucleotide Sequence, Alternative Start Sites 7 Figure 7D Predicted human GASP1 amino acid sequence encoded by SEQ ID NO: 6, alternative start site 8 Figure 8A Predicted mouse GASP2 nucleotide sequence 9 Figure 8B Predicted amino acid sequence of mouse GASP2 encoded by SEQ ID NO: 8 10 Figure 9A Predicted Nucleotide Sequence of Human GASP2 11 Figure 9B Predicted amino acid sequence of human GASP2 encoded by SEQ ID NO: 10 12 Example 2 competing peptide 13-20 Table 1, Embodiment 5, 6 mouse GDF-8 peptide 21-27 Table 1, Embodiment 5, 6 propeptide of mouse GDF-8 28-30 Table 1, Example 5 Peptide of mouse FLRG 31-35 Table 1, Example 5, 7 Peptide of mouse GASP1 36-37 Table 1, Example 8 Peptide of human GDF-8 38-39 Table 1, Example 8 Peptides of human GDF-8 propeptide 40-42 Table 1, Example 8 Peptide of human FLRG

43-45 Table 1, Example 8 Peptide of human GASP1 46 Example 7 forward primer 47 Example 7 reverse primer 48 Figure 13 Nucleotide sequence of cloned mouse GASP1 49 Figure 13 Amino acid sequence of cloned mouse GASP1 encoded by SEQ ID NO: 48 50 Example 9 forward primer 51 Example 9 reverse primer 52 Example 9 Sequences of illustrated N-terminal peptides 53 Example 11 synthetic oligonucleotides

Detailed description of the invention

Proteins comprising at least one follistatin domain

The present invention relates to proteins other than follistatin which have the unique structural feature of including at least one follistatin domain. Follistatin itself is not included in the present invention. It is believed that proteins comprising at least one follistatin domain will bind and inhibit GDF-8. Examples of proteins with at least one follistatin domain include, but are not limited to, follistatin-related genes (FLRG), FRP (flik, tsc36), agrin, osteonectin (SPARC , BM40), hevin (SC1, mast9, QR1), IGFBP7 (mac25) and U19878. GASP1 comprising the nucleotide and amino acid sequences provided in Figures 6 and 7, and GASP2 comprising the nucleotide and amino acid sequences provided in Figures 8 and 9 are other proteins comprising at least one follistatin domain instance.

As noted above, a follistatin domain is defined as an amino acid domain characterized by a cysteine-rich repeat unit or by nucleotide domains encoding the amino acid domain. The follistatin domain generally comprises a span of 65-90 amino acids and contains 10 conserved cysteine residues and a region similar to the Kazal serine protease inhibitor domain. In general, the loop regions between cysteine residues show sequence variability in the follistatin domain, but some conservation is still evident. The loop between the 4th and 5th cysteines is usually small, containing only 1 or 2 amino acids. Amino acids in the loop between the 7th and 8th cysteines are generally the most highly conserved, containing (G,A)-(S,N)-(S,N,T)-(D,N) - Consensus sequence of (G,N) followed by (T,S)-Y motif. The region between the 9th and 10th cysteine generally includes a motif comprising 2 hydrophobic residues (specifically V, I or L) separated by another amino acid.

A protein comprising at least one follistatin domain that binds GDF-8 can be isolated using a variety of methods. For example, affinity purification of GDF-8 can be utilized as exemplified in the present invention. In addition, low stringency screening of cDNA libraries can be used, or degenerate PCR techniques using probes targeting the follistatin domain can be used. When more genomic data are available, similarity searches using a number of sequence profiles and analysis programs such as MotifSearch (Genetics Computer Group, Madison, WI), ProfileSearch (GCG), and BLAST (NCBI) can be used to find Novel proteins with significant homology to known follistatin domains.

Those skilled in the art will recognize that GDF-8 or proteins comprising at least one follistatin domain may contain any number of conservative changes in their respective amino acid sequences without altering their biological properties. Such conservative amino acid changes are based on the relative similarity of amino acid side chain substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like. Representative conservative substitutions that take into account the various features described above are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamic acid aminoamide and asparagine; and valine, leucine and isoleucine. Furthermore, proteins comprising at least one follistatin domain can be used to generate functional fragments comprising at least one follistatin domain. This fragment is expected to bind and inhibit GDF-8. In an embodiment of the invention, the protein comprising at least one follistatin domain specifically binds mature GDF-8 or a fragment thereof, whether it is in monomeric form, active dimeric form or GDF- 8 Complex forms of potential complexes with affinities between 0.001-100 nM or 0.01-10 nM or 0.1-1 nM.

Nucleotide and Protein Sequences

Although not always necessary, one of ordinary skill in the art can, if desired, determine the amino acid or nucleic acid sequence of a novel protein comprising at least one follistatin domain. For example, the present invention provides the amino acid and nucleotide sequences of GASP1 and GASP2 as shown in Figures 6-9.

The invention also includes variants, homologues and fragments of the nucleic acid and amino acid sequences. For example, the nucleic acid or amino acid sequence may comprise at least 70%-79% identical, or at least 80%-89% identical, or at least 90%-95% identical, or at least 96%-100% identical to the nucleic acid or amino acid sequence of a native protein. the same sequence. Those skilled in the art will recognize that regions that bind GDF-8 may tolerate less sequence variation than other parts of the protein that are not involved in binding. Accordingly, these non-binding regions of the protein may contain substantial variations that do not significantly alter the binding properties of the protein. However, those skilled in the art will also recognize that many changes can be made to specifically increase the affinity of the protein for its target. Such affinity-increasing changes are typically determined empirically by altering the protein at the binding region and testing the ability to bind GDF-8, or the strength of that binding. All such changes, whether internal or external to the binding region, are included within the scope of the present invention.

Relative sequence similarity or identity can be determined using "Best Fit" or "Gap" of the Sequence Analysis Software Package ^™ (version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, WI). Program OK. "Gap" employs the algorithm of Needleman and Wunsch (Needleman and Wunsch, 1970) to find an alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "BestFit" performs a best-fit sequence alignment of the best fraction of similarity between two sequences. Optimal sequence alignments are found by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981; Smith et al., 1983).

The sequence analysis software packages described above contain a number of other useful sequence analysis tools for identifying homologues of the nucleotide and amino acid sequences disclosed herein. For example, the "BLAST" program (Altschul et al., 1990) searches for sequences similar to a query sequence (peptide or nucleic acid) in a specific database (e.g., the sequence database maintained by NCBI); "FastA" (Lipman and Pearson, 1985; See also Pearson and Lipman, 1988; Pearson et al., 1990) performs a Pearson and Lipman search for searching for similarities between a query sequence and a set of sequences of the same type (nucleic acid or protein); "TfastA" performs a Pearson and Lipman search Search, for searching for similarities between a protein query sequence and any set of nucleotide sequences that translates the nucleotide sequence by a total of 6 reading frames before comparison; "FastX" performs Pearson that takes frameshifts into account and Lipman search, for searching for similarities between a nucleotide query sequence and a set of protein sequences. "TfastX" performs a Pearson and Lipman search that takes into account frameshifts for similarity between a protein query sequence and any set of nucleotide sequences (it translates both strands of the nucleotide sequence before making a comparison).

modified protein

The present invention includes fragments of proteins comprising at least one follistatin domain. Such fragments may include all or part of the follistatin domain. Fragments may include all, part or none of the sequence between the follistatin domain and the N-terminus and/or between the follistatin domain and the C-terminus.

Those of ordinary skill in the art will appreciate that certain amino acids in the structure of a protein may be substituted by other amino acids without detrimentally affecting the activity of the protein, such as the binding properties of a protein comprising at least one follistatin domain. The inventors thus contemplate that various changes may be made in the amino acid sequence of proteins comprising at least one follistatin domain, or in the DNA sequence encoding the protein, without appreciable loss of their biological utility or activity. Such alterations may include deletions, insertions, truncations, substitutions, fusions, shuffing of motif sequences, and the like.

In producing such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on proteins is generally understood in the art (Kyte and Doolittle (1982) J. Mol. Biol., 157:105-132). It is recognized that the relatively hydrophilic character of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the protein's interaction with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. .

Each amino acid has been assigned a hydropathic index based on its hydrophobic and charge properties (Kyte and Doolittle, 1982); these are isoleucine (+4.5), valine (+4.2), leucine (+3.8 ), Phenylalanine (+2.8), Cysteine/Cystine (+2.5), Methionine (+1.9), Alanine (+1.8), Glycine (-0.4), Threonine (-0.7), Serine (-0.8), Tryptophan (-0.9), Tyrosine (-1.3), Proline (-1.6), Histidine (-3.2), Glutamic Acid (-3.5) , glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9) and arginine (-4.5). In making such changes, the hydropathic index of the substituted amino acid can be within ±2, within ±1, and within ±0.5.

It is also understood in the art that similar amino acid substitutions can be efficiently made based on hydrophilicity. US Patent No. 4,554,101 shows that the maximum local average hydrophilicity of a protein depends on the hydrophilicity of its adjacent amino acids, which is related to the biological properties of the protein.

As detailed in U.S. Patent 4,554,101, the following values of hydrophilicity have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0±1) , glutamic acid (+3.0±1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline ( -0.5±1), alanine (-0.5), histidine (-0.5), cysteine (-1.0), methionine (-1.3), valine (-1.5), leucine acid (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5) and tryptophan (-3.4). In making such changes, the substituted amino acid may have a hydrophilic value within ±2, within ±1, and within ±0.5.

Modifications may be conservative such that the protein's structure or biological function is not affected by the change. Such conservative amino acid modifications are based on the relative similarity of amino acid side chain substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like. Representative conservative substitutions that take into account the various features described above are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamic acid aminoamide and asparagine; and valine, leucine and isoleucine. The amino acid sequence of a protein comprising at least one follistatin domain can be altered with any number of conservative changes so long as binding of the protein to its target antigen is not deleteriously affected. Such changes can be introduced inside or outside of the protein binding portion comprising at least one follistatin domain. For example, changes introduced into the antigen-binding portion of a protein can be designed to increase the affinity of the protein for its target.

Stabilizing modification

Stabilizing modifications can stabilize proteins, increase protein half-life in vitro and/or in vivo, increase protein circulating half-life and/or reduce proteolytic degradation of proteins. Such stabilizing modifications include, but are not limited to, fusion proteins, modifications of glycosylation sites, and modifications of carbohydrate moieties. The stabilizer protein can be any protein that increases the overall stability of the modified GDF propeptide. Such fusion proteins may optionally include a linker peptide between the propeptide moiety and the stabilizing moiety, as will be recognized by one of ordinary skill in the art. It is well known in the art that fusion proteins are prepared such that a second protein is fused in-frame to a first protein so that the resulting translated protein comprises the first and second proteins. For example, in the present invention, fusion proteins can be prepared such that a protein comprising at least one follistatin domain is fused to a second protein (eg, a stabilizer protein moiety). This fusion protein is prepared such that the resulting translated protein contains a propeptide portion and a stabilizer portion.

Proteins comprising at least one follistatin domain may be glycosylated or linked to albumin or non-proteinaceous polymers. For example, proteins comprising at least one follistatin domain can be combined with one or more non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes as described in U.S. Pat. Nos. 4,640,835; 4,496,689 ; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. For example, proteins are chemically modified by covalent attachment to polymers to increase their circulating half-life. Polymers and methods of attaching them to peptides are also shown in US Patent Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546.

Proteins comprising at least one follistatin domain can be pegylated. PEGylation is the process of attaching polyethylene glycol (PEG) to proteins to prolong the half-life of proteins in the body. Pegylation of proteins comprising at least one follistatin domain can reduce the dose or frequency of protein administration required for optimal inhibition of GDF-8. A review of this technique is provided in Bhadra et al. (2002) Pharmazie, 57:5-29 and Harris et al. (2001) Clin. Pharmacokinet., 40:539-551.

A protein comprising at least one follistatin domain can be linked to the Fc region of an IgG molecule. A protein comprising at least one follistatin domain may be fused contiguously to the Fc region of an IgG molecule, or attached via a linker peptide to the Fc region of an IgG molecule. The use of such linker peptides is well known in the art of protein biochemistry. The Fc region may be derived, for example, from IgGl or IgG4.

A protein comprising at least one follistatin domain can be modified to have an altered glycosylation pattern (ie, different from the original or native glycosylation pattern). As used herein, "altered" refers to the deletion of one or more carbohydrate moieties, and/or the addition of one or more glycosylation sites to the original protein.

Glycosylation of proteins is generally either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain . Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline can also be used or 5-hydroxylysine.

A glycosylation site is added to a protein comprising at least one follistatin domain by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites point) and conveniently implemented. The change can also be produced by the addition or substitution of one or more serine or threonine residues (for O-linked glycosylation sites) to the sequence of the original protein. For convenience, the amino acid sequence of a protein can be altered by changes at the DNA level.

Another way to increase the number of carbohydrate moieties on a protein is by chemically or enzymatically coupling glycosides to amino acid residues of the protein. These methods are advantageous because they do not require inhibitors of GDF peptides to be produced in host cells with glycosylation capabilities for N- or O-linked glycosylation. Depending on the conjugation used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) such as serine, threonine or the free hydroxyl group of hydroxyproline, (e) an aromatic residue such as phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., 22:259-306.

Removal of any carbohydrate moieties present on a protein comprising at least one follistatin domain can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to triflic acid or an equivalent compound. This treatment results in the removal of most or all sugars except for the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the intact amino acid sequence.

Chemical deglycosylation is described by Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52; and Edge et al. (1981) Anal. Biochem., 118:131. Enzymatic cleavage of carbohydrate moieties on inhibitors of GDF peptides can be achieved by the use of various endo- and exoglycosidases as described by Thotakura et al. (1987) Meth. Enzymol., 138:350.

The protein comprising at least one follistatin domain may be linked to the protein albumin or a derivative of albumin. Methods for linking proteins and polypeptides to albumin or albumin derivatives are well known in the art. See, eg, US Patent No. 5,116,944.

pharmaceutical composition

The invention provides compositions comprising a protein comprising at least one follistatin domain. Such compositions may be suitable for pharmaceutical use and administration to patients. The composition generally comprises one or more proteins comprising at least one follistatin domain and a pharmaceutically acceptable excipient. As used herein, the phrase "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and and absorption delaying agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds that provide supplementary, additional or augmented therapeutic properties. The pharmaceutical composition can also be included in a container, pack or dispenser together with instructions for administration.

The pharmaceutical compositions of the invention are formulated to suit their intended route of administration. Methods for accomplishing the administration are well known to those of ordinary skill in the art. Administration can be, for example, intravenous, intramuscular or subcutaneous administration.

Solutions or suspensions for subcutaneous use generally contain one or more of the following: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial antioxidants, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; Reagents used to adjust osmolarity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The preparation can be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be liquid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, eg bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained by coating with eg lecithin, in the case of dispersions by maintaining the required particle size and by using surfactants. Protection against the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example sugars, polyalcohols, eg mannitol, sorbitol, sodium chloride, will in most cases be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent delaying absorption, for example, aluminum monostearate and gelatin.

In one embodiment, the protein comprising at least one follistatin domain is prepared with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microcapsules Sealed delivery system. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate copolymer, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. This material is also commercially available from Alza Ltd. and Nova Pharmaceuticals. Liposomal suspensions comprising proteins comprising at least one follistatin domain can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods well known to those skilled in the art, for example, as described in US Patent No. 4,522,811.

Therapeutically useful agents, such as growth factors (e.g., BMPs, TGF-β, FGF, IGF), cytokines (e.g., interleukins, and CDFs), antibiotics, and any other therapeutic agent effective for the condition being treated can optionally be Optionally included, or administered simultaneously or sequentially with a protein comprising at least one follistatin domain.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired dosage in association with the required pharmaceutical carrier. The corresponding desired therapeutic effect. The specification for the dosage unit forms of the invention are directly dependent on and are dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as limitations inherent in the field of compounding with such active compounds for individual therapy.

Treatment Indications

Proteins comprising at least one follistatin domain are useful for preventing, diagnosing, or treating a variety of human or animal medical conditions. Accordingly, the present invention provides for use in the treatment of diseases associated with muscle cells and tissues by administering to a subject an amount sufficient to ameliorate the symptoms of the disease comprising at least one protein comprising at least one follistatin domain and disease method. Such disorders include muscular dystrophies, including but not limited to severe or benign X-linked muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral dystrophy, myotonic dystrophy, distal muscular dystrophy, progressive dystrophic ophthalmoplegia, oculopharyngeal muscular dystrophy, Duchenne muscular dystrophy, and Fakuyama congenital muscular dystrophy); amyotrophic lateral sclerosis (ALS); intramuscular atrophy; organ atrophy; fragility; carpal tunnel syndrome; congestive obstructive pulmonary disease; congenital myopathy; myotonia congenita; familial periodic paralysis; sudden myoglobinuria; myasthenia gravis; Iran-Long syndrome; secondary myasthenia; denervation Innervation atrophy; sudden muscle wasting; and sarcopenia, cachexia, and other muscle wasting syndromes. The invention also relates to traumatic or chronic injuries of muscle tissue.

In addition to providing treatments for muscle diseases and disorders, the present invention also provides methods for preventing or treating metabolic diseases or disorders resulting from abnormal glucose homeostasis. Such diseases or conditions include metabolic diseases and conditions (eg, insulin-dependent (type 1) diabetes, non-insulin-dependent (type 2) diabetes), hyperglycemia, impaired glucose tolerance, metabolic syndrome (eg, syndrome X) , and insulin resistance induced by trauma (such as burns or nitrogen imbalance), adipose tissue disorders (such as obesity); or bone degenerative diseases (such as osteoporosis, especially in elderly and/or postmenopausal women osteoporosis; glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; and osteoporosis-related fractures). Further examples include low bone mass due to chronic glucocorticoid therapy, praecox gonadism, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa.

Normal glucose homeostasis requires a coordinated combination of pancreatic β-cells to fine-tune insulin secretion in response to small changes in blood glucose levels. A fundamental role of insulin is to stimulate the uptake of glucose from the blood into tissues, especially into muscle and adipose tissue.

Accordingly, the present invention provides for the treatment of diabetes and related disorders, such as obesity or hyperlipidemia, by administering to a subject a composition comprising at least one protein comprising at least one follistatin domain in an amount sufficient to ameliorate the symptoms. blood sugar method. Type 2 or non-insulin-dependent diabetes mellitus (NIDDM), in particular, is characterized by three: (1) resistance to the action of insulin on glucose uptake in peripheral tissues, especially skeletal muscle and adipocytes, (2) attenuated insulin suppression of hepatic Glucose-generating effects, and (3) abnormal regulation of insulin secretion (DeFronzo (1997) Diabetes Rev. 5: 177-269). Thus, a subject suffering from type 2 diabetes may be treated according to the invention by administering a protein comprising at least one follistatin domain, which protein increases sensitivity to insulin and glucose uptake by cells.

Similarly, other diseases and metabolic disorders characterized by insulin dysfunction (e.g. resistance, inactivation or deficiency) and/or insufficient glucose transport into cells can also be inhibited according to the invention by administering Therapy is a protein with a protein domain that increases insulin sensitivity and glucose uptake by cells.

Approaches to Using Protein Therapy

Proteins comprising at least one follistatin domain can be used to inhibit or reduce binding to GDF-8 proteins (whether monomeric, dimeric, active forms) relative to GDF-8 proteins that are not bound by the same protein. , or the complex form of the GDF-8 latent complex) related to one or more activities. In an embodiment, when bound to a protein comprising at least one follistatin domain, the mature GDF-8 protein relative to mature GDF-8 not bound to a protein having a follistatin domain a protein whose activity is inhibited by at least 50%, or at least 60, 62, 64, 66, 68, 70, 72, 72, 76, 78, 80, 82, 84, 86, or 88%, or at least 90, 91, 92, 93, or 94%, or at least 95% to 100%.

Pharmaceutical formulations comprising proteins comprising at least one follistatin domain are administered in a therapeutically effective amount. As used herein, an "effective amount" of a protein is a dose sufficient to reduce the activity of GDF-8 to achieve the desired biological result. The desired biological outcome can be any therapeutic benefit, including increased muscle mass, increased muscle strength, improved metabolism, reduced obesity, or improved glucose homeostasis. This improvement can be measured by a variety of methods, including measurement of lean and fat body mass (eg, dual x-ray scan analysis), muscle strength, serum lipids, serum leptin (leptin), serum glucose, glycated hemoglobin, glucose tolerance and methods of improving secondary complications of diabetes.

In general, a therapeutically effective amount will vary with the age, condition and sex of the subject and the severity of the subject's medical condition. The dosage can be determined by a physician and adjusted as necessary to suit the observed effects of the treatment. Suitable dosages for administering at least one protein comprising at least one follistatin domain may be in the range of 5 mg-100 mg, 15 mg-85 mg, 30 mg-70 mg, or 40 mg-60 mg. The protein may be administered in one dose, or at intervals such as daily, weekly and monthly. The dosage schedule can be adjusted according to the affinity of the protein for GDF-8, the half-life of the protein, or the severity of the patient's condition. Typically, the composition is administered as a bolus to maximize circulating levels of the protein comprising at least one follistatin domain for the longest period of time following administration. Continuous infusion can also be used after a bolus dose.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eg, determining the _LD50 (the dose lethal to 50% of the population) and the _ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the index of efficacy and it can be expressed as the ratio _LD50 / _ED50 . Proteins comprising at least one follistatin domain can be used which exhibit a large therapeutic index.

Dosage ranges for use in humans can be estimated using data obtained from assays of cell cultures and animal studies. The dosage of such compounds may lie within a range of circulating concentrations that include the _ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any protein comprising at least one follistatin domain used in the invention, the therapeutically effective dose can be estimated starting from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the _IC50 (ie, the concentration of the test protein which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dose can be monitored by suitable biological assays. Examples of suitable bioassays include GDF-8 protein/receptor binding assays, creatine kinase assays, assays based on glucose uptake in adipocytes, and immunological assays.

Methods of DNA Administration

The present invention also provides gene therapy for the in vivo production of a protein comprising at least one follistatin domain. Such treatment can achieve its therapeutic effect by introducing polynucleotide sequences into cells or tissues suffering from the conditions listed herein.

Polynucleotide sequences for delivery of proteins comprising at least one follistatin domain can be obtained using recombinant expression vectors such as chimeric viruses or colloidal dispersion systems. Therapeutic delivery of polynucleotide sequences can be performed using targeted liposomes. A variety of viral vectors that can be used in gene therapy include adenovirus, herpes virus, vaccinia or RNA viruses such as retroviruses. Retroviral vectors may be derivatives of murine or avian retroviruses. Examples of retroviral vectors into which a single foreign gene may be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV ), murine mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV). A number of additional retroviral vectors can be incorporated into the polygene. All of these vectors deliver or incorporate genes for selectable markers so that transduced cells can be identified and generated. For example, by inserting the GDF propeptide polynucleotide sequence of interest into the viral vector together with another gene encoding a ligand for a receptor on a specific target cell, the vector is now target specific.

Retroviral vectors can be made target specific by the attachment of, for example, sugars, glycolipids, or proteins. Targeting can be achieved through the use of antibodies. Those skilled in the art will recognize that specific polynucleotide sequences can be inserted into the retroviral genome or attached to the viral envelope such that polynucleotides comprising a protein comprising at least one follistatin domain Retroviral vectors target specific delivery. In one embodiment, the vector targets muscle cells or muscle tissue.

Since recombinant retroviruses are defective, they require a helper cell line containing a plasmid encoding all retroviral structural genes under the control of regulatory sequences in the LTR. These plasmids are missing the nucleotide sequence that initiates the packaging machinery that recognizes the RNA transcript for encapsidation. Helper cell lines deficient in packaging signals include, but are not limited to, eg, PSI.2, PA317, and PA12. These cell lines produced empty virions because the genome was not packaged. If a retroviral vector is introduced into such a cell in which the packaging signal is intact but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virions produced.

Alternatively, other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, polar env by conventional calcium phosphate transfection. These cells are then transfected with a vector plasmid containing the gene of interest. The resulting cells release the retroviral vector into the culture medium.

Another targeted delivery system for polynucleotides of proteins comprising at least one follistatin domain is a colloidal dispersion system. Colloidal dispersion systems include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that are useful delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated inside aqueous dispersions and delivered to cells in a biologically active form (see, eg, Fraley et al. (1981) Trends Biochem. Sci., 6:77). Methods for efficient gene transfer using liposome vectors are well known in the art (see, eg, Mannino et al. (1988) Biotechniques, 6:682). The composition of liposomes is usually a combination of phospholipids, usually with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical properties of liposomes depend on pH, ionic strength and the presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. Orientation of liposomes can also be based on, for example, organ specificity, cell specificity, and organelle specificity and are known in the art.

There are a variety of methods available for delivering cells expressing a protein comprising at least one follistatin domain to a site for modulation of a GDF-8 response. In one embodiment of the invention, cells expressing a follistatin protein may be delivered by direct application, eg, injecting a sample of such cells directly at the site of tissue damage. These cells can be purified. Such cells can be delivered in a medium or matrix that partially impedes their fluidity so that it localizes the cells to the site of the wound. Such a medium or matrix may be semi-solid, such as a paste or gel, including colloidal polymers. Alternatively, the medium or matrix may be in the form of a solid, a porous solid that enables cells to migrate into the solid matrix, and retains them therein while enabling cell proliferation.

Method for detecting and isolating GDF-8

The presence or level of GDF-8 can be detected in vivo or in vitro using a protein comprising at least one follistatin domain. By correlating the presence or levels of these proteins with the medical condition, one skilled in the art can diagnose the relevant medical condition. Medical conditions diagnosable by proteins comprising at least one follistatin domain are set forth herein.

Such detection methods are well known in the art and include ELISA, radioimmunoassay, immunoblotting, western blot, immunofluorescence, immunoprecipitation, and other comparable techniques. The protein comprising at least one follistatin domain may further be present in a diagnostic kit incorporating one or more techniques for detecting GDF-8. Such kits may contain other ingredients, packaging, instructions for use, or other substances to aid in the detection of proteins and the use of the kit.

When trying to use a protein comprising at least one follistatin domain for diagnostic purposes, it can be used, for example, with a ligand group (such as biotin) or a detectable labeling group (such as a fluorophore, radioisotopes or enzymes) to modify them. Proteins can be labeled, if desired, using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms, electron dense reagents, enzymes and ligands with specific binding partners. Enzymes are generally detected by their activity. For example, horseradish peroxidase is typically detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, which can be measured spectrophotometrically. Other suitable binding partners include biotin and avidin or streptavidin, IgG and protein A, and many receptor-ligand pairs known in the art. Other permutations and possibilities will be apparent to those of ordinary skill in the art and are considered equivalents within the scope of the present invention.

Proteins or fragments thereof comprising at least one follistatin domain may also be used to isolate GDF-8 during purification. In one class of methods, proteins can be immobilized, eg, by incorporation into a column or resin. The protein is used to bind GDF-8 and then administered to conditions that lead to the release of the bound GDF-8. GDF-8 can be produced commercially using this method.

The following examples provide embodiments of the invention. Those of ordinary skill in the art will recognize that many modifications and variations can be made without altering the spirit or scope of the invention. Such modifications and variations are considered to be within the scope of the present invention. The examples do not limit the invention in any way. It is to be understood that all quantities in the specification and claims which are approximately modified by the term, for example as minor variations in dosage, are considered to be within the scope of the present invention.

Example

Example 1: Purification of JA16-bound beads

N-hydroxysuccinimide-activated beads (4% agarose beads, Sigma H-8635, St Louis MO) were washed in MilliQ-H ₂ O and incubated with anti-GDF-8 JA16 monoclonal antibody at 4°C (3-4 μg/μl in 100 mM MOPS, pH 7.5) were incubated for 4 hours at a ratio of 10 mg JA16/ml final resin concentration. Beads were washed extensively with 100 mM MOPS pH 7.5 and phosphate buffered saline (PBS) (Ausubel et al., (1999) Current Protocols in Molecular Biology, John Wiley and Sons) and stored in PBS at 4°C until use. Control beads were prepared in the same manner but without the JA16 antibody.

Example 2: Affinity purification

A total of 40 μl of packaged JA16-conjugated beads or control beads were incubated with 15 ml of normal Balb/C mouse serum (Golden West Biologicals, Temecula CA) or 30 ml of collected normal human serum (ICN Biomedical, Aurora OH) at 4°C. Incubate for 3 hours. Beads were washed twice in -10 ml cold 1% Triton X-100/PBS, twice in -10 ml cold 0.1% Triton X-100/PBS, and twice in -1 ml cold PBS. Proteins were eluted from the beads in 3 subsequent steps. First, the beads were subjected to a 'mock elution' treatment where 100 μl PBS was added to the beads and incubated at 4°C for 30 minutes. The supernatant was collected and combined with 30 μl 4×LDS sample buffer (Invitrogen, Carlsbad CA). Second, the beads are subjected to 'peptide elution' by adding 1 μg/μl of the competing peptide (sequence:

DFGLDSDEHSTERSSRYPLTVDFEAFGWD-COOH (SEQ ID NO: 12) was added to the beads and incubated for an additional 30 minutes at 4°C. Supernatants were collected as described above. Third, the beads were treated with the 'SDS elution' technique, where 30 μl of 4×LDS buffer (Invitrogen) and 100 μl of PBS were added to the beads and heated at 80°C for 10 minutes before transferring the supernatant to a new tube .

A silver-stained gel of the proteins released in each elution step is shown in Figure 1. The two protein bands at approximately 12 and 36 kDa in the silver-stained gel shown in Figure 1 were specifically eluted from JA16-bound beads, but not from non-bound control beads of.

Example 3: Mass spectrometry

Samples were reduced with NuPage 10X Reducing Agent (Invitrogen) for 10 minutes at 80°C and alkylated with 110 μΜ iodoacetamide for 30 minutes at 22°C in the dark. Samples were immediately run on a 10% NuPage Bis-Tris gel according to the manufacturer's recommendation (Invitrogen) in the MES buffer system and silver stained using a glutaraldehyde-free system (Shevchenko et al., (1996) Anal.Chem. , 68: 850-858). Bands were excised and digested with sequencing-grade modified trypsin (Promega, Madison WI) in Abimed DigestPro (Langenfeld, Germany) or ProGest Investigator (Genomics Solutions, Ann Arbor MI). Digested samples were reduced in volume by evaporation and made up to a final volume of -20 μl with 1% acetic acid. Samples (5-10 μl) were loaded onto a 10 cm x 75 μm id C18 reverse phase column packed in Picofrit needles (New Objectives, Woburn MA). MS/MS data were collected using a LCQ Deca or LCQ Deca XP (Finnigan, San Jose CA) mass spectrometer and searches were performed against NCBI's non-redundant database using the Sequest program (Finnigan). Unless otherwise stated, all peptide sequences listed herein correspond to MS/MS spectra that were considered to be of high quality by manual inspection and yielded an X _corr score > 2.5 in the Sequest scoring system.

Example 4: Western Blot

Proteins were transferred onto 0.45 μm nitrocellulose membranes (Invitrogen) and blocked overnight at 4°C with blocking buffer (5% nonfat dry milk in Tris-buffered saline (TBS: 10 mM Tris-Cl, pH 7.5, 150 mM NaCl) . The blot was then probed with a 1:1000 dilution of primary antibody in blocking buffer for 1–3 hr at room temperature, washed with 5×TBS, and horseradish peroxidase-conjugated secondary antibody in blocking buffer at room temperature. Probe down for 1-3 hours and wash as above. Signals were detected by autoradiography using West Pico Substrate (Pierce).

Example 5: Isolation of GDF-8

Experiments using the methods described in the above examples resulted in the isolation of GDF-8. Since the reduced form of GDF-8 is 12 kDa, we speculated that the protein in the lower band on the silver-stained gel shown in Figure 1 is mature GDF-8. To confirm this hypothesis, we excised this band, digested it with trypsin, and obtained the MS/MS spectrum of the resulting peptide by LC-MS/MS. MS/MS spectra corresponding to the six tryptic peptides confirmed that mature GDF-8 was separated from this region of the gel, as shown in Figure 2A and Table 1.

Table 1 lists GDF-8 (SEQ ID NO: 13-20), GDF-8 propeptide (SEQ ID NO: 21-27), FLRG ( SEQ ID NOs: 28-30), and peptides of GASP1 (SEQ ID NOs: 31-35). The amino acids immediately preceding each peptide in the protein sequence are shown in parentheses, and the charge state (z) of the peptide and the correlation coefficient (X _corr , confidence measure) of the Sequest program are listed. The Sequence Listing numbers in the tables refer only to the isolated peptides and their sequences. The preceding amino acids stated in parentheses are not included in the peptide and are provided for reference only. All spectra are verified by manual inspection.

Interestingly, the western blot also contained a band corresponding to unprocessed full-length GDF-8 (43 kDa), implying that some part of this molecule was not proteolytically processed and secreted into serum (Fig. 2B). The presence of unprocessed GDF-8 was confirmed by mass spectrometry (data not shown). Therefore, the affinity purification method can effectively isolate GDF-8 from normal mouse serum.

Although the JA16 antibody recognizes GDF-8 and the highly related protein BMP/GDF-11, we noticed the absence of the BMP-11 peptide in our affinity purified samples by mass spectrometry.

Table 1: Peptides identified in JA16 immunoprecipitation mouse serum z X _corr GDF-8 (mature) (K) ANYCSGECEFVFLQK (SEQ ID NO: 13) 3+ 4.63 (K) MSPINMLYFNGK (SEQ ID NO: 14) 2+ 3.81 (R)DFGLDCDEHSTESR (SEQ ID NO: 15) 2+ 3.47 (K) ANYCSGECEFVFLQK (SEQ ID NO: 16) 2+ 3.31 (K)M ^* SPINMLYFNGK (SEQ ID NO: 17) 3+ 2.95 (R) YPLTVDFEAFGWDWIIAPK (SEQ ID NO: 18) 2+ 2.86 (K)M ^* SPINM ^* LYFNGK (SEQ ID NO: 19) 2+ 2.51 (R)GSAGPCCTPTK (SEQ ID NO: 20) 2+ 2.43 GDF-8 (propeptide) (K)LDM ^* SPGTGIWQSIDVK (SEQ ID NO: 21) 2+ 3.82 (K) ALDENGHDLAVTFPGPGEDGLNPFLEVK (SEQ ID NO: 22) 3+ 3.17 (K) LDMSPGTGIWQSIDVK (SEQ ID NO: 23) 2+ 2.98 (R)ELIDQYDVQR (SEQ ID NO: 24) 2+ 2.97 (K)TPTTVFVQILR (SEQ ID NO: 25) 2+ 2.91 (K) AQLWIYLRPVK (SEQ ID NO: 26) 2+ 2.77 (K)EGLCNACAWR (SEQ ID NO: 27) 2+ 2.75 Follistatin-like-related gene (FLRG) (R)PQSCLVDQTGSAHCVVCR (SEQ ID NO: 28) 3+ 3.34 (K) DSCDGVECGPGK (SEQ ID NO: 29) 2+ 2.99 (K) SCAQVVCPR (SEQ ID NO: 30) 2+ 2.59 A novel multidomain protease inhibitor (GASP1) (R)ECETDQECETYEK (SEQ ID NO: 31) 2+ 2.98 (R)ADFPLSVVR (SEQ ID NO: 32) 2+ 2.56 (R) EACEESCPFPR (SEQ ID NO: 33) 2+ 2.95 (R)SDFVILGR (SEQ ID NO: 34) 2+ 2.73 (R) VSELTEEQDSGR (SEQ ID NO: 35) 2+ 3.88

M ^* = oxidized methionine

Human Serum z X _corr GDF-8 (mature) (K) ANYCSGECEFVFLQK (SEQ ID NO: 36) 2+ 4.21 (R)DFGLDCDEHSTESR (SEQ ID NO: 37) 3+ 2.08 GDF-8 (propeptide) (K) ALDENGHDLAVTFPGPGEDGLNPFLEVK (SEQ ID NO: 38) 3+ 3.71 (R)ELIDQYDVQR (SEQ ID NO: 39) 2+ 3.01 Follistatin-like-related gene (FLRG) (R)PQSCVVDQTGSAHCVVCR (SEQ ID NO: 40) 3+ 3.37 (R) CECAPDCSGLPAR (SEQ ID NO: 41) 2+ 3.21 (R)LQVCGSDGATYR (SEQ ID NO: 42) 2+ 3.06 Multi-domain Protease Inhibitor (GASP1) (R) VSELTEEPDSGR (SEQ ID NO: 43) 2+ 2.44 (R)CYMDAEACSK (SEQ ID NO: 44) 2+ 2.69 (K)GITLAVVTCR (SEQ ID NO: 45) 2+ 2.42

Example 6: Isolation of proteins bound to GDF-8

Once the affinity purification technique was demonstrated to successfully isolate GDF-8 from normal mouse serum, we identified proteins that bind GDF-8 under native conditions. The 36 kDa band on the silver-stained gel shown in Figure 1 was analyzed as described above. Mass spectrometry identified 2 proteins in this region of the gel specific for JA16 immunopurified samples. These identified were the GDF-8 propeptide and the follistatin-like related gene (FLRG). Peptides identified from each of these proteins are shown in Table 1 (SEQ ID NOs: 13-27). High quality MS/MS spectra of 6 unique peptides found from GDF-8 propeptide, 3 unique peptides found from FLRG, representative peptides are shown in Figures 3A and 3C. Furthermore, the presence of these two proteins was confirmed by western blotting using polyclonal antibodies specific for GDF-8 propeptide and FLRG, respectively (Fig. 3B and 3D). Thus, circulating GDF-8 appears to bind GDF-8 propeptide and FLRG in vivo.

Example 7: Isolation of Novel Proteins Binding to GDF-8

To characterize the major components of the circulating GDF-8 complex in vivo, native GDF-8 and its associated proteins were isolated from wild-type mouse serum by affinity purification with agarose-conjugated anti-GDF-8 monoclonal antibody JA16 . Proteins bound to JA16 were then subjected to an elution step of PBS buffer alone (mock elution), peptides that compete with GDF-8 for binding to JA16, and SDS detergent. These samples were concentrated, run on a one-dimensional SDS-PAGE gel, and visualized by silver staining (Figure 4). Two bands unique to the JA16 purified sample were visible - a 12 kDa band identified as GDF-8 and a 36 kDa band containing the GDF-8 propeptide and FLRG.

To determine whether additional proteins that bind GDF-8 could be identified in vivo, we expanded the purification approximately 5-fold and used mass spectrometry to search for proteins present in the JA16 immune complex, but not in the negative control. To achieve this purpose, we excised the region corresponding to the molecular weight of 10-200kDa on the silver-stained gel to form 13 gel slices, as shown in Figure 4. Each of these sections was subjected to trypsinization in the gel and subjected to LC-MS/MS. Comparison of the resulting MS/MS spectra with the non-redundant NCBI database of known proteins did not reveal any additional proteins specific for JA16 immunoprecipitation, although previously described proteins (mature GDF-8, GDF-8 propeptide , unprocessed GDF-8 and FLRG) were all identified in these samples (Figure 4). Background proteins present in both the JA16 immune complex and the negative control samples included a number of serum proteins such as albumin, immunoglobulins and complement proteins. There was no evidence for the presence of other members of the TGF-β superfamily, including the highly related protein BMP-11/GDF-11, in the JA16 sample. Thus, the JA16 antibody specifically purified GDF-8 in these experiments.

Interestingly, we found that follistatin was absent in our GDF-8 immune complexes, although JA16 was able to immunoprecipitate GDF-8/follistatin complexes in vitro (data not shown). Follistatin has been shown to inhibit GDF-8 activity by antagonizing the association of GDF-8 with the ActRIIB receptor (Lee and McPherron (2001) Proc. Natl. Acad. Sci. U.S.A., 98:9306-9311). Our results suggest that follistatin does not play a major role in the regulation of the activity of the circulating GDF-8 complex under normal conditions.

Since the identification of proteins by MS/MS methods depends on the content of the databases searched, we further analyzed the MS/MS spectra collected from 13 samples with the predicted protein database from the Celera mouse genome sequence. The data. These analyzes identified an additional protein specific to JA16-purified samples, and was thus named GDF-associated serum protein 1 (GASP1). Since the initial identification of these proteins, these sequences have been added to NCBI's nr database through public genome sequencing efforts under accession number gi|20914039.

Five peptides corresponding to the GASP1 sequence were identified based on high quality MS/MS spectra (Table 1 (SEQ ID NO: 31-35); Figures 5A and B). A spectrum corresponding to the GASP1 peptide was found in band 3, which contained a protein of 70-80 kDa. However, a specific band corresponding to this protein was not visible, presumably due to the large amount of background immunoglobulin and albumin in this region (see Figure 4). A Sequest X _corr score above 2.3 is generally considered significant for 2+ ions. Fortuitously, one of the peptides identified in our experiments (sequence = ECETDQECETYEK (SEQ ID NO: 31)) spanned the junction between the 2 exons encoding the protein, in this case confirming the Celera gene prediction algorithm the accuracy.

The sequences of the GASP1 transcript and protein were predicted prior to the actual cloning of GASP1 ( FIG. 6 ). GASP1 is predicted to be a 571 amino acid protein with a predicted molecular weight of 63 kDa. It has a putative signal sequence/cleavage site at its N-terminus and two possible N-glycosylation sites at amino acids 314 and 514. Analysis of the GASP1 protein sequence by Pfam and BLAST (according to Altschul et al. (1990) J. Mol. Biol., 215: 403-410; Bateman et al. (2002) Nucleic Acids Res., 30: 276-280) showed that, GASP1 contains many conserved domains including WAP domain, follistatin/Kazal domain, immunoglobulin domain, 2 tandem Kunitz domains and netrin domain (Fig. 14A). The WAP domain, originally identified in whey acidic protein, contains 8 cysteines forming a core of 4 disulfide bonds and is frequently found in proteins with anti-protease activity (Hennighausen and Sippel (1982) Nucleic Acids Res., 10:2677-2684; Seemuller et al. (1986) FEBS Lett., 199:43-48). The follistatin domain is believed to mediate the interaction between GDF-8 and GASP1. The C-terminus of the follistatin domain contains similarity to the Kazal serine protease inhibitor domain. For GASP1, this region is more closely associated with the Kazal domain than in follistatin or FLRG, suggesting that this region may have additional protease inhibitor function. The Kunitz domain, originally identified in bovine pancreatic trypsin inhibitor, also inhibits serine proteases, thus identifying a possible role for GASP1 in the regulation of this class of proteins. Furthermore, netrin domains have been implicated in the inhibition of metalloproteases (Banyai and Patthy, 1999; Mott et al., 2000). Thus, based on the presence of these conserved regions, GASP1 may inhibit protease activity, perhaps regulate GDF-8 processing or activation of the underlying GDF-8 complex.

A BLAST search against the mouse Celera transcript database revealed a protein with >50% identity to GASP1, referred to herein as GASP2. GASP2 contains the same domain structure as GASP1, suggesting that these proteins define a 2-member family of multivalent protease inhibitors (Fig. 14B). Interestingly, only peptides corresponding to GASP1 but not GASP2 were found in our JA16 purified samples. This result suggests that GASP1 and GASP2 may have different biological specificities. Both GASP1 and GASP2 are conserved in humans (&gt;90% identity to mouse). The sequence of human GASP1 is now available under accession number gi|18652308 in the nr database of NCBI. Although the concentration of GDF-8 in human serum is quite low compared to that found in mouse serum (Hill et al. (2002) J. Biol. Chem., 277: 40735-40741), protein mass spectrometry analysis Sensitivity of <RTI ID=0.0>A</RTI> allowed us to identify 3 peptides corresponding to homologues of human GASP1 from human serum JA16 immunoprecipitated (Table 1). These peptides were not found in the corresponding negative controls. Furthermore, there was no evidence of the presence of human GASP2 in these experiments. Thus, the interaction between GASP1 and GDF-8 is conserved between mice and humans.

GDF-8 is produced almost exclusively in skeletal muscle. To determine the tissue distribution of GASP1 mRNA, a 551 bp fragment of GASP1 was amplified from first-strand cDNA generated from various mouse tissues and staged embryos (Figure 10). From the normalized mouse first-strand cDNA panel (Clontech, Palo Alto CA), the Advantage cDNA PCR Kit (Clontech) was used to amplify the mouse GASP1 fragment according to the manufacturer's recommendation (forward primer: 5'TTGGCCACTGCCACCACAATCTCAACCACTT 3' (SEQ ID NO: 46); reverse primer: 5'TCTCAGCATGGCCATGCCGCCGTCGA 3' (SEQ ID NO: 47)). GASP1 appears to be fairly broadly expressed, with particularly high expression in skeletal muscle and heart. Significant expression was also seen in brain, lung and testis. In contrast, liver and kidney expressed GASP1 mRNA at relatively low levels. During development, GASP1 mRNA levels remained fairly constant, perhaps with a slight increase between days 7 and 11 of mouse embryonic development.

Example 8: GDF-8 in human and mouse serum

The concentration of GDF-8 in human serum is considerably lower than that found in mouse serum. Since GDF-8 has potential as a therapeutic target, the aim was to determine the composition of the circulating GDF-8 complex in humans. This knowledge will determine the validity of the mouse model and potentially identify alternative therapeutic targets. Therefore, the JA16-based affinity purification of GDF-8 was repeated using human serum. Due to the lower level of GDF-8 in human serum compared to mice, no bands corresponding to mature GDF-8 and GDF-8 propeptide/FLRG were seen (FIG. 11A). However, western blotting using a polyclonal antibody recognizing the mature region of GDF-8 revealed the presence of both mature and unprocessed GDF-8 in JA16-purified samples (Fig. 1 IB).

We took advantage of the high sensitivity of mass spectrometry to identify proteins that co-purify with mature GDF8. Lanes corresponding to peptides eluted from negative control and JA16-bound bead samples were cut into 16 fragments. These gel sections were subjected to in-gel trypsin digestion, nanoflow LC-MS/MS and analysis with Sequest as previously described.

Interestingly, the only proteins specifically identified only in the JA16 samples but not in the negative control were mature GDF-8, GDF-propeptide, human FLRG and the human GASP1 homologue. The peptides found from each of these proteins are listed in Table 1 (SEQ ID NOs: 36-45) and their representative MS/MS spectra are shown in Figure 12. It thus appears that the GDF-8 complex in vivo is conserved between mice and humans.

Example 9: Cloning and Characterization of Mouse GASP1

After identifying the predicted GASP1 sequence, the goal was to determine the actual sequence of mouse GASP1. According to the sequence predicted by Celera, the GASP1 coding sequence was amplified from QUICKCLONE cDNA (Clontech) of mouse heart by PCR using PfuTurbo polymerase (Stratagene) with the following primers (fp: 5'CACCATGTGTGCCCCAGGGTATCATCGGTTCTGG3, (SEQ ID NO: 50); rp: 5'TTGCAAGCCCAGGAAGTCCTTGAGGAC3' (SEQ ID NO: 51)). The PCR product of this reaction was run on a 1% agarose gel as a single major band consisting of approximately 1700 base pairs. The amplified DNA was then cloned into the TOPO site of the pcDNA3.1D/V5-His-TOPO vector (Invitrogen) to contain the C-terminal V5-His tag in frame according to the manufacturer's recommendations. Sequencing of the full-length cDNA insert was performed on both strands. The nucleotide sequence of the mouse GASP1 clone is shown in FIG. 13 . Except for some base substitutions in the wobble codon that do not change the predicted amino acid sequence (i.e., 288C:G; 294G:A; 615G:A; 738A:G; 768C:T; 1407A:G; 1419A: G; and 1584C:G, where the first base at the indicated position was reported by Celera and the second base was obtained by sequencing the clone; see Figures 6A and B), corresponding to the predicted Celera sequence match.

To determine the N-terminal processing of the GASP1 protein, we transfected COS1 cells with a mammalian expression vector encoding mouse GASP1 cloned with a C-terminal V5-His (GASP1-VS-His) tag. Serum-free conditioned medium was harvested after 48 hours and detected by western blot analysis using anti-V5 polyclonal antibody (Sigma). More specifically, COS1 cells were transfected with GASP1-V5-His/pcDNA3.1D-V5-His-TOPO or empty vector using FuGENE6 reagent (Roche) in serum-free Dulbecco's modified Eagle's medium Conditioned medium was collected after 48 hours.

A single band electrophoresed at approximately 80 kDa was seen, confirming that GASP1 was secreted into the conditioned medium (data not shown). About 10 ml of this conditioned medium was passed through a His-affinity column and further purified by reverse phase chromatography. This purification scheme produced a band of the expected size for full-length GASP1 on a Coomassie-stained SDS-PAGE gel. The Edman sequencing of this band determines that the N-terminal sequence is L-P-P-I-R-Y-S-H-A-G-I (SEQ ID NO: 52). Thus, amino acids 1-29 of GASP1 constitute a signal sequence that is removed during processing and secretion.

Example 10: Binding of recombinantly produced GASP1 to GDF-8 propeptide and mature GDF-8

Next, it was determined that recombinantly produced GASP1 had the same pattern of binding GDF-8 as GASP1 isolated from mouse serum. For immunoprecipitation of recombinant proteins, 400 μl of conditioned medium from vector or GASP1 transfected cells was mixed with 1.2 μg of recombinant purified GDF-8 and/or GDF-8 propeptide protein (Thies et al., 2001). JA16 (10 μl packaging volume) or anti-V5 (30 μl)-conjugated agarose beads were incubated with supplemented conditioned medium for 2 hours at 4°C and washed twice in cold phosphate-buffered saline (PBS) containing 1% Triton. times, and washed twice in PBS. Resuspend the beads in 50 μl of 1×LDS buffer containing DTT. Western blots were performed as previously described (Hill et al., 2002).

To confirm and further characterize the interaction between GDF-8 and GASP1, we compared purified recombinant GDF-8 and purified recombinant GDF-8 propeptide with COS1 cells transfected with control vector or GASP1-V5-His. Conditioned medium incubation. We then immunoprecipitated GDF-8 with JA16-conjugated agarose beads and used western blotting to look for co-purification of GASP1 and GDF-8 propeptide (Fig. 15A). Co-immunoprecipitation of GASP1 (lane 3) and GDF-8 propeptide (lane 1 ) with GDF-8, demonstrating that GDF-8 can interact with both proteins. In the JA16 immunoprecipitation, neither GASP1 nor the propeptide was detected in the absence of GDF-8 (lane 4), eliminating the possibility of non-specific binding in these experiments. Both GASP1 and GDF-8 propeptide were eluted with GDF-8 when all three proteins were present, suggesting the possibility that these proteins could form a ternary complex (lane 5). However, this experiment does not eliminate the possibility that GASP1 and the propeptide bind to the same epitope on the isolated GDF-8 molecule.

To further confirm the interaction between GASP1 and GDF-8, we performed reverse immunoprecipitation by eluting GASP1 from conditioned medium supplemented with recombinant protein of GDF-8 and/or GDF-8 propeptide. To achieve this, we used an agarose-conjugated monoclonal antibody targeting the C-terminal V5-His-tagged V5 epitope on GASP1. As expected, GDF-8 co-immunoprecipitated with GASP1 (Figure 15B, lanes 3 and 5), further confirming the existence of a direct interaction between these proteins. Surprisingly, GDF-8 propeptide co-purifies with GASP1 even in the absence of GDF-8 (lane 4), suggesting that GDF-8 propeptide can directly bind to GASP1. Thus, GASP1 independently binds both GDF-8 and the GDF-8 propeptide. This is in contrast to FLRG, another follistatin-domain protein that specifically binds mature GDF-8 (Hill et al. (2002) J. Biol. Chem., 277:40735-40741). Simultaneous addition of GDF-8 and propeptide consistently showed less propeptide binding to GASP1 than when propeptide was added alone. This observation suggests that GASP1 may not bind to a small latent complex of GDF-8.

Example 11: GASP1-mediated inhibition of GDF-8 and BMP-11 activity, but not activin or TGF-β1 activity

The luciferase reporter construct, pGL3-(CAGA) ₁₂ (SEQ ID NO:53) (Dennler et al. (1998) EMBO J., 17:3091-3100), was transiently transfected into A204 or RD rhabdomyosarcoma cells. Dilutions of conditioned medium from vector or GASP1 transfected cells were mixed with 10 ng/ml GDF-8, 10 ng/ml BMP-11, 10 ng/ml rh Activin A (R&D Systems) or 0.5 ng/ml rh TGF- β1 (R&D Systems) was incubated at 37°C for 30 minutes. Luciferase activity was measured according to Thies et al. (2001) Growth Factors, 18: 251-259 and Zimmers et al. (2002) Science, 296: 1486-1488. In this assay, A204 cells responded to GDF-8, BMP-11 and activin, but not TGF-β1 well. RD cells respond to GDF-8 and TGF-β1. Therefore, we tested the ability of GASP1 to inhibit GDF-8, BMP-11 and activin using A204 cells, and monitored the activities of TGF-β and GDF-8 using RD cells. Results for GDF-8 were shown in A204 cells but were similar to those in RD cells. Concentration-dependent standard curves measuring luciferase activity induced by each of these growth factors were generated separately for each experiment (data not shown). The concentrations of growth factors used fell within the linear region of this curve such that small changes in concentration resulted in measurable changes in luciferase activity.

Two follistatin-domain proteins, follistatin and FLRG, inhibit GDF-8 _activity but also the associated Biological activity of the proteins activin and BMP-11. The ability of GASP1 to inhibit the activity of GDF-8, BMP-11, activin and TGF-β1 was also tested in the (CAGA) ₁₂ (SEQ ID NO:53) reporter assay.

Multiple dilutions of conditioned media from COS cells transfected with V5-His-tagged GASP1 or control vectors were mixed with purified recombinant GDF-8 (10 ng/ml), BMP-11 (10 ng/ml), activin (10 ng/ml) or TGF-β1 (0.5 ng/ml) and assayed for growth factor activity in rhabdomyosarcoma cells expressing the (CAGA) ₁₂ (SEQ ID NO: 53) reporter construct. GASP1 potently inhibited the activity of GDF-8 in a concentration-dependent manner (Fig. 16A). As expected, GASP1 also inhibited the activity of BMP-11 in this assay (Fig. 16B), since mature GDF-8 and BMP-11 are highly conserved and differ by only 11 amino acids. Surprisingly, GASP1 did not inhibit the activity of activin or TGF-β1 (Figure 16C and D), indicating an extremely high level of specificity that could not be demonstrated by follistatin itself. Thus, GASP1 shows specificity in its inhibition of GDF-8 and BMP-11.

The affinity of GASP1 for GDF-8 was assessed by determining the IC50 for inhibition of GDF-8 in a reporter gene assay. GASP1-V5-His protein was purified from conditioned medium on a cobalt affinity column and eluted as described above. Fractions containing GASP1 were further purified by size exclusion chromatography in PBS using a BioSepS3000 column (Phenomenex). As shown in Figure 17, GASP1 inhibits GDF-8 with an IC50 of approximately 3 nM.

Example 12: Treatment of Muscle Disorders

GASP1 can be administered according to Table 2 to a patient suffering from a disease or condition related to GDF-8 function. Patients ingest the composition at certain times or intervals, such as once a day, and the symptoms of their disease or disorder improve. For example, symptoms associated with a muscular disorder are improved, as shown by measurements of muscle mass, muscle activity, and/or muscle tone. It can be seen that the composition of the present invention can be used to treat diseases or disorders related to GDF-8 function, such as muscle disorders.

Table 2: Administration of GASP1 patient disease route of administration dose Dose frequency expected result 1 muscular dystrophy Subcutaneous 25mg once a day Increased muscle mass and improved muscle activity 2 " " 50mg " " 3 " " 50mg once a week " 4 " " 50mg once a month " 5 " intramuscular 25mg once a day " 6 " 50mg " 7 " " 50mg once a week " 8 " " 50mg once a month " 9 " Intravenous 25mg once a day " 10 " 50mg " " 11 " " 50mg once a week " 12 " 50mg once a month " 13 diabetes Subcutaneous 50mg once a day Improve regulation of blood sugar levels 14 " " 50mg once a week " 15 " intramuscular 50mg " " 16 " Intravenous 50mg " " 17 obesity Subcutaneous 50mg once a day Lose weight and gain muscle mass 18 " intramuscular 50mg once a week " 19 Intravenous 50mg "

The entire contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference. The foregoing detailed description of the invention has been given for the purpose of illustration only. Various changes and modifications can be made to the embodiments described above. It is therefore to be understood that the following claims, including all equivalents, are intended to define the scope of this invention.

Sequence Listing

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<213> mouse

<400>1

atgtgtgccc cagggtatca tcggttctgg tttcactggg ggctgctgtt gctgctgctc 60

ctcgaggctc cccttcgagg cctagcactg ccacccatcc gatactccca tgcgggcatc 120

tgccccaacg acatgaaccc caacctctgg gtggatgccc agagcacctg caagcgagag 180

tgtgaaacag accaggaatg tgagacctat gagaaatgct gccccaatgt gtgtgggacc 240

aagagctgtg tggcagcccg ctacatggat gtgaaaggga agaagggccc tgtgggcatg 300

cccaaggagg ccacatgtga ccatttcatg tgcctgcagc agggctctga gtgtgacatc 360

tgggacggcc agcccgtgtg taagtgcaaa gatcgctgtg agaaggagcc cagcttcacc 420

tgtgcctctg atggccttac ctactacaac cgttgcttca tggacgccga agcctgctcc 480

aagggcatca cactgtctgt ggtcacctgt cgttatcact tcacctggcc taacaccagc 540

cctccaccgc ctgagaccac ggtgcatccc accaccgcct ctccggagac tctcgggctg 600

gacatggcag ccccggccct gctcaaccac cctgtccatc agtcagtcac cgtgggtgag 660

actgtgagtt tcctctgtga cgtggtaggc cggcctcggc cagagctcac ttgggagaaa 720

cagctggagg accgagaaaa tgttgtcatg aggcccaacc acgtgcgcgg taatgtggtg 780

gtcactaaca ttgcccagct ggtcatctac aacgtccagc cccaggatgc tggcatatac 840

acctgtacag ctcgaaatgt cgctggtgtc ctgagggctg acttcccgtt gtcggtggtc 900

aggggtggtc aggccagggc cacttcagag agcagtctca atggcacagc ttttccagca 960

acagagtgcc tgaagccccc agacagtgag gactgtggag aggagcagac acgctggcac 1020

ttcgacgccc aggctaacaa ctgcctcact ttcacctttg gccactgcca ccacaatctc 1080

aaccactttg agacctacga ggcctgtatg ctggcttgta tgagtgggcc attggccacc 1140

tgcagcctgc ctgccctgca agggccttgc aaagcttatg tcccacgctg ggcctacaac 1200

agccagacag gcctatgcca gtccttcgtc tatggcggct gtgagggcaa cggtaacaac 1260

tttgaaagcc gtgaggcttg tgaggagtcg tgtcccttcc cgaggggtaa ccagcactgc 1320

cgggcctgca agccccggca aaaacttgtt accagcttct gtcggagtga ctttgtcatc 1380

ctgggcaggg tctctgagct gaccgaagag caagactcag gccgtgccct ggtgaccgtg 1440

gatgaggtct taaaagatga gaagatgggc ctcaagtttc tgggccggga gcctctggaa 1500

gtcaccctgc ttcatgtaga ctggacctgt ccttgcccca acgtgacagt gggtgagaca 1560

ccactcatca tcatggggga ggtcgacggc ggcatggcca tgctgagacc cgatagcttt 1620

gtgggggcat cgagcacacg gcgggtcagg aagctccgtg aggtcatgta caagaaaacc 1680

tgtgacgtcc tcaaggactt cctgggcttg caatga 1716

<210>2

<211>1716

<212>DNA

<213> mouse

<400>2

atgtgtgccc cagggtatca tcggttctgg tttcactggg ggctgctgtt gctgctgctc 60

ctcgaggctc cccttcgagg cctagcactg ccacccatcc gatactccca tgcgggcatc 120

tgccccaacg acatgaaccc caacctctgg gtggatgccc agagcacctg caagcgagag 180

tgtgaaacag accaggaatg tgagacctat gagaaatgct gccccaatgt gtgtgggacc 240

aagagctgtg tggcagcccg ctacatggat gtgaaaggga agaaggggcc tgtaggcatg 300

cccaaggagg ccacatgtga ccatttcatg tgcctgcagc agggctctga gtgtgacatc 360

tgggacggcc agcccgtgtg taagtgcaaa gatcgctgtg agaaggagcc cagcttcacc 420

tgtgcctctg atggccttac ctactacaac cgttgcttca tggacgccga agcctgctcc 480

aagggcatca cactgtctgt ggtcacctgt cgttatcact tcacctggcc taacaccagc 540

cctccaccgc ctgagaccac ggtgcatccc accaccgcct ctccggagac tctcgggctg 600

gacatggcag ccccagccct gctcaaccac cctgtccatc agtcagtcac cgtgggtgag 660

actgtgagtt tcctctgtga cgtggtaggc cggcctcggc cagagctcac ttgggagaaa 720

cagctggagg accgagagaa tgttgtcatg aggcccaacc acgtgcgtgg taatgtggtg 780

gtcactaaca ttgcccagct ggtcatctac aacgtccagc cccaggatgc tggcatatac 840

acctgtacag ctcgaaatgt cgctggtgtc ctgagggctg acttcccgtt gtcggtggtc 900

aggggtggtc aggccagggc cacttcagag agcagtctca atggcacagc ttttccagca 960

acagagtgcc tgaagccccc agacagtgag gactgtggag aggagcagac acgctggcac 1020

ttcgacgccc aggctaacaa ctgcctcact ttcacctttg gccactgcca ccacaatctc 1080

aaccactttg agacctacga ggcctgtatg ctggcttgta tgagtgggcc attggccacc 1140

tgcagcctgc ctgccctgca agggccttgc aaagcttatg tcccacgctg ggcctacaac 1200

agccagacag gcctatgcca gtccttcgtc tatggcggct gtgagggcaa cggtaacaac 1260

tttgaaagcc gtgaggcttg tgaggagtcg tgtcccttcc cgaggggtaa ccagcactgc 1320

cgggcctgca agccccggca aaaacttgtt accagcttct gtcggagtga ctttgtcatc 1380

ctgggcaggg tctctgagct gaccgaggag caagactcgg gccgtgccct ggtgaccgtg 1440

gatgaggtct taaaagatga gaagatgggc ctcaagtttc tgggccggga gcctctggaa 1500

gtcaccctgc ttcatgtaga ctggacctgt ccttgcccca acgtgacagt gggtgagaca 1560

ccactcatca tcatggggga ggtggacggc ggcatggcca tgctgagacc cgatagcttt 1620

gtgggggcat cgagcacacg gcgggtcagg aagctccgtg aggtcatgta caagaaaacc 1680

tgtgacgtcc tcaaggactt cctgggcttg caatga 1716

<210>3

<211>571

<212>PRT

<213> mice

<400>3

Met Cys Ala Pro Gly Tyr His Arg Phe Trp Phe His Trp Gly Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Glu Ala Pro Leu Arg Gly Leu Ala Leu Pro Pro

20 25 30

Ile Arg Tyr Ser His Ala Gly Ile Cys Pro Asn Asp Met Asn Pro Asn

35 40 45

Leu Trp Val Asp Ala Gln Ser Thr Cys Lys Arg Glu Cys Glu Thr Asp

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Gly Met Pro Lys Glu Ala Thr Cys Asp His Phe Met Cys Leu

100 105 110

Gln Gln Gly Ser Glu Cys Asp Ile Trp Asp Gly Gln Pro Val Cys Lys

115 120 125

Cys Lys Asp Arg Cys Glu Lys Glu Pro Ser Phe Thr Cys Ala Ser Asp

130 135 140

Gly Leu Thr Tyr Tyr Asn Arg Cys Phe Met Asp Ala Glu Ala Cys Ser

145 150 155 160

Lys Gly Ile Thr Leu Ser Val Val Thr Cys Arg Tyr His Phe Thr Trp

165 170 175

Pro Asn Thr Ser Pro Pro Pro Pro Glu Thr Thr Val His Pro Thr Thr

180 185 190

Ala Ser Pro Glu Thr Leu Gly Leu Asp Met Ala Ala Pro Ala Leu Leu

195 200 205

Asn His Pro Val His Gln Ser Val Thr Val Gly Glu Thr Val Ser Phe

210 215 220

Leu Cys Asp Val Val Gly Arg Pro Arg Pro Glu Leu Thr Trp Glu Lys

225 230 235 240

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

245 250 255

Gly Asn Val Val Val Thr Asn Ile Ala Gln Leu Val Ile Tyr Asn Val

260 265 270

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

275 280 285

Gly Val Leu Arg Ala Asp Phe Pro Leu Ser Val Val Arg Gly Gly Gln

290 295 300

Ala Arg Ala Thr Ser Glu Ser Ser Ser Leu Asn Gly Thr Ala Phe Pro Ala

305 310 315 320

Thr Glu Cys Leu Lys Pro Pro Asp Ser Glu Asp Cys Gly Glu Glu Gln

325 330 335

Thr Arg Trp His Phe Asp Ala Gln Ala Asn Asn Cys Leu Thr Phe Thr

340 345 350

Phe Gly His Cys His His Asn Leu Asn His Phe Glu Thr Tyr Glu Ala

355 360 365

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

370 375 380

Ala Leu Gln Gly Pro Cys Lys Ala Tyr Val Pro Arg Trp Ala Tyr Asn

385 390 395 400

Ser Gln Thr Gly Leu Cys Gln Ser Phe Val Tyr Gly Gly Cys Glu Gly

405 410 415

Asn Gly Asn Asn Phe Glu Ser Arg Glu Ala Cys Glu Glu Ser Cys Pro

420 425 430

Phe Pro Arg Gly Asn Gln His Cys Arg Ala Cys Lys Pro Arg Gln Lys

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Pro Leu Glu Val Thr Leu Leu His Val Asp Trp Thr Cys Pro Cys

500 505 510

Pro Asn Val Thr Val Gly Glu Thr Pro Leu Ile Ile Met Gly Glu Val

515 520 525

Asp Gly Gly Met Ala Met Leu Arg Pro Asp Ser Phe Val Gly Ala Ser

530 535 540

Ser Thr Arg Arg Val Arg Lys Leu Arg Glu Val Met Tyr Lys Lys Thr

545 550 555 560

Cys Asp Val Leu Lys Asp Phe Leu Gly Leu Gln

565 570

<210>4

<211>1923

<212>DNA

<213> people

<220>

<221> Modified bases

<222>(732)

<223> a, t, c or g

<400>4

atgaatccca acctctgggt ggacgcacag agcacctgca ggcgggagtg tgagacggac 60

caggagtgtg agatggacca ggtgagtggg atccagaagc cacagtgtga ggcagaccag 120

gtgaatgggg tccagaagcc gcaatgtgag atggaccaga agtgggagtg tgaggttgac 180

caggtgagtg gggtccagaa gccggtgtgt gaggcggacc aggtgagtgg ggtccagaag 240

ccacagtgtg agatggacca ggtgagtggg atccagaagc tggagtgtga ggcggaccag 300

aagtgggagt atgaggtgga ccaggtgagt ggggtccaga agccacagtg tgagatggac 360

caggtgagtg ggatccagaa gctggagtgt gaggcggacc aggagtgtga gacctatgag 420

aagtgctgcc ccaacgtatg tgggaccaag agctgcgtgg cggcccgcta catggacgtg 480

aaagggaaga agggcccagt gggcatgccc aaggaggcca catgtgacca cttcatgtgt 540

ctgcagcagg gctctgagtg tgacatctgg gatggccagc ccgtgtgtaa gtgcaaagac 600

cgctgtgaga aggagcccag ctttacctgc gcctcggacg gcctcaccta ctataaccgc 660

tgctacatgg atgccgaggc ctgctccaaa ggcatcacac tggccgttgt aacctgccgc 720

tatcacttca cntggcccaa caccagcccc ccaccacctg agaccaccat gcaccccacc 780

acagcctccc cagagacccc tgagctggac atggcggccc ctgcgctgct caacaaccct 840

gtgcaccagt cggtcaccat gggtgagaca gtgagcttcc tctgtgatgt ggtgggccgg 900

ccccggcctg agatcacctg ggagaagcag ttggaggatc gggagaatgt ggtcatgcgg 960

cccaaccatg tgcgtggcaa cgtggtggtc accaacattg cccagctggt catctataac 1020

gcccagctgc aggatgctgg gatctacacc tgcacggccc ggaacgtggc tggggtcctg 1080

agggctgatt tcccgctgtc ggtggtcagg ggtcatcagg ctgcagccac ctcagagagc 1140

agccccaatg gcacggcttt cccggcggcc gagtgcctga agcccccaga cagtgaggac 1200

tgtggcgaag agcagacccg ctggcacttc gatgcccagg ccaacaactg cctgaccttc 1260

accttcggcc actgccaccg taacctcaac cactttgaga cctatgaggc ctgcatgctg 1320

gcctgcatga gcgggccgct ggccgcgtgc agcctgcccg ccctgcaggg gccctgcaaa 1380

gcctacgcgc ctcgctgggc ttacaacagc cagacgggcc agtgccagtc ctttgtctat 1440

ggtggctgcg agggcaatgg caacaacttt gagagccgtg aggcctgtga ggagtcgtgc 1500

cccttcccca gggggaacca gcgctgtcgg gcctgcaagc ctcggcagaa gctcgttacc 1560

agcttctgtc gcagcgactt tgtcatcctg ggccgagtct ctgagctgac cgaggagcct 1620

gactcgggcc gcgccctggt gactgtggat gaggtcctaa aggatgagaa aatgggcctc 1680

aagttcctgg gccaggagcc attggaggtc actctgcttc acgtggactg ggcatgcccc 1740

tgccccaacg tgaccgtgag cgagatgccg ctcatcatca tgggggaggt ggacggcggc 1800

atggccatgc tgcgccccga tagctttgtg ggcgcatcga gtgcccgccg ggtcaggaag 1860

cttcgtgagg tcatgcacaa gaagacctgt gacgtcctca aggagtttct tggcttgcac 1920

tga 1923

<210>5

<211>640

<212>PRT

<213> people

<400>5

Met Asn Pro Asn Leu Trp Val Asp Ala Gln Ser Thr Cys Arg Arg Glu

1 5 10 15

Cys Glu Thr Asp Gln Glu Cys Glu Met Asp Gln Val Ser Gly Ile Gln

20 25 30

Lys Pro Gln Cys Glu Ala Asp Gln Val Asn Gly Val Gln Lys Pro Gln

35 40 45

Cys Glu Met Asp Gln Lys Trp Glu Cys Glu Val Asp Gln Val Ser Gly

50 55 60

Val Gln Lys Pro Val Cys Glu Ala Asp Gln Val Ser Gly Val Gln Lys

65 70 75 80

Pro Gln Cys Glu Met Asp Gln Val Ser Gly Ile Gln Lys Leu Glu Cys

85 90 95

Glu Ala Asp Gln Lys Trp Glu Tyr Glu Val Asp Gln Val Ser Gly Val

100 105 110

Gln Lys Pro Gln Cys Glu Met Asp Gln Val Ser Gly Ile Gln Lys Leu

115 120 125

Glu Cys Glu Ala Asp Gln Glu Cys Glu Thr Tyr Glu Lys Cys Cys Pro

130 135 140

Asn Val Cys Gly Thr Lys Ser Cys Val Ala Ala Arg Tyr Met Asp Val

145 150 155 160

Lys Gly Lys Lys Gly Pro Val Gly Met Pro Lys Glu Ala Thr Cys Asp

165 170 175

His Phe Met Cys Leu Gln Gln Gly Ser Glu Cys Asp Ile Trp Asp Gly

180 185 190

Gln Pro Val Cys Lys Cys Lys Asp Arg Cys Glu Lys Glu Pro Ser Phe

195 200 205

Thr Cys Ala Ser Asp Gly Leu Thr Tyr Tyr Asn Arg Cys Tyr Met Asp

210 215 220

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

225 230 235 240

Tyr His Phe Thr Trp Pro Asn Thr Ser Pro Pro Pro Pro Glu Thr Thr

245 250 255

Met His Pro Thr Thr Ala Ser Pro Glu Thr Pro Glu Leu Asp Met Ala

260 265 270

Ala Pro Ala Leu Leu Asn Asn Pro Val His Gln Ser Val Thr Met Gly

275 280 285

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

290 295 300

Ile Thr Trp Glu Lys Gln Leu Glu Asp Arg Glu Asn Val Val Met Arg

305 310 315 320

Pro Asn His Val Arg Gly Asn Val Val Val Thr Asn Ile Ala Gln Leu

325 330 335

Val Ile Tyr Asn Ala Gln Leu Gln Asp Ala Gly Ile Tyr Thr Cys Thr

340 345 350

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

355 360 365

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

370 375 380

Thr Ala Phe Pro Ala Ala Glu Cys Leu Lys Pro Pro Asp Ser Glu Asp

385 390 395 400

Cys Gly Glu Glu Gln Thr Arg Trp His Phe Asp Ala Gln Ala Asn Asn

405 410 415

Cys Leu Thr Phe Thr Phe Gly His Cys His Arg Asn Leu Asn His Phe

420 425 430

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

435 440 445

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

450 455 460

Arg Trp Ala Tyr Asn Ser Gln Thr Gly Gln Cys Gln Ser Phe Val Tyr

465 470 475 480

Gly Gly Cys Glu Gly Asn Gly Asn Asn Phe Glu Ser Arg Glu Ala Cys

485 490 495

Glu Glu Ser Cys Pro Phe Pro Arg Gly Asn Gln Arg Cys Arg Ala Cys

500 505 510

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

515 520 525

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

530 535 540

Ala Leu Val Thr Val Asp Glu Val Leu Lys Asp Glu Lys Met Gly Leu

545 550 555 560

Lys Phe Leu Gly Gln Glu Pro Leu Glu Val Thr Leu Leu His Val Asp

565 570 575

Trp Ala Cys Pro Cys Pro Asn Val Thr Val Ser Glu Met Pro Leu Ile

580 585 590

Ile Met Gly Glu Val Asp Gly Gly Met Ala Met Leu Arg Pro Asp Ser

595 600 605

Phe Val Gly Ala Ser Ser Ala Arg Arg Val Arg Lys Leu Arg Glu Val

610 615 620

Met His Lys Lys Thr Cys Asp Val Leu Lys Glu Phe Leu Gly Leu His

625 630 635 640

<210>6

<211>1731

<212>DNA

<213> people

<400>6

atgtgggccc caaggtgtcg ccggttctgg tctcgctggg agcaggtggc agcgctgctg 60

ctgctgctgc tactgctcgg ggtgcccccg cgaagcctgg cgctgccgcc catccgctat 120

tcccacgccg gcatctgccc caacgacatg aatcccaacc tctgggtgga cgcacagagc 180

acctgcaggc gggagtgtga gacggaccag gagtgtgaga cctatgagaa gtgctgcccc 240

aacgtatgtg ggaccaagag ctgcgtggcg gcccgctaca tggacgtgaa agggaagaag 300

ggcccagtgg gcatgcccaa ggaggccaca tgtgaccact tcatgtgtct gcagcagggc 360

tctgagtgtg acatctggga tggccagccc gtgtgtaagt gcaaagaccg ctgtgagaag 420

gagcccagct ttacctgcgc ctcggacggc ctcacctact ataaccgctg ctacatggat 480

gccgaggcct gctccaaagg catcacactg gccgttgtaa cctgccgcta tcacttcacc 540

tggcccaaca ccagcccccc accacctgag accaccatgc accccaccac agcctcccca 600

gagacccctg agctggacat ggcggcccct gcgctgctca acaaccctgt gcaccagtcg 660

gtcaccatgg gtgagacagt gagtttcctc tgtgatgtgg tgggccggcc ccggcctgag 720

atcacctggg agaagcagtt ggaggatcgg gagaatgtgg tcatgcggcc caaccatgtg 780

cgtggcaacg tggtggtcac caacattgcc cagctggtca tctataacgc ccagctgcag 840

gatgctggga tctacacctg cacggcccgg aacgtggctg gggtcctgag ggctgatttc 900

ccgctgtcgg tggtcagggg tcatcaggct gcagccacct cagagagcag ccccaatggc 960

acggctttcc cggcggccga gtgcctgaag cccccagaca gtgaggactg tggcgaagag 1020

cagacccgct ggcacttcga tgcccaggcc aacaactgcc tgaccttcac cttcggccac 1080

tgccaccgta acctcaacca ctttgagacc tatgaggcct gcatgctggc ctgcatgagc 1140

gggccgctgg ccgcgtgcag cctgcccgcc ctgcaggggc cctgcaaagc ctacgcgcct 1200

cgctgggctt acaacagcca gacgggccag tgccagtcct ttgtctatgg tggctgcgag 1260

ggcaatggca acaactttga gagccgtgag gcctgtgagg agtcgtgccc cttccccagg 1320

gggaaccagc gctgtcgggc ctgcaagcct cggcagaagc tcgttaccag cttctgtcgc 1380

agcgactttg tcatcctggg ccgagtctct gagctgaccg aggagcctga ctcgggccgc 1440

gccctggtga ctgtggatga ggtcctaaag gatgagaaaa tgggcctcaa gttcctgggc 1500

caggagccat tggaggtcac tctgcttcac gtggactggg catgcccctg ccccaacgtg 1560

accgtgagcg agatgccgct catcatcatg ggggaggtgg acggcggcat ggccatgctg 1620

cgccccgata gctttgtggg cgcatcgagt gcccgccggg tcaggaagct tcgtgaggtc 1680

atgcacaaga agacctgtga cgtcctcaag gagtttcttg gcttgcactg a 1731

<210>7

<211>576

<212>PRT

<213> people

<400>7

Met Trp Ala Pro Arg Cys Arg Arg Phe Trp Ser Arg Trp Glu Gln Val

1 5 10 15

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

20 25 30

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

35 40 45

Asp Met Asn Pro Asn Leu Trp Val Asp Ala Gln Ser Thr Cys Arg Arg

50 55 60

Glu Cys Glu Thr Asp Gln Glu Cys Glu Thr Tyr Glu Lys Cys Cys Pro

65 70 75 80

Asn Val Cys Gly Thr Lys Ser Cys Val Ala Ala Arg Tyr Met Asp Val

85 90 95

Lys Gly Lys Lys Gly Pro Val Gly Met Pro Lys Glu Ala Thr Cys Asp

100 105 110

His Phe Met Cys Leu Gln Gln Gly Ser Glu Cys Asp Ile Trp Asp Gly

115 120 125

Gln Pro Val Cys Lys Cys Lys Asp Arg Cys Glu Lys Glu Pro Ser Phe

130 135 140

Thr Cys Ala Ser Asp Gly Leu Thr Tyr Tyr Asn Arg Cys Tyr Met Asp

145 150 155 160

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

165 170 175

Tyr His Phe Thr Trp Pro Asn Thr Ser Pro Pro Pro Pro Glu Thr Thr

180 185 190

Met His Pro Thr Thr Ala Ser Pro Glu Thr Pro Glu Leu Asp Met Ala

195 200 205

Ala Pro Ala Leu Leu Asn Asn Pro Val His Gln Ser Val Thr Met Gly

210 215 220

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

225 230 235 240

Ile Thr Trp Glu Lys Gln Leu Glu Asp Arg Glu Asn Val Val Met Arg

245 250 255

Pro Asn His Val Arg Gly Asn Val Val Val Thr Asn Ile Ala Gln Leu

260 265 270

Val Ile Tyr Asn Ala Gln Leu Gln Asp Ala Gly Ile Tyr Thr Cys Thr

275 280 285

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

290 295 300

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

305 310 315 320

Thr Ala Phe Pro Ala Ala Glu Cys Leu Lys Pro Pro Asp Ser Glu Asp

325 330 335

Cys Gly Glu Glu Gln Thr Arg Trp His Phe Asp Ala Gln Ala Asn Asn

340 345 350

Cys Leu Thr Phe Thr Phe Gly His Cys His Arg Asn Leu Asn His Phe

355 360 365

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

370 375 380

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

385 390 395 400

Arg Trp Ala Tyr Asn Ser Gln Thr Gly Gln Cys Gln Ser Phe Val Tyr

405 410 415

Gly Gly Cys Glu Gly Asn Gly Asn Asn Phe Glu Ser Arg Glu Ala Cys

420 425 430

Glu Glu Ser Cys Pro Phe Pro Arg Gly Asn Gln Arg Cys Arg Ala Cys

435 440 445

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

450 455 460

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

465 470 475 480

Ala Leu Val Thr Val Asp Glu Val Leu Lys Asp Glu Lys Met Gly Leu

485 490 495

Lys Phe Leu Gly Gln Glu Pro Leu Glu Val Thr Leu Leu His Val Asp

500 505 510

Trp Ala Cys Pro Cys Pro Asn Val Thr Val Ser Glu Met Pro Leu Ile

515 520 525

Ile Met Gly Glu Val Asp Gly Gly Met Ala Met Leu Arg Pro Asp Ser

530 535 540

Phe Val Gly Ala Ser Ser Ala Arg Arg Val Arg Lys Leu Arg Glu Val

545 550 555 560

Met His Lys Lys Thr Cys Asp Val Leu Lys Glu Phe Leu Gly Leu His

565 570 575

<210>8

<211>1659

<212>DNA

<213> mouse

<400>8

atgcctgccc cacagccatt cctgcctctg ctctttgtct tcgtgctcat ccatctgacc 60

tcggagacca acctgctgcc agatcccgga agccatcctg gcatgtgccc caacgagctc 120

agcccccacc tgtgggtcga cgcccagagc acctgtgagc gtgagtgtac cggggaccag 180

gactgtgcgg catccgagaa gtgctgcacc aatgtgtgtg ggctgcagag ctgcgtggct 240

gcccgctttc ccagtggtgg cccagctgta cctgagacag cagcctcctg tgaaggcttc 300

caatgcccac aacagggttc tgactgtgac atctgggatg ggcagccagt ttgtcgctgc 360

cgtgaccgct gtgaaaaaga accccagcttc acatgtgctt ctgatggcct tacctattac 420

aaccgctgct acatggacgc agaagcctgc ctgcggggtc tccacctgca cgttgtaccc 480

tgtaagcaca ttctcagttg gccgcccagc agcccgggac cacccgagac cactgctcgc 540

ccaacccctg gggctgctcc catgccacct gccctgtaca acagcccctc accacaggca 600

gtgcatgttg gggggacagc cagcctccac tgtgatgtta gtggccgtcc accacctgct 660

gtgacctggg agaagcagag ccatcagcgg gagaacctga tcatgcgccc tgaccaaatg 720

tatggcaacg tggttgtcac cagtatcgga cagctagtcc tctacaatgc tcagttggag 780

gatgcgggcc tgtatacctg cactgcacga aacgctgccg gcctgctgcg ggccgacttt 840

cccctttccg ttttacagcg ggcaactact caggacaggg accccaggtat cccagccttg 900

gctgagtgcc aggccgacac acaagcctgt gttgggccac ctactcccca tcatgtcctt 960

tggcgctttg accccacagag aggcagctgc atgacattcc cagccctcag atgtgatggg 1020

gctgcccggg gctttgagac ctatgaggca tgccagcagg cctgtgttcg tggccccggg 1080

gatgtctgtg cactgcctgc agttcagggg ccctgccagg gctgggagcc acgctgggcc 1140

tacagcccac tgctacagca gtgccacccc tttgtataca gtggctgtga aggaaacagc 1200

aataactttg agacccggga gagctgtgag gatgcttgcc ctgtaccacg cacaccaccc 1260

tgtcgtgcct gccgcctcaa gagcaagctg gctctgagct tgtgccgcag tgactttgcc 1320

atcgtgggga gactcacaga ggtcctggag gagcccgagg ctgcaggcgg catagctcgt 1380

gtggccttgg atgatgtgct aaaggacgac aagatgggcc tcaagttctt gggcaccaaa 1440

tacctggagg tgacattgag tggcatggac tgggcctgcc catgccccaa cgtgacagct 1500

gtcgatgggc cactggtcat catgggtgag gttcgtgaag gtgtggctgt gttggacgcc 1560

aacagctatg tccgtgctgc cagcgagaag cgagtcaaga agattgtgga actgctcgag 1620

aagaaggctt gtgaactgct caaccgcttc caagactag 1659

<210>9

<211>552

<212>PRT

<213> mouse

<400>9

Met Pro Ala Pro Gln Pro Phe Leu Pro Leu Leu Phe Val Phe Val Leu

1 5 10 15

Ile His Leu Thr Ser Glu Thr Asn Leu Leu Pro Asp Pro Gly Ser His

20 25 30

Pro Gly Met Cys Pro Asn Glu Leu Ser Pro His Leu Trp Val Asp Ala

35 40 45

Gln Ser Thr Cys Glu Arg Glu Cys Thr Gly Asp Gln Asp Cys Ala Ala

50 55 60

Ser Glu Lys Cys Cys Thr Asn Val Cys Gly Leu Gln Ser Cys Val Ala

65 70 75 80

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

85 90 95

Cys Glu Gly Phe Gln Cys Pro Gln Gln Gly Ser Asp Cys Asp Ile Trp

100 105 110

Asp Gly Gln Pro Val Cys Arg Cys Arg Asp Arg Cys Glu Lys Glu Pro

115 120 125

Ser Phe Thr Cys Ala Ser Asp Gly Leu Thr Tyr Tyr Asn Arg Cys Tyr

130 135 140

Met Asp Ala Glu Ala Cys Leu Arg Gly Leu His Leu His Val Val Pro

145 150 155 160

Cys Lys His Ile Leu Ser Trp Pro Pro Ser Ser Pro Gly Pro Pro Glu

165 170 175

Thr Thr Ala Arg Pro Thr Pro Gly Ala Ala Pro Met Pro Pro Ala Leu

180 185 190

Tyr Asn Ser Pro Ser Pro Gln Ala Val His Val Gly Gly Thr Ala Ser

195 200 205

Leu His Cys Asp Val Ser Gly Arg Pro Pro Pro Ala Val Thr Trp Glu

210 215 220

Lys Gln Ser His Gln Arg Glu Asn Leu Ile Met Arg Pro Asp Gln Met

225 230 235 240

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

245 250 255

Ala Gln Leu Glu Asp Ala Gly Leu Tyr Thr Cys Thr Ala Arg Asn Ala

260 265 270

Ala Gly Leu Leu Arg Ala Asp Phe Pro Leu Ser Val Leu Gln Arg Ala

275 280 285

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

290 295 300

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

305 310 315 320

Trp Arg Phe Asp Pro Gln Arg Gly Ser Cys Met Thr Phe Pro Ala Leu

325 330 335

Arg Cys Asp Gly Ala Ala Arg Gly Phe Glu Thr Tyr Glu Ala Cys Gln

340 345 350

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

355 360 365

Gln Gly Pro Cys Gln Gly Trp Glu Pro Arg Trp Ala Tyr Ser Pro Leu

370 375 380

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

385 390 395 400

Asn Asn Phe Glu Thr Arg Glu Ser Cys Glu Asp Ala Cys Pro Val Pro

405 410 415

Arg Thr Pro Pro Cys Arg Ala Cys Arg Leu Lys Ser Lys Leu Ala Leu

420 425 430

Ser Leu Cys Arg Ser Asp Phe Ala Ile Val Gly Arg Leu Thr Glu Val

435 440 445

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

450 455 460

Asp Val Leu Lys Asp Asp Lys Met Gly Leu Lys Phe Leu Gly Thr Lys

465 470 475 480

Tyr Leu Glu Val Thr Leu Ser Gly Met Asp Trp Ala Cys Pro Cys Pro

485 490 495

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

500 505 510

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

515 520 525

Glu Lys Arg Val Lys Lys Ile Val Glu Leu Leu Glu Lys Lys Ala Cys

530 535 540

Glu Leu Leu Asn Arg Phe Gln Asp

545 550

<210>10

<211>1695

<212>DNA

<213> people

<400>10

atgcccgccc tacgtccact cctgccgctc ctgctcctcc tccggctgac ctcggggggct 60

ggcttgctgc cagggctggg gagccacccg ggcgtgtgcc ccaaccagct cagccccaac 120

ctgtgggtgg acgcccagag cacctgtgag cgcgagtgta gcagggacca ggactgtgcg 180

gctgctgaga agtgctgcat caacgtgtgt ggactgcaca gctgcgtggc agcacgcttc 240

cccggcagcc cagctgcgcc gacgacagcg gcctcctgcg agggctttgt gtgcccacag 300

cagggctcgg actgcgacat ctgggacggg cagcccgtgt gccgctgccg cgaccgctgt 360

gagaaggagc ccagcttcac ctgcgcctcg gacggcctca cctactacaa ccgctgctat 420

atggacgccg aggcctgcct gcggggcctg cacctccaca tcgtgccctg caagcacgtg 480

ctcagctggc cgcccagcag cccggggccg ccggagacca ctgcccgccc cacacctggg 540

gccgcgcccg tgcctcctgc cctgtacagc agcccctccc cacaggcggt gcaggttggg 600

ggtacggcca gcctccactg cgacgtcagc ggccgcccgc cgcctgctgt gacctgggag 660

aagcagagtc accagcgaga gaacctgatc atgcgccctg atcagatgta tggcaacgtg 720

gtggtcacca gcatcgggca gctggtgctc tacaacgcgc ggcccgaaga cgccggcctg 780

taacacctgca ccgcgcgcaa cgctgctggg ctgctgcggg ctgacttccc actctctgtg 840

gtccagcgag agccggccag ggacgcagcc cccagcatcc cagccccggc cgagtgcctg 900

ccggatgtgc aggcctgcac gggccccact tccccacacc ttgtcctctg gcactacgac 960

ccgcagcggg gcggctgcat gaccttcccg gcccgtggct gtgatggggc ggcccgcggc 1020

tttgagacct acgaggcatg ccagcaggcc tgtgcccgcg gccccggcga cgcctgcgtg 1080

ctgcctgccg tgcagggccc ctgccggggc tgggagccgc gctgggccta cagcccgctg 1140

ctgcagcagt gccatccctt cgtgtacggt ggctgcgagg gcaacggcaa caacttccac 1200

agccgcgaga gctgcgagga tgcctgcccc gtgccgcgca caccgccctg ccgcgcctgc 1260

cgcctccgga gcaagctggc gctgagcctg tgccgcagcg acttcgccat cgtggggcgg 1320

ctcacggagg tgctggagga gcccgaggcc gccggcggca tcgcccgcgt ggcgctcgag 1380

gacgtgctca aggatgacaa gatgggcctc aagttcttgg gcaccaagta cctggaggtg 1440

acgctgagtg gcatggactg ggcctgcccc tgccccaaca tgacggcggg cgacgggccg 1500

ctggtcatca tgggtgaggt gcgcgatggc gtggccgtgc tggacgccgg cagctacgtc 1560

cgcgccgcca gcgagaagcg cgtcaagaag atcttggagc tgctggagaa gcaggcctgc 1620

gagctgctca accgcttcca ggactagccc ccgcaggggc ctgcgccacc ccgtcctggt 1680

gaataaacgc actcc 1695

<210>11

<211>548

<212>PRT

<213> people

<400>11

Met Pro Ala Leu Arg Pro Leu Leu Pro Leu Leu Leu Leu Leu Arg Leu

1 5 10 15

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

20 25 30

Cys Pro Asn Gln Leu Ser Pro Asn Leu Trp Val Asp Ala Gln Ser Thr

35 40 45

Cys Glu Arg Glu Cys Ser Arg Asp Gln Asp Cys Ala Ala Ala Glu Lys

50 55 60

Cys Cys Ile Asn Val Cys Gly Leu His Ser Cys Val Ala Ala Arg Phe

65 70 75 80

Pro Gly Ser Pro Ala Ala Pro Thr Thr Ala Ala Ser Cys Glu Gly Phe

85 90 95

Val Cys Pro Gln Gln Gly Ser Asp Cys Asp Ile Trp Asp Gly Gln Pro

100 105 110

Va1 Cys Arg Cys Arg Asp Arg Cys Glu Lys Glu Pro Ser Phe Thr Cys

115 120 125

Ala Ser Asp Gly Leu Thr Tyr Tyr Asn Arg Cys Tyr Met Asp Ala Glu

130 135 140

Ala Cys Leu Arg Gly Leu His Leu His Ile Val Pro Cys Lys His Val

145 150 155 160

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

165 170 175

Pro Thr Pro Gly Ala Ala Pro Val Pro Pro Ala Leu Tyr Ser Ser Pro

180 185 190

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

195 200 205

Val Ser Gly Arg Pro Pro Pro Ala Val Thr Trp Glu Lys Gln Ser His

210 215 220

Gln Arg Glu Asn Leu Ile Met Arg Pro Asp Gln Met Tyr Gly Asn Val

225 230 235 240

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

245 250 255

Asp Ala Gly Leu Tyr Thr Cys Thr Ala Arg Asn Ala Ala Gly Leu Leu

260 265 270

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

275 280 285

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

290 295 300

Ala Cys Thr Gly Pro Thr Ser Pro His Leu Val Leu Trp His Tyr Asp

305 310 315 320

Pro Gln Arg Gly Gly Cys Met Thr Phe Pro Ala Arg Gly Cys Asp Gly

325 330 335

Ala Ala Arg Gly Phe Glu Thr Tyr Glu Ala Cys Gln Gln Ala Cys Ala

340 345 350

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

355 360 365

Arg Gly Trp Glu Pro Arg Trp Ala Tyr Ser Pro Leu Leu Gln Gln Cys

370 375 380

His Pro Phe Val Tyr Gly Gly Cys Glu Gly Asn Gly Asn Asn Asn Phe His

385 390 395 400

Ser Arg Glu Ser Cys Glu Asp Ala Cys Pro Val Pro Arg Thr Pro Pro

405 410 415

Cys Arg Ala Cys Arg Leu Arg Ser Lys Leu Ala Leu Ser Leu Cys Arg

420 425 430

Ser Asp Phe Ala Ile Val Gly Arg Leu Thr Glu Val Leu Glu Glu Pro

435 440 445

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

450 455 460

Asp Asp Lys Met Gly Leu Lys Phe Leu Gly Thr Lys Tyr Leu Glu Val

465 470 475 480

Thr Leu Ser Gly Met Asp Trp Ala Cys Pro Cys Pro Asn Met Thr Ala

485 490 495

Gly Asp Gly Pro Leu Val Ile Met Gly Glu Val Arg Asp Gly Val Ala

500 505 510

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

515 520 525

Lys Lys Ile Leu Glu Leu Leu Glu Lys Gln Ala Cys Glu Leu Leu Asn

530 535 540

Arg Phe Gln Asp

545

<210>12

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Exemplary Competing Peptides

<400>12

Asp Phe Gly Leu Asp Ser Asp Glu His Ser Thr Glu Ser Arg Ser Ser

1 5 10 15

Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp

20 25 30

<210>13

<211>15

<212>PRT

<213> mouse

<400>13

Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys

1 5 10 15

<210>14

<211>12

<212>PRT

<213> mouse

<400>14

Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys

1 5 10

<210>15

<211>14

<212>PRT

<213> mouse

<400>15

Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg

1 5 10

<210>16

<211>15

<212>PRT

<213> mouse

<400>16

Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys

1 5 10 15

<210>17

<211>12

<212>PRT

<213> mouse

<400>17

Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys

1 5 10

<210>18

<211>19

<212>PRT

<213> mouse

<400>18

Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile

1 5 10 15

Ala Pro Lys

<210>19

<211>12

<212>PRT

<213> mouse

<400>19

Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys

1 5 10

<210>20

<211>11

<212>PRT

<213> mouse

<400>20

Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys

1 5 10

<210>21

<211>16

<212>PRT

<213> mouse

<400>21

Leu Asp Met Ser Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val Lys

1 5 10 15

<210>22

<211>28

<212>PRT

<213> mouse

<400>22

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

1 5 10 15

Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys

20 25

<210>23

<211>16

<212>PRT

<213> mice

<400>23

Leu Asp Met Ser Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val Lys

1 5 10 15

<210>24

<211>10

<212>PRT

<213> mouse

<400>24

Glu Leu Ile Asp Gln Tyr Asp Val Gln Arg

1 5 10

<210>25

<211>11

<212>PRT

<213> mice

<400>25

Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg

1 5 10

<210>26

<211>11

<212>PRT

<213> mouse

<400>26

Ala Gln Leu Trp Ile Tyr Leu Arg Pro Val Lys

1 5 10

<210>27

<211>10

<212>PRT

<213> mice

<400>27

Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg

1 5 10

<210>28

<211>18

<212>PRT

<213> mice

<400>28

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

1 5 10 15

Cys Arg

<210>29

<211>12

<212>PRT

<213> mouse

<400>29

Asp Ser Cys Asp Gly Val Glu Cys Gly Pro Gly Lys

1 5 5 10

<210>30

<211>9

<212>PRT

<213> mouse

<400>30

Ser Cys Ala Gln Val Val Cys Pro Arg

1 5

<210>31

<211>13

<212>PRT

<213> mouse

<400>31

Glu Cys Glu Thr Asp Gln Glu Cys Glu Thr Tyr Glu Lys

1 5 10

<210>32

<211>9

<212>PRT

<213> mouse

<400>32

Ala Asp Phe Pro Leu Ser Val Val Arg

1 5

<210>33

<211>11

<212>PRT

<213> mouse

<400>33

Glu Ala Cys Glu Glu Ser Cys Pro Phe Pro Arg

1 5 10

<210>34

<211>8

<212>PRT

<213> mouse

<400>34

Ser Asp Phe Val Ile Leu Gly Arg

1 5

<210>35

<211>12

<212>PRT

<213> mouse

<400>35

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

1 5 10

<210>36

<211>15

<212>PRT

<213> people

<400>36

Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys

1 5 10 15

<210>37

<211>14

<212>PRT

<213> people

<400>37

Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg

1 5 10

<210>38

<211>28

<212>PRT

<213> people

<400>38

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

1 5 10 15

Gly Glu Asp Gly Le uAsn Pro Phe Leu Glu Val Lys

20 25

<210>39

<211>10

<212>PRT

<213> people

<400>39

Glu Leu Ile Asp Gln Tyr Asp Val Gln Arg

1 5 10

<210>40

<211>18

<212>PRT

<213> people

<400>40

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

1 5 10 15

Cys Arg

<210>41

<211>13

<212>PRT

<213> people

<400>41

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

1 5 10

<210>42

<211>12

<212>PRT

<213> people

<400>42

Leu Gln Val Cys Gly Ser Asp Gly Ala Thr Tyr Arg

1 5 10

<210>43

<211>12

<212>PRT

<213> people

<400>43

Val Ser Glu Leu Thr Glu Glu Pro Asp Ser Gly Arg

1 5 10

<210>44

<211>10

<212>PRT

<213> people

<400>44

Cys Tyr Met Asp Ala Glu Ala Cys Ser Lys

1 5 10

<210>45

<211>10

<212>PRT

<213> people

<400>45

Gly Ile Thr Leu Ala Val Val Thr Cys Arg

1 5 10

<210>46

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>46

ttggccactg ccaccacaat ctcaaccact t 31

<210>47

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>47

tctcagcatg gccatgccgc cgtcga 26

<210>48

<211>1716

<212>DNA

<213> mouse

<220>

<221> CDS

<222>(1)..(1713)

<400>48

atg tgt gcc cca ggg tat cat cgg ttc tgg ttt cac tgg ggg ctg ctg 48

Met Cys Ala Pro Gly Tyr His Arg Phe Trp Phe His Trp Gly Leu Leu

1 5 10 15

ttg ctg ctg ctc ctc gag gct ccc ctt cga ggc cta gca ctg cca ccc 96

Leu Leu Leu Leu Leu Glu Ala Pro Leu Arg Gly Leu Ala Leu Pro Pro

20 25 30

atc cga tac tcc cat gcg ggc atc tgc ccc aac gac atg aac ccc aac 144

Ile Arg Tyr Ser His Ala Gly Ile Cys Pro Asn Asp Met Asn Pro Asn

35 40 45

ctc tgg gtg gat gcc cag agc acc tgc aag cga gag tgt gaa aca gac 192

Leu Trp Val Asp Ala Gln Ser Thr Cys Lys Arg Glu Cys Glu Thr Asp

50 55 60

cag gaa tgt gag acc tat gag aaa tgc tgc ccc aat gtg tgt ggg acc 240

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

65 70 75 80

aag agc tgt gtg gca gcc cgc tac atg gat gtg aaa ggg aag aag ggg 288

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

85 90 95

cct gta ggc atg ccc aag gag gec aca tgt gac cat ttc atg tgc ctg 336

Pro Val Gly Met Pro Lys Glu Ala Thr Cys Asp His Phe Met Cys Leu

100 105 110

cag cag ggc tct gag tgt gac atc tgg gac ggc cag ccc gtg tgt aag 384

Gln Gln Gly Ser Glu Cys Asp Ile Trp Asp Gly Gln Pro Val Cys Lys

115 120 125

tgc aaa gat cgc tgt gag aag gag ccc agc ttc acc tgt gcc tct gat 432

Cys Lys Asp Arg Cys Glu Lys Glu Pro Ser Phe Thr Cys Ala Ser Asp

130 135 140

ggc ctt acc tac tac aac cgt tgc ttc atg gac gcc gaa gcc tgc tcc 480

Gly Leu Thr Tyr Tyr Asn Arg Cys Phe Met Asp Ala Glu Ala Cys Ser

145 150 155 160

aag ggc atc aca ctg tct gtg gtc acc tgt cgt tat cac ttc acc tgg 528

Lys Gly Ile Thr Leu Ser Val Val Thr Cys Arg Tyr His Phe Thr Trp

165 170 175

cct aac acc agc cct cca ccg cct gag acc acg gtg cat ccc acc acc 576

Pro Asn Thr Ser Pro Pro Pro Pro Glu Thr Thr Val His Pro Thr Thr

180 185 190

gcc tct ccg gag act ctc ggg ctg gac atg gca gcc cca gcc ctg ctc 624

Ala Ser Pro Glu Thr Leu Gly Leu Asp Met Ala Ala Pro Ala Leu Leu

195 200 205

aac cac cct gtc cat cag tca gtc acc gtg ggt gag act gtg agt ttc 672

Asn His Pro Val His Gln Ser Val Thr Val Gly Glu Thr Val Ser Phe

210 215 220

ctc tgt gac gtg gta ggc cgg cct cgg cca gag ctc act tgg gag aaa 720

Leu Cys Asp Val Val Gly Arg Pro Arg Pro Glu Leu Thr Trp Glu Lys

225 230 235 240

cag ctg gag gac cga gag aat gtt gtc atg agg ecc aac cac gtg cgt 768

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

245 250 255

ggt aat gtg gtg gtc act aac att gcc cag ctg gtc atc tac aac gtc 816

Gly Asn Val Val Val Thr Asn Ile Ala Gln Leu Val Ile Tyr Asn Val

260 265 270

cag ccc cag gat gct ggc ata tac acc tgt aca gct cga aat gtc gct 864

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

275 280 285

ggt gtc ctg agg gct gac ttc ccg ttg tcg gtg gtc agg ggt ggt cag 912

Gly Val Leu Arg Ala Asp Phe Pro Leu Ser Val Val Arg Gly Gly Gln

290 295 300

gcc agg gcc act tca gag agc agt ctc aat ggc aca gct ttt cca gca 960

Ala Arg Ala Thr Ser Glu Ser Ser Ser Leu Asn Gly Thr Ala Phe Pro Ala

305 310 315 320

aca gag tgc ctg aag ccc cca gac agt gag gac tgt gga gag gag cag 1008

Thr Glu Cys Leu Lys Pro Pro Asp Ser Glu Asp Cys Gly Glu Glu Gln

325 330 335

aca cgc tgg cac ttc gac gcc cag gct aac aac tgc ctc act ttc acc 1056

Thr Arg Trp His Phe Asp Ala Gln Ala Asn Asn Cys Leu Thr Phe Thr

340 345 350

ttt ggc cac tgc cac cac aat ctc aac cac ttt gag acc tac gag gcc 1104

Phe Gly His Cys His His Asn Leu Asn His Phe Glu Thr Tyr Glu Ala

355 360 365

tgt atg ctg gct tgt atg agt ggg cca ttg gcc acc tgc agc ctg cct 1152

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

370 375 380

gcc ctg caa ggg cct tgc aaa gct tat gtc cca cgc tgg gcc tac aac 1200

Ala Leu Gln Gly Pro Cys Lys Ala Tyr Val Pro Arg Trp Ala Tyr Asn

385 390 395 400

agc cag aca ggc cta tgc cag tcc ttc gtc tat ggc ggc tgt gag ggc 1248

Ser Gln Thr Gly Leu Cys Gln Ser Phe Val Tyr Gly Gly Cys Glu Gly

405 410 415

aac ggt aac aac ttt gaa agc cgt gag gct tgt gag gag tcg tgt ccc 1296

Asn Gly Asn Asn Phe Glu Ser Arg Glu Ala Cys Glu Glu Ser Cys Pro

420 425 430

ttc ccg agg ggt aac cag cac tgc cgg gcc tgc aag ccc cgg caa aaa 1344

Phe Pro Arg Gly Asn Gln His Cys Arg Ala Cys Lys Pro Arg Gln Lys

435 440 445

ctt gtt acc agc ttc tgt cgg agt gac ttt gtc atc ctg ggc agg gtc 1392

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

450 455 460

tct gag ctg acc gag gag caa gac tcg ggc cgt gcc ctg gtg acc gtg 1440

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

465 470 475 480

gat gag gtc tta aaa gat gag aag atg ggc ctc aag ttt ctg ggc cgg 1488

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

485 490 495

gag cct ctg gaa gtc acc ctg ctt cat gta gac tgg acc tgt cct tgc 1536

Glu Pro Leu Glu Val Thr Leu Leu His Val Asp Trp Thr Cys Pro Cys

500 505 510

ccc aac gtg aca gtg ggt gag aca cca ctc atc atc atg ggg gag gtg 1584

Pro Asn Val Thr Val Gly Glu Thr Pro Leu Ile Ile Met Gly Glu Val

515 520 525

gac ggc ggc atg gcc atg ctg aga ccc gat agc ttt gtg ggg gca tcg 1632

Asp Gly Gly Met Ala Met Leu Arg Pro Asp Ser Phe Val Gly Ala Ser

530 535 540

agc aca cgg cgg gtc agg aag ctc cgt gag gtc atg tac aag aaa acc 1680

Ser Thr Arg Arg Val Arg Lys Leu Arg Glu Val Met Tyr Lys Lys Thr

545 550 555 560

tgt gac gtc ctc aag gac ttc ctg ggc ttg caa tga 1716

Cys Asp Val Leu Lys Asp Phe Leu Gly Leu Gln

565 570

<210>49

<211>571

<212>PRT

<213> mouse

<400>49

Met Cys Ala Pro Gly Tyr His Arg Phe Trp Phe His Trp Gly Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Glu Ala Pro Leu Arg Gly Leu Ala Leu Pro Pro

20 25 30

Ile Arg Tyr Ser His Ala Gly Ile Cys Pro Asn Asp Met Asn Pro Asn

35 40 45

Leu Trp Val Asp Ala Gln Ser Thr Cys Lys Arg Glu Cys Glu Thr Asp

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Gly Met Pro Lys Glu Ala Thr Cys Asp His Phe Met Cys Leu

100 105 110

Gln Gln Gly Ser Glu Cys Asp Ile Trp Asp Gly Gln Pro Val Cys Lys

115 120 125

Cys Lys Asp Arg Cys Glu Lys Glu Pro Ser Phe Thr Cys Ala Ser Asp

130 135 140

Gly Leu Thr Tyr Tyr Asn Arg Cys Phe Met Asp Ala Glu Ala Cys Ser

145 150 155 160

Lys Gly Ile Thr Leu Ser Val Val Thr Cys Arg Tyr His Phe Thr Trp

165 170 175

Pro Asn Thr Ser Pro Pro Pro Pro Glu Thr Thr Val His Pro Thr Thr

180 185 190

Ala Ser Pro Glu Thr Leu Gly Leu Asp Met Ala Ala Pro Ala Leu Leu

195 200 205

Asn His Pro Val His Gln Ser Val Thr Val Gly Glu Thr Val Ser Phe

210 215 220

Leu Cys Asp Val Val Gly Arg Pro Arg Pro Glu Leu Thr Trp Glu Lys

225 230 235 240

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

245 250 255

Gly Asn Val Val Val Thr Asn Ile Ala Gln Leu Val Ile Tyr Asn Val

260 265 270

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

275 280 285

Gly Val Leu Arg Ala Asp Phe Pro Leu Ser Val Val Arg Gly Gly Gln

290 295 300

Ala Arg Ala Thr Ser Glu Ser Ser Ser Leu Asn Gly Thr Ala Phe Pro Ala

305 310 315 320

Thr Glu Cys Leu Lys Pro Pro Asp Ser Glu Asp Cys Gly Glu Glu Gln

325 330 335

Thr Arg Trp His Phe Asp Ala Gln Ala Asn Asn Cys Leu Thr Phe Thr

340 345 350

Phe Gly His Cys His His Asn Leu Asn His Phe Glu Thr Tyr Glu Ala

355 360 365

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

370 375 380

Ala Leu Gln Gly Pro Cys Lys Ala Tyr Val Pro Arg Trp Ala Tyr Asn

385 390 395 400

Ser Gln Thr Gly Leu Cys Gln Ser Phe Val Tyr Gly Gly Cys Glu Gly

405 410 415

Asn Gly Asn Asn Phe Glu Ser Arg Glu Ala Cys Glu Glu Ser Cys Pro

420 425 430

Phe Pro Arg Gly Asn Gln His Cys Arg Ala Cys Lys Pro Arg Gln Lys

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Glu Pro Leu Glu Val Thr Leu Leu His Val Asp Trp Thr Cys Pro Cys

500 505 510

Pro Asn Val Thr Val Gly Glu Thr Pro Leu Ile Ile Met Gly Glu Val

515 520 525

Asp Gly Gly Met Ala Met Leu Arg Pro Asp Ser Phe Val Gly Ala Ser

530 535 540

Ser Thr Arg Arg Val Arg Lys Leu Arg Glu Val Met Tyr Lys Lys Thr

545 550 555 560

Cys Asp Val Leu Lys Asp Phe Leu Gly Leu Gln

565 570

<210>50

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>50

caccatgtgt gccccagggt atcatcggtt ctgg 34

<210>51

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Primers

<400>51

ttgcaagccc aggaagtcct tgaggac 27

<210>52

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Exemplary N-terminal Peptide Sequences

<400>52

Leu Pro Pro Ile Arg Tyr Ser His Ala Gly Ile

1 5 10

<210>53

<211>48

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequences: Synthetic Oligonucleotides

<400>53

cagacagaca gacagacaga cagacagaca gacagacaga cagacaga 48

Claims (47)

1. A pharmaceutical composition comprising: i) at least one protein comprising at least one follistatin domain, wherein the protein is not follistatin, and ii) at least one pharmaceutically acceptable carrier. 2. The composition of claim 1, wherein said protein is selected from the group consisting of FLRG, FRP, agrin, osteonectin, hevin, IGFBP7, U19878 and GASP2. 3. The composition of claim 1, wherein said protein has a stabilizing modification. 4. The composition of claim 3, wherein said modification is a fusion to the Fc region of an IgG molecule. 5. The composition of claim 4, wherein the IgG molecule is IgGl or IgG4, or a derivative thereof. 6. The composition of claim 5, wherein the IgG molecule is IgGl or a derivative thereof. 7. The composition of claim 4, wherein the IgG molecule is fused via a linker peptide to a protein comprising at least one follistatin domain. 8. The composition of claim 3, wherein said modification comprises an altered glycosylation site. 9. The composition of claim 3, wherein said modification comprises at least one carbohydrate moiety. 10. The composition of claim 3, wherein said modification comprises albumin or a derivative of albumin. 11. The composition of claim 3, wherein said modification comprises a non-proteinaceous polymer. 12. The composition of claim 3, wherein said modification comprises pegylation. 13. A diagnostic kit comprising at least one protein comprising at least one follistatin domain, wherein the protein is not follistatin and at least one additional kit component is selected from: i) at least one protein-binding agent; ii) at least one buffer and/or solution; and iii) at least one structural ingredient. 14. The kit of claim 13, wherein said protein is selected from the group consisting of FLRG, FRP, agrin, osteonectin, hevin, IGFBP7, U19878 and GASP2. 15. A recombinant cell comprising a nucleic acid encoding a protein comprising at least one follistatin domain, wherein the protein is not follistatin. 16. The recombinant cell of claim 15, wherein said protein has a stability modification. 17. The recombinant cell of claim 15 or 16, wherein said protein is selected from the group consisting of FLRG, FRP, agrin, osteonectin, hevin, IGFBP7, U19878 and GASP2. 18. A method of modulating GDF-8 comprising administering at least one protein comprising at least one follistatin domain, wherein the protein is not follistatin, and enabling the protein to interact with GDF-8 . 19. A method of treating a patient with a medical condition comprising administering an effective dose of at least one protein comprising at least one follistatin domain, wherein the protein is not follistatin, and the protein is capable of Interacts with GDF-8. 20. A method of treating a patient with a medical condition comprising administering a nucleic acid encoding a protein comprising at least one follistatin domain, wherein the protein is not follistatin, enabling the nucleic acid to be translated into a protein , and enables the translated protein to interact with GDF-8. 21. A method of expressing a nucleic acid, comprising: i) administering to the cell a nucleic acid encoding a protein comprising at least one follistatin domain, wherein the protein is not follistatin, ii) enabling the nucleic acid to enter the cell, and iii) enabling the cell to express the protein. 22. The method of claim 18, 19, 20 or 21, wherein said protein is selected from the group consisting of FLRG, FRP, agrin, osteonectin, hevin, IGFBP7, U19878 and GASP2. 23. The method of claim 18, 19, 20 or 21, wherein said protein has a stabilizing modification. 24. The method of claim 23, wherein said modification is a fusion to the Fc region of an IgG molecule. 25. The method of claim 24, wherein the IgG molecule is IgGl or IgG4 or a derivative thereof. 26. The method of claim 25, wherein the IgG molecule is IgGl or a derivative thereof. 27. The method of claim 24, wherein the IgG molecule is fused via a linker peptide to a protein comprising at least one follistatin domain. 28. The method of claim 23, wherein said modification comprises an altered glycosylation site. 29. The method of claim 23, wherein said modification comprises at least one carbohydrate moiety. 30. The method of claim 23, wherein said modification comprises albumin or a derivative of albumin. 31. The method of claim 23, wherein said modification comprises a non-proteinaceous polymer. 32. The method of claim 23, wherein said modification comprises pegylation. 33. The method of claim 19, wherein said patient obtains a therapeutic benefit from an increase in muscle tissue mass and quantity. 34. The method of claim 19, wherein said disorder is a muscular disorder. 35. The method of claim 34, wherein said muscular disorder is muscular dystrophy. 36. The method of claim 35, wherein said muscle atrophy is selected from the group consisting of severe or benign X-linked muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral dystrophy, myotonic dystrophy, distal muscular dystrophy, Dystrophic, progressive dystrophic ophthalmoplegia, oculopharyngeal muscular dystrophy, and Fakuyama congenital muscular dystrophy. 37. The method of claim 34, wherein said condition is selected from the group consisting of congenital myopathy, myotonia congenita, familial periodic paralysis, paroxysmal myoglobinuria, myasthenia gravis, Iran-Long syndrome, secondary Episodic myasthenia, denervated atrophy, sudden muscle atrophy, muscle wasting syndrome, sarcopenia, and cachexia. 38. The method of claim 34, wherein said condition is a muscle condition selected from trauma to muscle tissue and chronic damage to muscle tissue. 39. The method of claim 19, wherein said disorder is a metabolic disease or disorder. 40. The method of claim 39, wherein said condition is type 2 diabetes, non-insulin dependent diabetes, hyperglycemia or obesity. 41. The method of claim 19, wherein said disorder is an adipose tissue disorder, such as obesity. 42. The method of claim 19, wherein said condition is a bone degenerative disease, such as osteoporosis. 43. The method of claim 19, wherein said protein is administered at regular intervals or at daily, weekly or monthly intervals. 44. The method of claim 19, wherein said protein is administered at a dose of 5 mg to 100 mg. 45. The method of claim 19, wherein said protein is administered at a dose of 15 mg to 85 mg. 46. The method of claim 19, wherein said protein is administered at a dose of 3 mg to 70 mg. 47. The method of claim 19, wherein said protein is administered at a dose of 40 mg to 60 mg.

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