WO1997039122A9

WO1997039122A9 - ISOLATED AND CLONED MAST CELL 78 kDa PHOSPHOPROTEIN (MAST CELL DEGRANULATION INHIBITORY AGENT) AND USE THEREOF

Info

Publication number: WO1997039122A9
Application number: PCT/US1997/006042
Authority: WO
Filing date: 1997-04-11
Publication date: 1998-03-26
Anticipated expiration: 1998-10-12

Abstract

A 78 kDa mast cell protein (moesin) has been cloned from rats, and its cDNA and amino acid sequence determined. This protein is phosphorylated on specific serine and threonine residues by the action in mast cells of one or more protein kinase C isozymes, especially the κ isozyme, thereby producing a phosphoprotein that inhibits mast cell degranulation (Mast Cell Degranulation Inhibitor Agent). In vivo phosphorylation of this protein in mast cells can be stimulated by drugs such as cromolyn, nedocromil, flavonoids such as quercetin and kaempherol, and lodoxamide. Mast cell degranulation can be inhibited by administration of phosphomoesin phosphatase inhibitors. Tissues deficient in mast cell moesin can be identified with a labeled anti-moesin or anti-phosphomoesin antibody and treated by transfecting such mast cells with moesin cDNA in a viral vector such as an influenza virus vector.

Description

ISOLATED AND CLONED MAST CELL 78 kDa PHOSPHOPROTEIN (MAST CELL DEGRANULATION INHIBITORY AGENT) AND USE THEREOF

Field of the Invention

This application relates in general to regulatory proteins that link the cytoskeleton to the plasma membrane in mast cells

("MC") . More particularly, this invention relates to a specific

MC 78 kDa cytoskeleton-plasma membrane linkage protein whose enzymatic phόsphorylation on specific threonine and/or serine residues inhibits MC degranulation and, by so doing, prevents or reverses human disorders that result from release of preformed MC components (e.g., histamine, heparin, proteases and cytokines) and those generated de novo (prostaglandins, leukotrienes, lipoxins and platelet activating factor) as a result of the secretogue stimulus.

Background of the Invention

Mast cells express on their surface the IgE binding protein (FceRI) which has high affinity for the Fc portion of IgE. Receptor-bound IgE, when bridged by multivalent antigen, triggers an integrated non-cytolytic series of biochemical reactions which involve activation of protein tyrosine kinases . These include the strc-related p56^lyn and p72^syk, of which the first phosphorylates the β and γ subunits of FceRI and the syk tyrosine kinase itself. In addition, possibly syk then leads to phosphorylation of phospholipase C γl and 2 (PLC) which are then translocated from the cytosolic to the membranous fraction and lead to production of 1, 4, 5-inositol triphosphate and diacylglycerol . These events then lead to an increase in free intracellular calcium and activation of PKC which leads to further phosphorylation of FceRI on threonine of the γ chains and serine on the _/3-subunits. The net result is the release of many mediators stored in MC cytoplasmic granules by degranulation or exocytosis. These mediators include histamine, heparin, proteases and cytokines . In addition, de novo synthesis and release of mediators such as prostaglandins, leukotrienes, lipoxins and platelet activating factor are stimulated.

Mast cells are also activated by diverse stimuli such as lymphokines and cytokines, naturally occurring or synthetic basic peptides, lectins, hormones, some neuropeptides and neurotransmitters , as well as direct nerve stimulation. Mast cells are, therefore, important not only for allergic and late phase reactions, but also for many conditions such as migraines, interstitial cystitis and irritable bowel syndrome which are linked to mast cell degranulation. "Degranulation" as used herein is defined as the biochemical and morphological state associated with secretion of some or all MC mediators from MC granules, whether by exocytosis or some other mechanism of activation.

In MC, secretogues induce phosphorylation of specific proteins. Sieghart et al . , Na ture 235:329 (1978); Wells et al . , Biochem. Pharm . , 32: 837 (1983). Four such proteins have been detected. These have molecular masses of 42 kDa, 59 kDa, 68 kDa and 78 kDa. Phosphorylation of MC protein 78 kDa is stimulated by disodium cromoglycate ( "cromolyn" ) , a commercially available "antiallergic" drug, which inhibits MC secretion. Theoharides et al . , Science, 202: 80 (1980) . This phosphorylation is apparently carried out by protein kinase C ("PKC") -catalyzed phosphorylation of serine and/or threonine residues of the protein. The first three proteins are rapidly phosphorylated (within 10 seconds of challenge) - the last, several minutes thereafter.

It has previously been determined that the 78 kDa protein is highly homologous to human moesin (Ankes et al . , PNAS 88:8297 (1991); Correia et al . , Biochem . Pharm . , 1996, in press). Therefore, this protein will, hereinafter, be referred to interchangeably as "78 kDa protein" or "moesin". Moesin, in addition, has high similarity to the proteins ezrin, radixin and merlin which belong to the erythrocyte band 4.1 superfamily considered to link the plasma membrane to cytoskeletal components .

Moesin is evidently not phosphorylated in any cell type other than MC (Krieg et al . , J. Biol . Chem . 2662:19258 (1992), Franck et al . , J. Cel l Sci . , 105:219 (1993)); which distinguishes moesin from ezrin which is phosphorylated on tyrosine residues, not serine or threonine, by epidermal growth factor (EGF) in human carcinoma A-431 cells. The tyrosine phosphorylation is also stimulated by platelet-derived growth factor (PDGF) . These tyrosine factors do not stimulate the phosphorylation of moesin. Franck et al . above.

As noted above, proteins, particularly moesin, are phosphorylated when MC are treated with cromolyn. Shapiro et al . , Pharmacotherap . , 5:156 (1985). Cromolyn is a "membrane MC stabilizer" which does not cross the plasma membrane, but which can inhibit both antigen and C48/80- induced mast cell secretion. Ennis et al . , Nature, 289:186 (1981) The concentration range at which cromolyn facilitates the incorporation of phosphate into the 78 kDa moesin corresponds to that which inhibits histamine release. Theoharides et al . , Science, 202:80 (1980). Such results are also seen with the cromolyn analogue, nedocromil, (Cairns et al . , J. Med . Chem. , 28:1832 (1985)) and with structurally related compounds such as the flavonoid quercetin. Sieghart et al . , Biochem . Pharm. , 30:2737 (1981).

Because of the potential for linkage between phosphorylation of moesin and inhibition of MC degranulation, an important need exists to identify the exact species of phosphorylated moesin that may be responsible for this inhibition of MC degranulation, to clone and express the gene for this species of protein, and to use the thus isolated or recombinant phosphomoesin in the design of pharmaceuticals for inhibiting MC degranulation and secretion where desired, e.g., to avoid or treat allergic reactions, migraine headaches, irritable bowel syndrome, intestinal cystitis and the like. Moesin itself has diagnostic utility in, for example, the production of anti -moesin antibodies that are useful in diagnosing for the presence of moesin in MC, e.g, in nasal polyps in which the level of this protein may be low to absent. These needs have been addressed and fulfilled by the invention whose embodiments are described below.

Summary of the Invention

It is an object of this invention to isolate and purify from mast cells moesin and phosphomoesin .

It is another object to clone the cDNA for rat MC moesin, to sequence this cDNA, and to deduce from this sequence the amino acid sequence of the protein, including those sequences arising from the degeneracy of the genetic code .

It is still another object to insert the cDNA of moesin into an expression vector and to express the protein in an expression system.

It is a further object to determine the pattern of phosphorylation of the serine, threonine and tyrosine residues of moesin that results in Mast Cell Degranulation Inhibitory Agent activity.

It is another object to provide a pharmaceutical composition for delivering to a subject in need of same a phosphomoesin so as to inhibit or reverse MC degranulation.

It is still another object to produce an anti-moesin antibody for diagnostic uses or for the purpose of immunoprecipitating moesin.

These objects have been achieved by the invention embodiments described below. In one embodiment of the invention, an isolated homogeneous, MC 78 kDa phosphoprotein with cDNA and amino acid sequences as shown in Fig. 2 is provided, and uses described.

In a further embodiment of the invention, isolated rat MC moesin cloned cDNA is provided, along with its sequence, and the deduced corresponding amino acid sequence. The substrates for PKC-catalyzed phosphorylation in this amino acid sequence are identified.

In another embodiment of the invention, anti -moesin antibodies that recognize the 78 kDa phosphoprotein are provided, and used to immunoprecipitate the phosphoprotein and to screen patients suspected of lacking MC moesin.

In still another embodiment of the invention, an expression vector incorporating moesin cDNA is provided, as is an E. coli in which this cDNA is expressed.

Still another embodiment relates to the use of moesin cDNA in a viral vector for administration to patients where MC are deficient in moesin.

In yet another embodiment of the invention, drugs and other agents that regulate the phosphorylation of the 78 kDa protein, and thereby regulate MC degranulation and secretion, are identified, and protocols for their use are provided.

Brief Description of the Figures

Fig. 1 shows the size and overlapping nature of moesin cDNA fragments obtained from a library screening. The coding region is boxed.

Fig. 2 shows the nucleotide sequence of rat moesin cDNA and deduced amino acid sequence (SEQ ID NOS: 1-2). Fig. 3 shows the homology of amino acid sequences of moesin from different species. The underlined amino acids are the putative sites phosphorylated by PKC (consensus: K/R X X T/S) . The amino acid residues in bold face are essential to the recognition and phosphorylation. The consensus sequence of moesin was generated by the PRETTY program. Only the amino acid residues different among the species are shown vertically, in addition to the consensus sequence (SEQ ID NOS: 3-7 are shown in this figure) .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The cDNA has been cloned for a 78 kDa rat mast cell protein (also referred to as moesin) whose in vivo phosphorylation on specific serine and threonine residues by protein kinase C ("PKC") produces a phosphorylated form of the 78 kDa protein. It has also been discovered that the phosphorylated form of the 78 kDa protein inhibits mast cell degranulation. This inhibition of degranulation, in turn, inhibits secretion of mast cell granular substances such as preformed substances (histamine, heparin, protease and . cytokines) and de novo synthesized mediators (prostaglandins, leukotrienes, lipoxins, platelet activating factor, etc.) . Inhibition of the secretion of these substances has clinical consequences. For example, inhibition of histamine secretion has an anti -allergic effect. Other released substances produce migraine headaches, interstitial cystitis and irritable bowel syndrome. Clearly, the phosphorylated 78 kDa protein has surprising and profound clinical effects.

A cDNA library for moesin may be constructed by purifying RNA from MC-like RBL2H3 cells grown in D-MEM growth medium supplemented with fetal bovine serum.

To screen the cDNA library for the moesin cDNA, primers corresponding to human cDNA sequences may be synthesized, e.g. sequences 1269-1292 or 1604-1579. A portion of human moesin cDNA (1269-1604) may be amplified from a human fetal brain library, labeled with ³²P-d CTP, and used as a probe to screen the RBL cDNA libraries. Positive clones may be sequenced (e.g., with BioRad's Bst Polymerase system) , and T3 and T7 primers, as well as synthesized primers corresponding to the region already sequenced, may be used to sequence the gene.

The RBL moesin cDNA may be expressed in E. coli as follows. A cloned cDNA is amplified with a primer, subcloned into a plasmid, cleaved by a restriction enzyme, and subcloned into a T7 expression vector (e.g., pET 28) . This recombinant plasmid may then be transformed into an E. coli strain, and expression induced by IPTG (Stadier et al . , Enzymol . , 185:60 (1990). The expressed moesin may be purified to homogeneity using a nickel column under denaturing conditions (Invitrogen) . Moesin is then dialyzed against a buffer-detergent mixture, then against just buffer. cDNA expressed in E. col i by the T7 expression systems produces a fusion protein with 6 histidine residues at the N- terminus . With pET-28 expression vector and IPTG, E. coli produces a protein which has a mass of 78 kDa by SDS-PAGE and represents about 50% of E. coli ' s protein content.

Purified, expressed moesin can be phosphorylated in vi tro or in vivo . For in vi tro phosphorylation, the protein may be incubated in a reaction mixture containing, inter alia, PKC, activators (e.g., diolein and phosphatdyeserime) and ³²P [ATP]. For dephosphorylation, the phosphorylated moesin is incubated with a phosphatase, and the products separated by SDS page. For in vivo phosphorylation MC may be isolated, purified, loaded with ³²Pi, then treated with cromolyn (Theoharides et al . , Science, 207:80 (1980) . The labeled cells may be permeablized (e.g., with digitonin) , the mixture separated into cytosolic and membrane fractions by differential centrifugation, and the samples run on SDS-PAGE for Western blotting and autoradiography . In vivo phosphorylation of MC moesin may be accomplished by administering to a subject an effect amount of a PKC-stimulating agent such as cromalyn, nedocromil (a cromalyn analogue) , a flavonoid such as quercetin and kaempherol, and lodoxamide . These agents may be used in a derivatized form such as esters that penetrate MC more readily and remain longer within these cells.

To detect PKC isozymes in purified peritoneal MC and RBL cells, immunoblot analyses may be carried out using rabbit polyclonal antibodies to six different isoforms { , β , γ, δ, e and f) , and as secondary antibody, goat anti-rabbit - HRP .

The in vivo phosphorylated moesin may be detected by treating purified mast cells with cromolyn, nedocromil, a flavonoid such a quercetin or kaempherol, or lodoxamide, ar a derivative thereof such as an ester, fixing and sectioning the cells, incubating sections first with rabbit anti -moesin polyclonal antibody and then with goat anti-rabbit IgG-biotin, followed by exposure to streptavidine-rhodamine to complete the immunocytochemistry .

To determine a partial internal amino acid sequence of phosphomoesin, following treatment of purified mast cells with ³²Pi and cromolyn and purification of the phosphoprotein to homogeneity, the phosphoprotein may be cleaved by cyanogen bromide and the fragments thus obtained separated by SDS-PAGE, electroblotted onto a membrane, and autoradiographed. Radiolabeled fragments may then cut out and sequenced by automatic Edman degradation reactions (e.g., using an Applied Biosystems sequencer) .

To analyze for phosphoamino acids in phosphorylated 78 kDa protein, tryptic/chymotryptic digests of the protein may be completely digested with HCl at 100 °C, and the individual amino acids separated on electro TLC plates and phosphorylated amino acids identified.

Protein kinase (PKC, PKG, CAMKII) inhibitors chelerythrine, H-8, H-89, KN-62 and staurosporine may be tested by preparing radiolabeled MC as above, incubating the cells with the putative inhibitor, followed by a moesin phosphorylation stimulator (e.g. cromolyn, nedocromil, quercetin, or other flavonoids such as kaempherol, lysing the cells, and running the products on one- dimensional SDS-PAGE.

To obtain phosphorylated moesin for administration to patients, purified human MC may be treated with a phosphorylation stimulator (e.g., cromolyn), and the phosphorylated 78 kDa phosphoprotein isolated and purified to homogeneity (as described above and below) to a state suitable for administration to humans. Alternately, the human moesin cDNA portion critical for inhibition of degranulation may be expressed in bacteria, and then phosphorylated in vi tro . Phosphomoesin, in an appropriate pharmaceutic vehicle, as in liposomes, may be administered routinely to patients by appropriate routes leading to inhibition of MC degranulation stimulated by allergic or other means. The high homology between human and animal moesins suggests that the latter may be used in humans without untoward effects.

Phosphorylation of moesin by f isozyτne PKC may be induced in vivo by administration of the agents described above, e.g., (a) cromolyn, flavonoids, or other specific isozyτne zeta activator; (b) inhibitors of the phosphatase calcineurin.

Production of polyclonal and monoclonal antibodies directed against the 78 kDa phosphoprotein may be accomplished by routine methods, and may be used to screen patients with atopic and other neuroinflammatory disease for the possible absence of phosphomoesin. For example, it has been recently observed that nasal polyps (a site of MC) in allergic rhinitis patients lack moesin. Transfection of MC in patients lacking phosphomoesin may be accomplished with a vector carrying the moesin cDNA, such as influenza virus vectors.

Probes modeled after the moesin cDNA can also be constructed and used to determine the presence, absence or amount of phosphomoesin in a particular tissue of a body. The phosphomoesin may be used in therapy for preventing or inhibiting disorders characterized by excessive MC degranulation. The phosphoprotein may be administered to a patient parenterally in an appropriate pharmaceutical carrier such as are described in Remington's Pharmaceutical Sciences, 18th ed. , 1990, Mack Publishing Co., Easton, PA. It would not take undue experimentation for the physician skilled in the art to determine the dosage to use for a particular patient. In addition, the phosphoprotein can be delivered to the patient encapsulated in liposomes composed of pharmaceutically acceptable biomaterials .

The embodiments of the invention described below are merely to exemplify the invention, and should not be construed as limiting the invention which is described in the specification and claims.

EXAMPLES

EXAMPLE 1

Materials and Methods

1. Reagents . All reagents were obtained from Sigma Chemical Co. (St. Louis, MO), unless stated otherwise . Cromolyn, a bischromone [1 , 3 -bis - ( 2 -carboxychromone - 5 -yloxy) -2 hydropropane] was also purchased from Sigma.

2 • Purification of peritoneal mast cells. Mast^' cells were obtained by lavage of the peritoneum of male 225 g rats (Taconic, Germantown, NY) injected with Locke's buffer (150 mM NaCl, 5 mM KC1, 2 mM CaCl₂, 5 mM Hepes , 1 g/liter dextrose and 1 g/liter BSA, pH 7.2) . Cells were then purified to greater than 90% purity by centrifugation at 350xg for 10 min at room temperature through metrizamide (Accurate Chemical and Scientific Corp, Westbury, NY) Shapiro et al . , above.

3. Culture of rat basophilic leukemia cells (RBL) . RBL cells were obtained from Dr. H. Metzger (NIH) and were maintained as described before. Tamir et al . , J. Cell Biol . , 93:638 (1980) . 4. Protein Assay. The Pierce BCA Protein Assay kit was used for the spectrophotometric determination (at 562 nm) of protein concentration. For small samples (5-250 μg range) , the enhanced protocol using 60°C for 30 min was utilized.

5. Radiolabeling of cells with f³²1 Pi ■ Cells were labeled at 10 μCi/million cells/ml with [³²] P-carrier free orthophosphoric acid (ICN-Biomedicals, CA) in phosphate-free Locke's buffer for 1 h at 37°C, were washed twice with Locke's without BSA and were resuspended in the same buffer. They were treated with 10^M cromolyn for 30 sec at 37°C. Following prelabeling, cells were incubated with cromolyn (100 μM) for 30 sec and 5 min, immediately lysed, and the lysate run on one dimension SDS-PAGE. Exposure of film was for 24 hr at -70°C. Arrows indicate the position of 78 kDa protein. The 78 kDa protein is phosphorylated within 30 sec in the presence of cromolyn and completely dephosphorylated by 5 min.

Mast cells were prepared and treated as described in Methods except that the SDS gel was 7.5%. A23187 was used at 0.1 μg/ l for 1 min and C48/80 at 0.5 μg/ml for 1 min. No calcium indicates that no extracellular calcium was added.

Purified mast cells (10⁶ cells per lane), lysed and immediately boiled for 5 min were loaded on 10% SDS gels and were run as described in Methods . The separated proteins were then blotted on nitrocellulose for 20 min at 15 U. The blots were cut in single lanes and were incubated overnight with the antisera.

RBL mast cells were permeabilized with digitonin (25 μg) in HEPES buffer (20 mM HEPES, pH 7.4, 10% sucrose, 1 mM PMSF) plus indicated NaCl, centrifuged at 1000 x g for 60 min to separate the membrane (pellet) and cytosol (supernatant) fractions, subjected to SDS-PAGE, transferred onto nitrocellulose and probed with rabbit anti-rat moesin serum. 6. One dimensional electrophoresis (SDS-PAGE) .

A. The discontinuous buffer system developed by Laemmli, Nature, 277:680 (1970).

B. The method of Schagger and Von Jagow for separation of peptides of low molecular weight

(5-20 kDa) .

7. Two Dimensional Electrophoresis for Isoelectric focusing (IEF) A. O'Farrell protocol (O'Farrell, J^". Biol . Chem. ,

250:4007 (1975) ) .

Sample preparation with phenol extraction: Cells were first lysed in 0.5 ml of Extraction Buffer (0.7M Sucrose, 0.5M Tris, 30mM HCl, 50mM EDTA, 0.1M KC1 , 2% BME and 2 mM PMSF) and were sonicated with multiple short bursts of maximum intensity. The volume was adjusted to 1.5 ml, incubated for 10 min on ice and an equal volume of water-saturated phenol was then added. After 10 min with shaking at room temperature, the phases were separated by centrifugation. Proteins were precipitated from the phenol phase with 5 volumes of 0.1 M ammonium acetate in methanol and kept at - 20°C overnight. The precipitate was washed 3 times with ammonium acetate and once with acetone . The pellet was then dried under nitrogen gas and was solubilized in O'Farrell 's buffer [0.5% SDS, 9.5 M urea, 2% ampholytes (pH 3.5-10) from Bio-Rad Labs

(Hercules, CA) and 5% BME] . After 10 min, an equal volume of Garrel's buffer [9.5 M urea, 2% ampholytes (pH 3.5-10), 4% NP-40 and 5% BME] was added.

Composition of first dimension gels: IEF gels were composed of 4% Bis-Acrylamide, 8.0 M Urea, 4% NP-

40, 2% Ampholytes (pH 3.5 - 10), 0.14% TEMED and 0.14% ammonium persulfate. Protein samples (about 100 μg) were loaded per cylinder gel at the basic end and allowed to electrophorese at 400V for 9000 VHrs . The voltage was then increased to 800 Volts for 1 h. The gels were then extruded and equilibrated in 10 ml of Equilibration buffer for 15 min and were either placed directly onto second dimension gel or frozen at -20°C. Two additional tube gels were also run along with the samples . They were extruded and cut at 1 cm intervals. Each piece was equilibrated in distilled water and the pH of these solutions was then measured and plotted relative to the length of the tube gel to determine the pi's of the proteins.

Resolution in the second dimension: Resolution was on 7.5% SDS-PAGE gels overlaid with 1% agarose stacking containing 0.125 M Tris (pH 6.8) . The second dimension gels were run overnight at 50 V (constant voltage) until the tracking dye ran off the bottom. The gels were dried immediately and placed against film. The molecular weights of the protein was determined by prestained molecular weight markers (Sigma Chemicals, St. Louis, MO).

B. Imada et al . , BBA, 625:179 (1980) discontinuous gel system designed for the separation of poorly soluble, hydrophobic cell surface proteins using detergents SDS and Triton CF10, and urea in the first dimension. In the second dimension proteins were separated by their molecular weights.

8. Composition of the first dimension gel. The detergents 0.1% SDS and 0.3% Triton CF10 (alkylaryl ether) were present in both the Stacking gel (1.8% acrylamide, 0.18% Bis, Tris 0.125 M pH 6.8) and the Separating gel (3% acrylamide, 0.3% Bis, urea 9 M, Tris 0.375 M, pH 9) . Th anode buffer contained (375 mM Tris, pH 9) and the cathode buffer (50 mM glycine-NaOH, pH 10.5) .

9. Sample preparation. Cells were lysed and solubilized in SDS Solubilizing buffer (2% SDS, 0.0625 M Tris, 10% Glycerol, 5% BME) and the samples boiled for 3-5 min. They were, then, directly applied to the gel and overlaid with the Cathode buffer which was cooled before use. Further separation was carried out at constant voltage of 200-300 V for 3-4 h. The gels were extruded and immediately analyzed in the second dimension as described for IEF method.

10. Electroelution. The autoradiograph was matched with the gel and the desired molecule to be electroeluted marked out. The excised gel was briefly suspended in water to separate gel from backing paper and to rehydrate the gel. Tris-Glycine-SDS buffer was used to elute proteins into centricon-30 tubes (Amicon, Beverly, MA) . Voltage of 150-250 V was applied for 20 hr . The sample was then washed twice using 1.5 ml of Volatile Buffer (50 mM Ammonium Bicarbonate and 0.1% SDS) .

11. Chemical cleavage bv cyanogen bromide. The protein sample was washed twice with Volatile buffer (5 mM NH₄HC0₃, 0.1% SDS) in a final volume of 75 μl. An amount of 40 μl of 0.15 M CNBr in 70% formic acid was added and incubated overnight in the dark at room temperature. The sample was dried on a Speed-Vac and solubilized in Sample Buffer (50 μl) .

12. Western blotting. Transfer of proteins from gel to nitrocellulose membranes was by semi -dry blotting. The Transfer buffer contained 25 mM Tris, 190 mM glycine and 20% methanol. Transfer was typically carried out at 20 V for 25 min. Blocking non specific binding sites on the membrane was carried out using 3% BSA in PBS. Primary rabbit anti-rat moesin antibody was typically used at a dilution of 1:2000 with overnight shaking. Secondary antibody used was anti -rabbit IgG Horse Radish Peroxidase (Zymed, San Francisco, CA) . Detection was carried out with diamino benzidine .

13. Electroblottin . The method of Paul Matsudaira, JBC 262:10035 (1987) was used where proteins were directly electroblotted onto polyvinylidene difluoride (PVDF) . The gel was first soaked in Transfer Buffer (10 mM 3 - (cyclohexylamino) -1- propane sulfonic acid 10% methanol, pH 11, for 5 min to reduce the amount of Tris and glycine. PVDF membrane was then rinsed in 100% methanol and stored in Transfer Buffer. The proteins were electroblotted onto PVDF. The PVDF membrane was then washed in deionized water for 5 min, membrane stained with 0.1% Coomassie Blue R-250 in 50% methanol for 5 min and then destained in 50% methanol, 10% acetic acid for 5-10 min. The membrane was rinsed in water and air dried at room temperature . The membrane was autoradiographed and the spot of interest cut out for sequencing.

14. Peptide seσuencinα. This was carried out by automated cycles of the Edman degradation reaction using the Applied Biosystems Model 477 Pulsed Liquid Sequencer at the Tufts University Protein Sequence Facility.

15. Phosphoamino acid analysis. The 78 kDa phosphoprotein was exhaustively digested with 50 μg/ml trypsin, 50 μg/ml chymotrypsin (Boehringer-Mannheim Biochemicals, Indianapolis, IN) in 25 mM ammonium bicarbonate for 18 hr at 37°C. The supernatant was then lyophilized, resuspended in about 20 μl of water and digested in a Kimble tube with 6.0 N HCl at 100°C for 2-3 hr . The digested sample was resuspended in the pH 1.9 buffer tank (2% formic acid, 8% acetic acid) and spotted on TLC plates along with phenol red as a tracer. Also spotted was about 1 μl of a 10 mg/ml stock solution of phospho-thr/ser/tyr along with phenol red. The TLC plate was then wetted with the pH 1.9 buffer. The first "half" dimension was run at 500 V until tracer dye was 2 cm short of the apex of the bent TLC plate. At this point, the plate was transferred to the pH 3.5 tank (10% acetic acid, 1% pyridine) and run at 400 V until tracer dye was about 3 cm from the plate edge. The plate was then removed and left to dry. Ninhydrin (0.25%) in n-butanol was used to develop the standards. The plates were then autoradiographed and the sample compared to the standards.

16. Kinase inhibitors. Radiolabeled mast cells were pretreated with different concentrations and for different times with various cell permeable serine/threonine kinase inhibitors which included inhibitors of PKA, PKG, PKC and calcium/calmodulin kinase II (CAMK IIMBIOMOL Research Labs, PA), followed by cromolyn for 30 sec at 37°C, lysed and run on one dimension SDS- PAGE. The relative specificity (Ki in μM) of these inhibitors was as follows: chelerythrine (PKC, 0.66), H-8 (PKA, 1.2; PKG, 0.48; PKC, 15), H-89 (PKA, 0.048; PKG, 0.48; PKC, 31.7; CAMK II, 29.7), KN-62 (CAMK II, 0.9), Staurosporine (PKA, 0.007; PKG, 0.0085; PKC, 0.0007) .

17. Cloning of moesin gene and production of antibodies against the expressed protein. The RBL moesin gene was subcloned into pET-28 vector and transformed into E . coli JM109 (DE3); after IPTG induction, a fusion protein with polyhistidine on the N-terminus was expressed. The protein was purified to homogeneity using a nickel column under denaturing conditions. The purified protein was injected into rabbits and polyclonal antisera were generated by I muno Dynamics (San Diego, Ca) and were used without further purification. Rabbit polyclonal anti- calf moesin serum was kindly supplied by Dr. Furthmayr. It crossreacted with both ezrin and moesin and recognized two bands in RBL cell lysate. A monoclonal antibody which recognizes both ezrin and moesin was purchased from Zymed.

18. Immunoprecipitation. Mast cells were loaded with [³²] Pi , treated with cromolyn and boiled for 5 min in 1% SDS. Double strength Inhibitor solution (PBS containing 20 mM NaPP, 100 mM NaF, 2 mM EGTA, 2 mM EDTA, 5% NP40 and protease inhibitors at a final concentration of 10 μg/ml) was added to the sample at 1:1 dilution. Proteins in the lysate were precleared by incubation for 20 min with 50 μl of Protein A linked to Sepharose . The clear supernatant was transferred to 10 μl of the rabbit anti-rat moesin polyclonal antibody and incubated for 30 more min. To the reaction mixture 50 μl of Protein A linked to Sepharose was added and incubated for another 30 min and spun at 10,000 xg for 1 min. The pellet was washed twice with 1 ml of SII (NaCl 150 mM, Hepes 15 mM, EDTA 1 mM, NP40 0.5%, pH 7.4) was added. To the pellet, 100 μl of Solubilizing SDS buffer was added, boiled for 5 min and loaded onto SDS PAGE gels.

19. Immunocvtochemistr . Purified mast cells were treated with 100 μM cromolyn for 30 sec and were immediately fixed in 4% paraformaldehyde. Frozen sections were cut at 7 μm and treated with 1:200 dilution of rabbit anti-rat moesin polyclonal antibody at room temperature for 1 hr . The sections were then incubated with 1:200 dilution of goat anti-rabbit IgG-biotin (Vector Labs, CA) for 30 min, followed by a further exposure to streptavidine- rhodamine (Pierce, Rockford, IL) for 30 min. The sections were then mounted in aqueous mounting medium and observed under a light microscope (Nikon, Don Santo Corp, Natick, MA) .

20. Rat PKC (a . β and γ mixture) . The isozyme mixture was purified from rat brain. Recombinant rabbit PKC a and γ expressed in Baculovirus system and purified to >90%, were all purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Phosphotatases were also purchased from Upstate Biotechnology Inc. Horseradish peroxidase conjugated-goat anti -rabbit IgG

(heavy and light chains) was purchased from Zymed Laboratories

(San Francisco, CA) . γ-³²P-ATP and α-³⁵S-dATP were purchased from

Amersham Co. (Arlington Heights, IL) . Cromolyn, the cation ionophore A23187, compound 48/80 (C48/80) and quercetin were purchased from Sigma (St. Louis, MO).

21. RBL 2H3 cell culture. RBL cells were kindly provided by Dr. Henry Metzger (NIH) and were grown in stationary cultures in D-MEM medium supplemented with 15% fetal bovine serum (Gibco BRL, Grand Island, NY), as previously described Tamir et al . , above .

22. Cloning the cDNA for the 78 kDa MC protein

A. Construction of cDNA Library. mRNA was purified from 5 x 10⁸ RBL cells. Random hexanucleotide primers and oligo (dT) primers were used for first strand synthesis. The cDNA library was constructed with λ ZapII vector at EcoR I site.

B. cDNA Library Screening. Two primers corresponding to human moesin cDNA sequences 1269-1292 and 1604-1579, respectively, were synthesized. The portion of human moesin cDNA (1269-1604) was amplified from human fetal brain library, labeled with ²P-dCTP (Amersham) using Radprime DNA labeling System (Gibco) and used as probe to screen RBL cDNA libraries. In brief, pBluescript SK plaques were transferred onto nylon filters (Amersham) and screened at 65 °C in Rapid-hyb buffer (Amersham) . The filters were washed with a final stringency of 0.1 x SSC, 0.1% SDS at 65°C. Positive plaques were converted to pBluescript SK using helper phage R408 according to the manufacturer's protocol.

C. Sequencing. Positive clones were sequenced with Bst

Polymerase (BioRad) . T3 and T7 primers, as well as synthesized primers according to the region already sequenced, were used to sequence the gene.

D. Expression of cDNA RBL moesin in E. coli .

One of the RBL moesin cDNA sequences cloned was amplified with primer CAC CAT GCC GAA GAC GAT C (SEQ ID NO: 8) and T3 primer, purified and subcloned into pCR II, then cleaved by EcoR I and subcloned into T7 expression vector pET28. The recombinant plasmid was then transformed into E. coli JM109(DE3). The expression was induced by IPTG, as previously described (20) . The moesin expressed was purified using a nickel column (Invitrogene) under denaturing conditions according to the manufacturer's protocol.

The purified moesin was dialyzed against phosphate- buffered saline (PBS) containing 1% Triton X-100, then against PBS without the detergent.

23. Cell Permeabilization. The RBL cells were harvested with a single trypsin treatment. The cells were washed three times with HEPES buffer (20 mM HEPES, pH 7.5, 10% sucrose, 1 mM PMSF) and resuspended in the buffer at 5xl0⁶ cells/ml. Digitonin was added at final concentration of 25 μg/ml. The cell suspension was kept on ice for 5 min, then centrifuged at top speed in a Brinkman centrifuge 5415. Digitonin generates lesions on the plasma membrane which are big enough to permit cytosol proteins to leak out of the cell, while structural elements like cytoskeleton and membrane proteins remain in place. Thus, after centrifugation, the supernatants represent cytosol contents, while pellets washed once with the HEPES buffer represent the membrane components. The permeabilization was monitored by trypan blue exclusion.

24. Western Blotting and Immunoprecipitation. Polyclonal antiserum was generated by immunizing rabbits with purified moesin which was expressed in E. coli . The Western blotting and immunoprecipitation were performed as described Correia et al . ,

Biochem . Pharm . Ill: in press (1996) .

25. Phosphorylation and Dephosphorylation in vi tro . Purified moesin expressed in E. coli was used in a reaction mixture containing 20 mM Tris-HCI (pH 7.5), 0.1 mM CaCl₂, 0.5 mM MgCl₂, 0.03% Triton X-100, 60 μg/ml diolein, 0.31 μg/ml phosphatidylserine, 25 ng PKC and 0.2 mCi/ml ³²P [ATP] (3000 Ci/mmol, Amersham). The mixture was incubated at 30°C for 10 min. For dephosphorylation, 0.1 unit of the phosphatase was added and incubated for another 10 min. The reaction was terminated by adding SDS-PAGE sample buffer and boiled for 5 min. The samples were analyzed by SDS-PAGE, the gel was dried and autoradiographed. 26. Phosphorylation in vivo . Mast cells were purified and loaded with [³²] Pi and treated with cromolyn as previously described. The cells were permeabilized with 25 μg/ml digitonin following which cytosol and membrane fractions were separated by centrifugation. The samples were divided and run on two separate SDS-PAGE, one for Western blotting and another for autoradiography .

27. Immunoblot analysis of PKC isozvmes. Immunoblot analysis was carried out on purified rat peritoneal mast cells and RBL cells using rabbit polyclonal antibodies to six different isoforms {a , β , y , δ e and f) at a concentration of 1:2000 (GIBCO BRL) . Mast cells or RBL cells (10⁶ cells per lane) were lysed, immediately boiled for 5 min, loaded on 7.5% SDS gels and were run overnight at 60V. The blots were cut in single lanes and were incubated overnight with 5 μl of the primary antibody (in 5 ml total volume) , followed by 20 μl of the secondary antibody, goat anti -rabbit -HRP (1:500 in 5 ml). Detection was with diaminobenzidine .

28. Immunocytochemistrv . Purified mast cells were treated with 100 μM cromolyn for 30 sec and were immediately fixed in 4% paraformaldehyde. Frozen sections were cut at 7 μm and treated with 1:200 dilution of rabbit anti -rat moesin polyclonal antibody at room temperature for 1 hr . The sections were then incubated with 1:200 dilution of goat anti-rabbit IgG-biotin (Vector Labs, CA) for 30 min, followed by a further exposure to streptavidine- rhodamine (Pierce, Rockford, IL) for 30 min. The sections were then mounted in aqueous mounting medium and observed under a light microscope (Nikon, Don Santo Corp, Natick, MA) .

EXAMPLE 2 Cloning of the RBL moesin cDNA

From 5 x 10⁵ plaques screened, 11 positive clones were obtained. The insertion sizes of the clones were between 1.5 to 3.5 kb (Fig. 1). When the ends of those clones were sequenced, 7 of them showed high homology with the human moesin cDNA. Those 7 clones overlapped and spanned the whole human moesin cDNA. Moreover, one of the clones, pRM9 had a poly (A:T) end, an indication that it was the 3' end non translation region of the corresponding mRNA (Fig. 1) .

EXAMPLE 3

Purification for sequencing of the 78 kDa phosphoprotei .

In radiolabeled MC treated with cromolyn (100 μM) , a 78 kDa protein was rapidly phosphorylated within 30 sec and was completely dephosphorylated in 5 min) . Two dimensional IEF was first used to purify this protein, but the 78 kDa phosphoprotein demonstrated considerable charge heterogeneity. Solubilization with NP-40 and CHAPS was not adequate, while loading the protein sample at the acidic end and reversing the polarity of the electrophoretic run (NEPHGE gels) also failed to introduce the protein fully into the gel. Extraction in phenol prevented aggregation and permitted solubilization in SDS, but due to the numerous steps involved, the 78 kDa phosphoprotein was finally directly solubilized in SDS and purified on an alternate two dimensional gel system. This protocol separated proteins primarily on the basis of their hydrophobicity in the first dimension and molecular weight in the second dimension. Sufficient quantity (40-60 pmols) of protein was obtained for sequencing.

EXAMPLE 4 Partial amino acid sequence.

Internal sequence was obtained by electroelution of the phosphoprotein after cyanogen bromide treatment. The fragments generated were then separated on a 10% Tricine-SDS-PAGE system, electroblotted onto PVDF, stained and autoradiographed. Two radiolabeled fragments of molecular weights 18 and 24 kDa were cut out and sequenced. Twelve amino acids from the N terminus of the 24 kDa fragment (M, D, A, E, L, E, F, A, I, Q, P, N SEQ ID NO:9) showed 100% homology to the N terminus 12-23 of mouse moesin residues 12-23 of SEQ ID NO: 2) , ezrin and radixin. Sixteen amino acids from the N terminus of the 18 kDa fragment (M, E, R, A, L, L, E, N, E, K, K, K, R, E, L, A SEQ ID NO:10) showed 100% homology to region 318-333 of mouse moesin residues 318-333 of SEQ ID N0:2), 81% to mouse radixin and 64% to mouse ezrin.

EXAMPLE 5 Phosphoa ino acid analysis.

Phosphoamino acid analysis revealed that after cromolyn treatment, moesin was phosphorylated primarily on serine (with much less on threonine) residues, thus implicating a serine/threonine kinase and phosphatase in regulation of its phosphorylation .

EXAMPLE 6 Sequencing of the rat moesin cDNA

The nucleotide sequence and deduced amino acid sequence of the rat moesin cDNA (SEQ ID NOS: 1-2) are shown on Fig. 2. The protein contains 577 amino acids, same as human moesin, and has 99% homology with human moesin. The calculated molecular mass of the protein was 67.3 kDa and the pi was 6.37.

PKC can recognize specific motifs and phosphorylate serine and threonine residues within them. These are (R/Kl-3, X2-0)- S/T-(X²-⁸, R/K^1"3), S/T(X²-°, R/K^1"3) and R/K^1"3, X₂^)-S/T. When the deduced amino acid sequence was searched for those phosphorylation sites, twelve sites were found, which are shown in Fig. 3. These twelve sequences are apparently conserved between the species, as sequences from different species published showed little variation in those regions. It is worth noticing that one of the putative phosphorylation sites, 558*' is located on the consensus actin binding site KYKXL (SEQ ID NO: 11) , suggesting a possible effect of phosphorylation on actin binding. EXAMPLE 7 Expression of the RBL moesin cDNA in E. coli

The coding region of the moesin cDNA was cloned in expression vector pET-28. When E. coli JM109(DE3) with the recombinant plasmid (pET28-RMl) were treated with IPTG, which induced synthesis of T7 RNA polymerase and in turn led to the expression of cloned gene, the E. coli produced a protein that appeared by SDS-PAGE to have a molecular mass of about 78 kDa. This protein represented almost 50% of total E . col i protein and was purified to homogeneity with a nickel column under denaturing conditions .

EXAMPLE 8 Phosphorylation and dephosphorylation of moesin in vi tro.

Moesin purified from E. coli did not have any major hydrophobic regions according to its sequence, suggesting that it probably would not refold properly in solution. It was dissolved in PBS containing 1% Triton X-100 but precipitated in PBS without detergent, suggesting that the solubility of the protein was low. The precipitated moesin was again dissolved in PBS containing 1% Triton, was dialyzed against PBS without detergent, then centrifuged to remove the precipitant. About 75% of precipitated moesin was dissolved by this method.

When the dissolved protein was incubated with purified PKC (a , β , and γ isozyme mixture) utilizing the Triton X-100 micelle method, a strong phosphorylated band representing a protein with molecular mass about 78 kDa could be seen by autoradiography . Since PKC autophosphorylates itself and its molecular mass is similar to moesin, immunoprecipitation was performed after the reaction and confirmed that the phosphorylated protein was moesin (data not shown) .

In order to investigate which isozyme of PKC may be able to phosphorylate moesin in vi tro, we used the only two commercially available isozymes of rabbit PKC, isozymes a and γ. Both isozymes could phosphorylate moesin in vi tro without any obvious difference .

To explore the phosphatase responsible for the dephosphorylation of moesin in vi tro, we tested phosphatase- 1, phosphatase-2A and phosphatase-2B (calcineurin) . Moesin phosphorylated in vi tro could be dephosphorylated by all of them again with no apparent obvious difference.

EXAMPLE 9 Effect of calcium ions on phosphorylation of moesin in rat mast cells

Phosphorylation of moesin in rat mast cells was compared under different conditions in the presence or absence of calcium ions. The cation ionophore A23187, which is a powerful stimulus of mast cell secretion, induced phosphorylation of the 68, 59 and 42 kD proteins associated with secretion but not the 78 kD protein (moesin) which may be linked to termination of secretion. Addition of 6 μM cromolyn which is sufficient to induce phosphorylation of moesin could not do so in the presence of A23187. C48/80 clearly induced phosphorylation of moesin both in the presence and absence of calcium. Interestingly, addition of cromolyn with C48/80 could not increase the phosphorylation of moesin any more than C48/80 alone. It was, however, evident that cromolyn could induce phosphorylation of moesin in the absence of extracellular calcium.

In order to confirm that phosphorylation of moesin was in fact calcium- independent , mast cells were treated with 6 μM cromolyn for 30 sec in the absence of added calcium, after increasing time of pre-incubation with 1 mM EDTA. Phosphorylation of moesin was present even after 30 min pre- incubation with EDTA to chelate intracellular calcium ions. Phosphorylation of moesin could not be induced after 2 hr pre- incubation with EDTA by which time more than 50% of the mast cells had lost viability, as judged by Trypan blue exclusion.

EXAMPLE 10 Presence of PKC isozymes in rat mast cells

The presence of PKC isozymes was explored using Western blot analysis in purified rat peritoneal mast cells and the a , β , δ and f isozymes were identified. The γ and e isozymes could not be identified in rat peritoneal mast cells even though the e isozyme was present in RBL cells (results not shown) . The α and β isozymes are calcium-dependent, while the others are calcium- independent. Ozawa et al . , J. Biol . Chem . 268:1749 (1993). However, the β and δ isozymes are linked to stimulation cf secretion, while the α-^* and e to its inhibition. Ozawa et al . , (1993) above. Consequently, it must be the zeta isozyme that is involved with the phosphorylation of moesin in MC, an action not reported previously. Zhou et al . , Exp . Cell Res . , 214:1 (1994).

EXAMPLE 11

Inhibitors of Serine/Threonine protein kinases.

Pretreatment with chelerythrine which is selective for PKC inhibition prevented incorporation of phosphate into the 78 kDa protein by cromolyn. Pretreatment at 100 μM fo.r 1 hr with H-8 which is selective for PKA and PKG, H-89 which is selective for PKA and KN-62 which is selective for CAMK II had no effect on 78 kDa phosphorylation. Pretreatment with sphingosine (100 μM for 2 min), a specific inhibitor of PKC, and staurosporine (0.1 μM for 15 min) , a frequently used potent but non specific PKC inhibitor, also completely prevented the incorporation of phosphate into the 78 kDa protein. These results implicated PKC in cromolyn' s phosphorylation of the 78 kDa protein. EXAMPLE 12 Specificity of anti-rat moesin polyclonal antibody.

Western analysis performed on total RBL lysate with anti -rat moesin serum recognized a single band. Immunoprecipitation of RBL cell lysate with anti-rat moesin and probing with a polyclonal anti-calf moesin/ezrin serum (supplied by Dr. Furthmayr) demonstrated that the anti -rat moesin serum immunoprecipitated only moesin. In contrast, the anti-calf moesin/ezrin serum recognized two bands in RBL cell lysate. Western blotting of total MC lysate with a monoclonal serum which recognizes both moesin and ezrin showed the prominent band in RBL cells was ezrin, while in MC it was moesin.

EXAMPLE 13

Immunoprecipitation.

Radiolabeled control or cromolyn- treated MC extracts were first immunoprecipitated with anti-rat moesin serum and the precipitate was analyzed by SDS-PAGE. Autoradiography revealed a single phosphorylated 78 kDa protein. Immunoprecipitation removed all of the 78 kDa phosphoprotein from the supernatant fluid, thus indicating that the phosphoprotein could not be simply "co-precipitated" along with moesin.

EXAMPLE 14 Western blot analysis of the distribution of moesin in permeabilized RBL and rat mast cells

When RBL cells were permeabilized with digitonin (25 μg/ml) , about 50% of moesin was in the soluble fraction. Similar results were obtained with rat peritoneal mast cells. Increasing concentrations of NaCl (0, 150, 500 mM, respectively) resulted in more moesin recovered in the soluble fraction, suggesting that moesin was associated with the membrane/cytoskeleton non- covalently which could be disrupted by high salt. The influence of Mg²⁺ and Ca²⁺ on the binding of moesin to the membranous fraction was also investigated, but there was had no obvious effect (data not shown). In mast cells loaded with [³²] Pi and treated with cromolyn, moesin identified by Western blot analysis was present in both the cytosol and membrane fractions without any obvious difference between control samples or those treated with cromolyn. However, phosphorylated moesin identified by autoradiography was primarily found in the precipitable fraction suggesting that it binds firmly to the membrane/cytoskeleton.

EXAMPLE 15

Localization

The specific anti-rat moesin clearly localized the 78 kDa protein at discrete punctate structures at the plasma membrane and no cytoplasmic staining was observed. Treatment with cromolyn did not alter its distribution.

EXAMPLE 16 Immunocytochemical localization of moesin in rat peritoneal mast cells and RBL cells

Using the polyclonal rabbit anti-rat moesin serum, moesin was localized at the cytoplasmic side of the plasma membrane. The distribution was not homogeneous, but had a rather punctate appearance. Two-day old RBL cells had weak cytoplasmic positivity but failed to show any moesin associated with the plasma membrane. Cell -surface associated moesin was present in about 20% of six day-old confluent cells, and increased to over 50% in six day-old cells grown in the presence of 5 x 10^"5 M quercetin, a phosphorylation situation for moesin. These results indicate that proper localization can be stimulated in mature differentiated MC as maturation is known to be induced by quercitin. Tronsky et al . , Biochem. Pharm . , 46: 2315 (1993). EXAMPLE 17 Immunocytochemical localization of moesin and _/8-actin in rat mast cells

Double immunocytochemistry was used with the polyclonal rabbit anti-moesin serum ( fluorescein) and anti-jS-actin serum (rhodamine) . The distribution of moesin and 3-actin was almost identical suggesting that there may also be some functional relationship between the two.

EXAMPLE 18 Mechanism of action

Phosphorylation of MC moesin by PKC isozyme theta occurs on specific serine/threonine residues (Ser₅₆, Thr₆₆, Ser₇₄, Ser₃₇₄, and

Thr_5Sg) permitting a change in the configuration of the protein that allows an interaction with actin-containing cytoskeleton, thus forming a physical barrier to exocytosis and secretion

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: THEOHARIDES, Thβoharis C.

(ii) TITLE OF INVENTION: ISOLATED AND CLONED MAST CELL 78 KDA

PHOSPHOPROTEIN (MAST CELL DEGRANULATION INHIBITORY AGENT) AND USE THEREOF

(iii) NUMBER OF SEQUENCES: 11

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Foley & Lardner

(B) STREET: 3000 K Street, N.W. , Suite 500

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: USA (F) ZIP: 20007-5109

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/631,184 (B) FILING DATE: 12-APR-1996

(C) CLASSIFICATION:

(viii) ATTORNE /AGENT INFORMATXON: (A) NAME: BLECHER, Melvin (B) REGISTRATION NUMBER: 33,649

(C) REFERENCE/DOCKET NUMBER: 74579/101

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (202)672-5300 (B) TELEFAX: (202)672-5399

(C) TELEX: 904136

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2099 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 99..1829

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCACCCGGTT CTCCCGAGTC CTTGCCGAAG TGTACTGCGC GCTCAGAGCA GCCCGACAGT 60

TTCCAGCCAA ACTTCAGCAA CTCCTCCCTT GCCGCACC ATG CCG AAG ACG ATC 113

Met Pro Lys Thr Ile 1 5 AGT GTT CGT GTA ACC ACC ATG GAT GCA GAG CTG GAG TTT GCC ATT CAG 161 Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu Glu Phe Ala Ile Gin 10 15 20 CCC AAC ACC ACT GGC AAG CAG CTG TTT GAC CAG GTG GTG AAA ACT ATT 209 Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin Val Val Lys Thr Ile 25 30 35

GGT TTG AGA GAA GTT TGG TTC TTT GGT CTG CAG TAC CAG GAC ACA AAA 257 Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin Tyr Gin Asp Thr Lys 40 45 50

GCT TTC TCT ACT TGG CTG AAA CTC AAT AAG AAG GTG ACT GCA CAG GAT 305 Ala Phe Ser Thr Trp Leu Lys Leu Asn Lye Lys Val Thr Ala Gin Asp 55 60 65

GTC CGG AAG GAA AGT CCG TTG CTC TTC AAG TTC CGG GCC AAG TTC TAT 353

Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe Arg Ala Lys Phe Tyr

70 75 80 85

CCA GAG GAT GTG TCT GAA GAA CTG ATT CAG GAT ATC ACC CAG CGC CTG 401

Pro Glu Asp Val Ser Glu Glu Leu lie Gin Asp Ile Thr Gin Arg Leu

90 95 100 TTC TTT CTG CAA GTG AAG GAG GGC ATT CTC AAT GAT GAC ATT TAT TGT 449

Phe Phe Leu Gin Val Lys Glu Gly Ile Leu Asn Asp Asp Ile Tyr Cys 105 110 115

CCA CCT GAA ACC GCT GTG CTC TTG GCG TCC TAT GCC GTC CAG TCT AAG 497 Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr Ala Val Gin Ser Lys

120 125 130

TAT GGT GAC TTC AAC AAG GAA GTG CAC AAG TCT GGC TAC CTG GCT GGA 545 Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser Gly Tyr Leu Ala Gly 135 140 145

GAT AAG TTG CTT CCC CAA AGA GTC TTG GAG CAG CAC AAA CTC AAC AAG 593

Asp Lys Leu Leu Pro Gin Arg Val Leu Glu Gin His Lys Leu Asn Lys 150 155 160 165

GAC CAG TGG GAA GAG CGG ATC CAG GTA TGG CAT GAG GAA CAC CGG GGC 641

Asp Gin Trp Glu Glu Arg Ile Gin Val Trp His Glu Glu His Arg Gly 170 175 180 ATG CTC AGG GAG GAT GCT GTC CTG GAA TAT CTC AAG ATT GCT CAG GAC 689

Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu Lys Ile Ala Gin Asp 185 190 195

CTG GAG ATG TAT GGT GTG AAC TAT TTC AGC ATC AAG AAC AAG AAA GGC 737 Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile Lys Asn Lys Lys Gly 200 205 210

TCA GAG CTG TGG CTG GGT GTG GAT GCC TTG GGT CTC AAC ATC TAT GAG 785 Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly Leu Asn Ile Tyr Glu 215 220 225

CAG AAT GAC AGA TTG ACT CCT AAG ATT GGC TTC CCT TGG AGT GAA ATC 833

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

230 235 240 245

AGG AAC ATC TCT TTC AAT GAT AAG AAA TTT GTC ATC AAG CCT ATT GAC 881

Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val Ile Lys Pro Ile Asp

250 255 260 AAA AAG GCC CCG GAC TTT GTG TTC TAT GCT CCT CGG CTT CGG ATT AAC 929 Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro Arg Leu Arg Ile Asn 265 270 275 AAA AGG ATC TTG GCC CTG TGC ATG GGG AAT CAT GAG CTG TAC ATG CGT 977 Lys Arg Ile Leu Ala Leu Cys Met Gly Asn His Glu Leu Tyr Met Arg 280 285 290 CGG CGA AAG CCT GAC ACC ATT GAG GTG CAG CAG ATG AAG GCC CAG GCT 1025 Arg Arg Lys Pro Asp Thr Ile Glu Val Gin Gin Met Lys Ala Gin Ala 295 300 305

CGA GAA GAG AAG CAC CAG AAG CAG ATG GAG CGT GCT CTA CTG GAA AAT 1073 Arg Glu Glu Lys His Gin Lys Gin Met Glu Arg Ala Leu Leu Glu Asn 310 315 320 325

GAG AAG AAG AAG CGT GAG CTG GCT GAA AAA GAG AAG GAG AAG ATT GAA 1121 Glu Lys Lys Lys Arg Glu Leu Ala Glu Lys Glu Lys Glu Lys Ile Glu 330 335 340

CGG GAG AAG GAA GAG CTG ATG GAG AAG CTG AAG CAG ATT GAG GAG CAG 1169 Arg Glu Lys Glu Glu Leu Met Glu Lys Leu Lys Gin Ile Glu Glu Gin 345 350 355

ACC AAG AAG GCT CAG CAA GAA CTG GAA GAA CAG ACC CGC AGG GCC CTA 1217 Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin Thr Arg Arg Ala Leu 360 365 370 GAA CTT GAG CAG GAA CGG AAG CGT GCC CAG AGT GAG GCT GAA AAG CTG 1265

Glu Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser Glu Ala Glu Lys Leu 375 380 385

GCC AAG GAG CGT CAA GAA GCT GAA GAA GCC AAG GAG GCC CTG CTG CAG 1313 Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys Glu Ala Leu Leu Gin

390 395 400 405

GCT TCT CGG GAC CAG AAG AAG ACT CAG GAA CAG CTG GCT TCA GAA ATG 1361 Ala Ser Arg Asp Gin Lys Lys Thr Gin Glu Gin Leu Ala Ser Glu Met 410 415 420

GCA GAG CTG ACA GCA CGG GTC TCC CAG TTG GAA ATG GCT CGA AAG AAG 1409

Ala Glu Leu Thr Ala Arg Val Ser Gin Leu Glu Met Ala Arg Lys Lys 425 430 435

AAG GAG AGT GAG GCT GAG GAA TGC CAC CAA AAG GCC CAG ATG GTC CAG 1457

Lys Glu Ser Glu Ala Glu Glu Cys His Gin Lys Ala Gin Met Val Gin 440 445 450 GAA GAC TTG GAG AAG ACT CGC GCT GAG CTG AAG ACA GCC ATG AGT ACC 1505

Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys Thr Ala Met Ser Thr 455 460 465

CCT CAT GTG GCA GAG CCT GCT GAG AAT GAA CAT GAT GAG CAG GAT GAG 1553 Pro His Val Ala Glu Pro Ala Glu Asn Glu His Asp Glu Gin Asp Glu 470 475 480 485

AAT GGA GCC GAG GCC AGT GCT GAG CTG CGG GCT GAT GCT ATG GCC AAG 1601 Asn Gly Ala Glu Ala Ser Ala Glu Leu Arg Ala Asp Ala Met Ala Lys 490 495 500

GAC CGC AGT GAG GAA GAA CGT ACC ACT GAG GCA GAG AAG AAT GAG CGT 1649 Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala Glu Lys Asn Glu Arg 505 510 515

GTG CAG AAG CAT CTT AAG GCC CTT ACT TCA GAG CTG GCC AAT GCC CGA 1697 Val Gin Lys His Leu Lys Ala Leu Thr Ser Glu Leu Ala Asn Ala Arg 520 525 530 GAT GAG TCC AAG AAG ACT ACC AAT GAC ATG ATC CAT GCT GAG AAC ATG 1745 Asp Glu Ser Lys Lys Thr Thr Asn Asp Met Ile His Ala Glu Asn Met 535 540 545 CGA CTG GGA CGA GAC AAA TAC AAG ACC CTG CGC CAG ATC CGG CAG GGC 1793 Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg Gin Ile Arg Gin Gly 550 555 560 565

AAT ACC AAA CAG CGC ATT GAT GAA TTT GAG TCC ATG TAGTGGGCGC 1839

Asn Thr Lys Gin Arg Ile Asp Glu Phe Glu Ser Met 570 575

GCAGCCGTTA GGGACCCCTC CTCCTTCTTC CTTGTCCCCC ACACTCCTAT AGTTTTGCCT 1899

AACTAACACC CTGCTGGAGC CACTAACTAG AAAAGCCTTG GAGCCATACC AAACATTCAG 1959

TATGACCATG GGACCAAATT TAGTTTCCCT ATTCATATCC TTGGGCAAAC AAATGGCCCA 2019 CCTGTGTAGC CAATGGAACC TCCTCTTCCT TCTTTGTCAC ACTCATTCAA CATAGCTCTC 2079

TAGAATAGAC CATTTCCCCC 2099

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 577 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Pro Lys Thr He Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15

Glu Phe Ala Ile Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin 20 25 30

Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin

35 40 45 Tyr Gin Asp Thr Lys Ala Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys

50 55 60

Val Thr Ala Gin Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe

65 70 75 80

Arg Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gin Asp

85 90 95

Ile Thr Gin Arg Leu Phe Phe Leu Gin Val Lys Glu Gly Ile Leu Asn 100 105 110

Asp Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr 115 120 125 Ala Val Gin Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser 130 135 140

Gly Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gin Arg Val Leu Glu Gin 145 150 155 160

His Lys Leu Asn Lys Asp Gin Trp Glu Glu Arg Ile Gin Val Trp His 165 170 175

Glu Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu 180 185 190 Lys Ile Ala Gin Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile 195 200 205

Lys Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly 210 215 220

Leu Asn Ile Tyr Glu Gin Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe 225 230 235 240 Pro Trp Ser Glu Ile Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val

245 250 255

Ile Lys Pro Ile Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro 260 265 270

Arg Leu Arg Ile Asn Lys Arg Ile Leu Ala Leu Cys Met Gly Asn His 275 280 285

Glu Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr lie Glu Val Gin Gin 290 295 300

Met Lys Ala Gin Ala Arg Glu Glu Lys His Gin Lys Gin Met Glu Arg

305 310 315 320 Ala Leu Leu Glu Asn Glu Lys Lys Lys Arg Glu Leu Ala Glu Lys Glu

325 330 335

Lys Glu Lys Ile Glu Arg Glu Lys Glu Glu Leu Met Glu Lys Leu Lys 340 345 350

Gin Ile Glu Glu Gin Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin 355 360 365

Thr Arg Arg Ala Leu Glu Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser 370 375 380

Glu Ala Glu Lys Leu Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys 385 390 395 400 Glu Ala Leu Leu Gin Ala Ser Arg Asp Gin Lys Lys Thr Gin Glu Gin

405 410 415

Leu Ala Ser Glu Met Ala Glu Leu Thr Ala Arg Val Ser Gin Leu Glu 420 425 430

Met Ala Arg Lys Lys Lys Glu Ser Glu Ala Glu Glu Cys His Gin Lys* 435 440 445

Ala Gin Met Val Gin Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys 450 455 460

Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu His 465 470 475 480 Asp Glu Gin Asp Glu Asn Gly Ala Glu Ala Ser Ala Glu Leu Arg Ala

485 490 495

Asp Ala Met Ala Lys Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala 500 505 510

Glu Lys Asn Glu Arg Val Gin Lys His Leu Lys Ala Leu Thr Ser Glu 515 520 525

Leu Ala Asn Ala Arg Asp Glu Ser Lys Lys Thr Thr Asn Asp Met Ile 530 535 540 His Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg 545 550 555 560

Gin Ile Arg Gin Gly Asn Thr Lys Gin Arg Ile Asp Glu Phe Glu Ser 565 570 575

Met

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 576 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Pro Lys Thr Ile Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15

Glu Phe Ala Ile Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin 20 25 30 Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin

35 40 45

Tyr Gin Asp Thr Lys Ala Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys 50 55 60

Val Thr Ala Gin Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe 65 70 75 80

Arg Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu lie Gin Asp 85 90 95 lie Thr Gin Arg Leu Phe Phe Leu Gin Val Lys Glu Gly Ile Leu Asn

100 105 110 Asp Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr

115 120 125

Ala Val Gin Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser

130 135 140

Gly Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gin Arg Val Leu Glu Gin

145 150 155 160

His Lys Leu Asn Lys Asp Gin Trp Glu Glu Arg Ile Gin Val Trp His 165 170 175

Glu Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu 180 185 190 Lys Ile Ala Gin Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile

195 200 205

Lys Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly 210 215 220

Leu Aβn Ile Tyr Glu Gin Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe

225 230 235 240 Pro Trp Ser Glu Ile Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val 245 250 255

Ile Lys Pro Ile Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro 260 265 270

Arg Leu Arg Ile Asn Lys Arg Ile Leu Ala Leu Cys Met Gly Asn His 275 280 285 Glu Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr Ile Glu Val Gin Gin

290 295 300

Met Lys Ala Gin Ala Arg Glu Glu Lys His Gin Lys Gin Met Glu Arg 305 310 315 320

Ala Leu Leu Glu Asn Glu Lys Lys Lys Arg Glu Val Ala Glu Lys Glu 325 330 335

Lys Glu Lys lie Glu Arg Glu Lys Glu Glu Leu Met Glu Lys Leu Lys 340 345 350

Gin Ile Glu Glu Gin Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin 355 360 365 Thr Arg Ser Pro Leu Glu Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser

370 375 380

Glu Ala Glu Lys Leu Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys 385 390 395 400

Glu Ala Leu Leu Gin Ala Ser Arg Asp Gin Lys Lys Thr Gin Glu Gin 405 410 415

Leu Ala Ser Glu Met Ala Glu Leu Thr Ala Arg lie Ser Gin Leu Glu 420 425 430

Met Ala Arg Lys Lys Lys Glu Ser Glu Ala Val Glu Trp Gin Gin Lys

435 440 445 Ala Gin Met Val Gin Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys

450 455 460

Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu His 465 470 475 480

Asp Glu Gin Asp Glu Asn Gly Ala Glu Ala Ser Ala Leu Arg Ala Asp 485 490 495

Ala Met Ala Lys Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala Glu 500 505 510

Lys Asn Glu Arg Val Gin Lys His Leu Lys Ala Leu Thr Ser Glu Leu 515 520 525 Ala Asn Ala Arg Asp Glu Ser Lys Lys Thr Ala Asn Asp Met Ile His

530 535 540

Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg Gin 545 550 555 560

Ile Arg Gin Gly Asn Thr Lys Gin Arg Ile Asp Glu Phe Glu Ser Met 565 570 575

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 576 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Pro Lys Thr Ile Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15 Glu Phe Ala lie Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin

20 25 30

Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin 35 40 45

Tyr Gin Asp Thr Lys Ala Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys 50 55 60

Val Thr Ala Gin Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe 65 70 75 80

Arg Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gin Asp 85 90 95 Ile Thr Gin Arg Leu Phe Phe Leu Gin Val Lys Glu Gly Ile Leu Asn

100 105 110

Asp Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr 115 120 125

Ala Val Gin Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser 130 135 140

Gly Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gin Arg Val Leu Glu Gin 145 150 155 160

His Lys Leu Asn Lys Asp Gin Trp Glu Glu Arg Ile Gin Val Trp His

165 170 175 Glu Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu

180 185 190

Lys Ile Ala Gin Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile 195 200 205

Lys Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly 210 215 220

Leu Asn Ile Tyr Glu Gin Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe 225 230 235 240

Pro Trp Ser Glu Ile Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val 245 250 255 Ile Lys Pro Ile Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro

260 265 270

Arg Leu Arg Ile Asn Lys Arg lie Leu Ala Leu Cys Met Gly Asn His 275 280 285

Glu Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr Ile Glu Val Gin Gin 290 295 300 Met Lys Ala Gin Ala Arg Glu Glu Lys His Gin Lys Gin Met Glu Arg 305 310 315 320

Ala Leu Leu Glu Asn Glu Lys Lys Lys Arg Glu Met Ala Glu Lys Glu 325 330 335

Lys Glu Lys Ile Glu Arg Glu Lys Glu Glu Leu Met Glu Lys Leu Lys 340 345 350 Gin Ile Glu Glu Gin Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin

355 360 365

Thr Arg Arg Ala Leu Glu Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser 370 375 380

Glu Ala Glu Lys Leu Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys 385 390 395 400

Glu Ala Leu Leu Gin Ala Ser Arg Asp Gin Lys Lys Thr Gin Glu Gin 405 410 415

Leu Ala Ser Glu Met Ala Glu Leu Thr Ala Arg lie Ser Gin Leu Glu 420 425 430 Met Ala Arg Lys Lys Lys Glu Ser Glu Ala Glu Glu Cys His Gin Lys

435 440 445

Ala Gin Met Val Gin Glu Asp Leu Glu Lye Thr Arg Ala Glu Leu Lys 450 455 460

Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu His 465 470 475 480

Asp Glu Gin Asp Glu Asn Gly Ala Glu Ala Ser Ala Leu Arg Ala Asp 485 490 495

Ala Met Ala Lys Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala Glu

500 505 510 Lys Asn Glu Arg Val Gin Lys His Leu Lys Ala Leu Thr Ser Glu Leu

515 520 525

Ala Asn Ala Arg Asp Glu Ser Lys Lys Thr Thr Asn Asp Met Ile His 530 535 540

Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg Gin 545 550 555 560 lie Arg Gin Gly Asn Thr Lys Gin Arg He Asp Glu Phe Glu Ser Met 565 570 575

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 576 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Pro Lys Thr Ile Asn Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15

Glu Phe Ala lie Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin 20 25 30

Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin 35 40 45

Tyr Gin Asp Thr Lys Gly Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys 50 55 60 Val Thr Ala Gin Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe

65 70 75 80

Arg Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gin Asp

85 90 95

Ile Thr Gin Arg Leu Phe Phe Leu Gin Val Lys Glu Gly Ile Leu Asn

100 105 110

Asp Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr 115 120 125

Ala Val Gin Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser 130 135 140 Gly Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gin Arg Val Leu Glu Gin

145 150 155 160

His Lys Leu Asn Lys Asp Gin Trp Glu Glu Arg Ile Gin Val Trp His 165 170 175

Glu Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu 180 185 190

Lys lie Ala Gin Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ser 195 200 205

Lys Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly

210 215 220 Leu Asn lie Tyr Glu Gin Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe

225 230 235 240

Pro Trp Ser Glu Ile Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val 245 250 255

Ile Lys Pro lie Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro 260 265 270

Arg Leu Arg Ile Asn Lys Arg Ile Leu Ala Leu Cys Met Gly Asn His 275 280 285

Glu Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr Ile Glu Val Gin Gin 290 295 300 Met Lys Ala Gin Ala Arg Glu Glu Lys His Gin Lys Gin Met Glu Arg

305 310 315 320

Ala Leu Leu Glu Asn Glu Lye Lys Lys Arg Glu Met Ala Glu Lys Glu 325 330 335

Lys Glu Lys Ile Glu Arg Glu Lys Glu Glu Leu Met Glu Arg Leu Lys 340 345 350 Gln Ile Glu Glu Gin Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin 355 360 365

Thr Arg Arg Ala Leu Ala Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser 370 375 380

Glu Ala Glu Lys Leu Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys 385 390 395 400 Glu Ala Leu Leu Lys Ala Ser Arg Asp Gin Lys Lys Thr Gin Glu Gin

405 410 415

Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg Ile Ser Gin Leu Glu 420 425 430

Met Ala Arg Gin Lys Lys Glu Ser Glu Ala Ala Glu Trp Gin Gin Lys 435 440 445

Ala Gin Met Val Gin Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys 450 455 460

Thr Ala Met Ser Thr Pro His Gly Ala Glu Pro Ala Glu Asn Glu Gin 465 470 475 480 Asp Glu Gin Asp Glu Asn Gly Ala Glu Ala Ser Ala Leu Arg Ala Asp

485 490 495

Ala Met Ala Lys Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala Glu 500 505 510

Lys Asn Glu Arg Val Gin Lys His Leu Lys Ala Leu Thr Ser Glu Leu 515 520 525

Ala Asn Ala Arg Asp Glu Ser Lys Lys Thr Ala Asn Asp Met Ile His 530 535 540

Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg Gin 545 550 555 560 Ile Arg Gin Gly Asn Thr Lys Gin Arg Ile Asp Glu Phe Glu Ser Met

565 570 575

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 576 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Pro Lys Thr Ile Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15

Glu Phe Ala Ile Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin 20 25 30

Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin 35 40 45 Tyr Gin Asp Thr Lys Gly Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys 50 55 60

Val Thr Ala Gin Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe 65 70 75 80

Arg Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gin Asp 85 90 95 He Thr Gin Arg Leu Phe Phe Leu Gin Val Lys Glu Gly Ile Leu Asn

100 105 110

Asp Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr

115 120 125

Ala Val Gin Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser 130 135 140

Gly Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gin Arg Val Leu Glu Gin 145 150 155 160

His Lys Leu Asn Lys Asp Gin Trp Glu Glu Arg Ile Gin Val Trp His 165 170 175 Glu Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu

180 185 190

Lys Ile Ala Gin Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile 195 200 205

Lys Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly 210 215 220

Leu Asn Ile Tyr Glu Gin Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe 225 230 235 240

Pro Trp Ser Glu Ile Arg Asn lie Ser Phe Asn Asp Lys Lys Phe Val 245 250 255 Ile Lys Pro Ile Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro

260 265 270

Arg Leu Arg Ile Asn Lys Arg Ile Leu Ala Leu Cys Met Gly Asn His 275 280 285

Glu Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr Ile Glu Val Gin Gin 290 295 300

Met Lys Ala Gin Ala Arg Glu Glu Lys His Gin Lys Gin Met Glu Arg 305 310 315 320

Ala Leu Leu Glu Asn Glu Lys Lys Lys Arg Glu Met Ala Glu Lys Glu 325 330 335 Lys Glu Lys lie Glu Arg Glu Lys Glu Glu Leu Met Glu Arg Leu Lys

340 345 350

Gin Ile Glu Glu Gin Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin 355 360 365

Thr Arg Arg Ala Leu Glu Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser 370 375 380

Glu Ala Glu Lys Leu Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys 385 390 395 400 Glu Ala Leu Leu Gin Ala Ser Arg Asp Gin Lys Lys Thr Gin Glu Gin 405 410 415

Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg Ile Ser Gin Leu Glu 420 425 430

Met Ala Arg Gin Lys Lys Glu Ser Glu Ala Val Glu Trp Gin Gin Lys 435 440 445 Ala Gin Met Val Gin Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys

450 455 460

Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu Gin

465 470 475 480

Asp Glu Gin Asp Glu Asn Gly Ala Glu Ala Ser Ala Leu Arg Ala Asp

485 490 495

Ala Met Ala Lye Asp Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala Glu 500 505 510

530 535 540

Ala Glu Asn Met Arg Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg Gin 545 550 555 560 lie Arg Gin Gly Asn Thr Lys Gin Arg lie Asp Glu Phe Glu Ser Met 565 570 575

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 571 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Pro Lys Thr Ile Ser Val Arg Val Thr Thr Met Asp Ala Glu Leu 1 5 10 15

Glu Phe Ala lie Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin 20 25 30 Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin

35 40 45

Tyr Gin Asp Thr Lys Phe Ser Thr Trp Leu Lys Leu Asn Lys Lys Val 50 55 60

Thr Ala Gin Asp Val Arg Lys Glu Ser Pro Leu Leu Phe Lys Phe Arg 65 70 75 80

Ala Lys Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gin Asp Ile 85 90 95 Thr Gin Arg Leu Phe Phe Leu Gin Val Lys Glu Gly lie Leu Asn Asp 100 105 110

Asp Ile Tyr Cys Pro Pro Glu Thr Ala Val Leu Leu Ala Ser Tyr Ala 115 120 125

Val Gin Ser Lys Tyr Gly Asp Phe Asn Lys Glu Val His Lys Ser Gly 130 135 140 Tyr Leu Ala Gly Asp Lys Leu Leu Pro Gin Arg Val Leu Glu Gin His

145 150 155 160

Lys Leu Asn Lys Asp Gin Trp Glu Glu Arg Ile Gin Val Trp His Glu

165 170 175

Glu His Arg Gly Met Leu Arg Glu Asp Ala Val Leu Glu Tyr Leu Lys

180 185 190 lie Ala Gin Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ile Lys 195 200 205

Asn Lys Lys Gly Ser Glu Leu Trp Leu Gly Val Asp Ala Leu Gly Leu 210 215 220 Asn Ile Tyr Glu Gin Asn Asp Arg Leu Thr Pro Lys Ile Gly Phe Pro

225 230 235 240

Trp Ser Glu Ile Arg Asn Ile Ser Phe Asn Asp Lys Lys Phe Val Ile

245 250 255

Lys Pro Ile Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro Arg

260 265 270

Leu Arg Ile Asn Lys Arg lie Leu Ala Leu Cys Met Gly Asn His Glu 275 280 285

Leu Tyr Met Arg Arg Arg Lys Pro Asp Thr Ile Glu Val Gin Gin Met 290 295 300 Lys Ala Gin Ala Arg Glu Glu Lys His Gin Lys Gin Met Glu Arg Ala

305 310 315 320

Leu Leu Glu Asn Glu Lys Lys Lys Arg Glu Met Ala Glu Lys Glu Lys 325 330 335

Glu Lys Ile Glu Arg Glu Lys Glu Glu Leu Met Glu Leu Lys Gin Ile 340 345 350

Glu Glu Gin Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin Thr Arg 355 360 365

Arg Ala Leu Glu Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser Glu Ala 370 375 380 Glu Lys Leu Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys Glu Ala

385 390 395 400

Leu Leu Gin Ala Ser Arg Asp Gin Lys Lys Thr Gin Glu Gin Leu Ala 405 410 415

Glu Met Ala Glu Leu Thr Ala Arg Ile Ser Gin Leu Glu Met Ala Arg 420 425 430

Lys Lys Glu Ser Glu Ala Val Glu Trp Gin Gin Lys Ala Gin Met Val 435 440 445 Gln Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu Lys Thr Ala Met Ser 450 455 460

Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu Asp Glu Gin Asp Glu 465 470 475 480

Asn Gly Ala Glu Ala Ser Ala Leu Arg Ala Asp Ala Met Ala Lys Asp 485 490 495 Arg Ser Glu Glu Glu Arg Thr Thr Glu Ala Glu Lys Asn Glu Arg Val

500 505 510

Gin Lys His Leu Lys Ala Leu Thr Ser Glu Leu Ala Asn Ala Arg Asp 515 520 525

Glu Ser Lys Lys Thr Ala Asn Asp Met Ile His Ala Glu Asn Met Arg 530 535 540

Leu Gly Arg Asp Lys Tyr Lys Thr Leu Arg Gin Ile Arg Gin Gly Asn 545 550 555 560

Thr Lys Gin Arg Ile Asp Glu Phe Glu Ser Met 565 570 ( 2 ) INFORMATION FOR SEQ ID NO : 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CACCATGCCG AAGACGATC 19

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Met Asp Ala Glu Leu Glu Phe Ala Ile Gin Pro Asn

1 5 10

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Glu Arg Ala Leu Leu Glu Asn Glu Lys Lys Lys Arg Glu Leu Ala 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Lys Tyr Lys Xaa Leu 1 5

Claims

hat is claimed is:

1. An isolated, homogeneous rat mast cell protein of molecular mass about 78 kDa, deduced amino acid sequence as shown in Figure 2, a minimum molecular weight of about 67-68 kDa, and a pi of between 6 and 7.

2. A protein of claim 1, further comprising said protein phosphorylated on at least amino acids residues 56 ^Scr and 66 ^Thr or 74 ^Ser and 374 ^Ser, said phosphorylated protein having the property of inhibiting mast cell degranulation.

3. A protein of claim 1, further comprising phosphorylation of said protein at amino acid residue 558 ^Tr, wherein said protein has the property of binding to actin.

4. A protein of claim 1, wherein said protein is a recombinant protein produced by a process comprising the steps of: a) preparing a cDNA library complementary to the mRNA encoding said protein,- b) screening said cDNA library for the cDNA encoding said protein; c) expressing said protein cDNA in E. coli ; d) isolating said protein from said E. coli ; and e) purifying said recombinant protein to homogeneity.

5. A protein of claim 1 or claim 4, wherein said protein is phosphorylated in vi tro by incubation with protein kinase C plus ATP and a phosphorylation activator.

6. A recombinant protein of claim 5, wherein said protein kinase C is the f" isozyme .

7. A protein of claim 1, wherein said protein is phosphorylated in vivo by the administration to a subject of an effective amount of a drug that stimulates endogenous phosphorylation of said protein.

8. A protein of claim 7, wherein said drug is selected from the group consisting of cromolyn, nedocromil, a flavonoid, and lodoxamide .

9. A method of inhibiting mast cells degranulation in a patient, comprising the step of administering to said patient an effective amount of a phosphomoesin or fragment thereof in a pharmaceutically acceptable carrier.

10. A method of claim 9, wherein said carrier is a liposome.

11. A method of inhibiting mast cell degranulation in a patient, comprising the step of administering to said patient an effective amount of a stimulator of the phosphorylation of serine and threonine residues of mast cell moesin or an inhibitor of phosphomoesin phosphatase, in a pharmaceutically acceptable carrier .

12. A method of claim 11, wherein said stimulator is selected from the group consisting of cromolyn, nedocromil and a flavonoid, or a derivative thereof that increases penetration into mast cells by said stimulator.

13. A method of claim 12, wherein said derivative in an ester.

14. A method for screening a patient for a deficiency of moesin or phosphomoesin, comprising the steps of: a. contacting an extract of a tissue of said patient with a labeled antibody directed against moesin or phosphomoesin;

b. determining the amount of moesin or phosphomoesin bound to the respective antibody; and, c. comparing the amount of moesin or phosphomoesin in said extract with the respective amounts in the counterpart tissue from a normal control subject.

15. A method for producing moesin in a mast cell of a subject comprising the step of administrating to said patient moesin cDNA inserted in a viral vector in a pharmaceutically acceptable carrier.