Abstract:
This invention relates generally to methods of producing peptide-based anti-angiogenesis compounds using plasmin reductases, and specifically to methods of producing an A 61  anti-angiogenic plasmin fragment using an annexin II heterotetramer or subunit thereof. This invention also relates to anti-angiogenesis methods and compositions comprising a plasmin reductase or polynucleotides encoding subunits thereof. Compositions useful for the inhibition or promotion of angiogenesis are also disclosed.

Description:
PARENT CASE TEXT  
       [0001]    This application claims benefit of priority to U.S. Provisional Patent Application No. 60/333,866, filed Nov. 28, 2001. 
     
    
     GOVERNMENT SUPPORT  
       [0002] This work was supported in part by a grant from the National Institutes of Health (CA78639). The United States Government has certain rights in this invention. 
     
    
     
       SEQUENCE LISTING  
         [0003]    A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f). The sequence listing contains sequences, which serve to illustrate the art-recognized plasmin/plasminogen amino acid number system, and which are not meant to limit the scope of the invention to those particular sequences. Other plasmin/plasminogen sequences, as well as p36 and p11 sequences are known in the art and can be readily obtained by one skilled in the art.  
         BACKGROUND OF THE INVENTION  
         [0004]    1. Field of the Invention  
           [0005]    This invention relates to methods of producing peptide-based anti-angiogenesis compounds using plasmin reductases, and specifically to methods of producing an A 61  anti-angiogenic plasmin fragment using an annexin II heterotetramer. This invention also relates to compositions and methods useful for modulating angiogenesis.  
           [0006]    2. Description of the Related Art  
           [0007]    The following references listed below as part of this paragraph are cited throughout this disclosure using the associated numerical identifiers. Applicant makes no statement, inferred or direct, regarding the status of these references as prior art. These references are incorporated herein by reference:  
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           [0024]    17. Kassam, G., Choi, K. S., Ghuman, J., Kang, H. M., Fitzpatrick, S. L., Zackson, T., Zackson, S., Toba, M., Shinomiya, A., and Waisman, D. M. (1998)  J. Biol. Chem.  273, 4790- 
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           [0036]    29. Soderberg, A., Sahaf, B., and Rosen, A. (2000)  Cancer Res.  60, 2281-2289  
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           [0044]    Annexin II heterotetramer (“AIIt”) is a Ca 2+ -binding protein complex that binds tPA, plasminogen and plasmin and stimulates both the formation and autoproteolysis of plasmin at the cell surface (17-19) (reviewed in (20)). The protein consists of two copies of an annexin II 36 kDa subunit (p36) called annexin II and two copies of an 11 kDa subunit (p11) called S100A10. It is known in the art that the carboxyl-terminal lysines of the p11 subunit plays a key role in plasminogen binding and activation (18).  
           [0045]    Angiostatin was originally identified in the urine of mice bearing Lewis lung carcinoma (LLC) as a 38 kDa proteolytically-derived fiagment of plasminogen which encompassed the first four kringle domains of plasminogen (Lys 78 -Ala 440  according to SEQ ID NO:1). Angiostatin was shown to be a potent antiangiogenic protein that inhibited the growth of human and murine carcinomas and also induced dormancy in their metastases. Angiostatin was also characterized as a specific antiangiogenic protein that blocked microvascular endothelial cell proliferation but not the proliferation of nonendothelial cells (1).  
           [0046]    Angiostatin is a member of a family of antangiogenic plasminogen fragments (“AAPFs”). Physiologically relevant AAPFs include a 38 kDa AAPF isolated from the conditioned media of tumor-infiltrating mnacrophages (2), a 43 kDa and 38 kDa AAPF identified in the conditioned media of Chinese hamster ovary and HT1080 fibrosarcoma cells and a 48 kDa AAPF present in macrophage conditioned media (3). Other AAPFs include a 43 kDa and a 38 kDa AAPF isolated from the conditioned media of human prostrate carcinoma PC-3 cells (4; 5) and AAPFs of 66, 60 and 57 kDa detected in the conditioned media of HT1080 and Chinese hamster ovary cells (6). Since the carboxyl-terminus of most of these AAPFs was not determined, the exact primary sequence of most of the AAPFs is unknown.  
           [0047]    Two distinct pathways have been identified for the formation of AAPFs. First, certain proteinases can directly cleave plasminogen into AAPFs. These proteinases include metalloelastase, gelatinase B (MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7), cathepsin D and prostate-specific antigen (7-11). The source of these proteinases may be tumor-infiltrating macrophages (2) or the cancer cells themselves. For example, the conversion of plasminogen to angiostatin by macrophages is dependent on the release of metalloelastase from these cells. In comparison, Lewis lung carcinoma cells release MMP-2 which also cleaves plasminogen to angiostatin (12). Second, AAPFs are also generated by a three step mechanism which involves the conversion of plasminogen to plasmin by urokinase-type plasminogen activator (“uPA”), the autoproteolytic cleavage of plasmin and the release of the resultant plasmin fragment by cleavage of disulfide bonds. The cleavage of the plasmin disulfide bonds can be accomplished by free sulthydryl group donors (FSD) such as glutathione or by hydroxyl ions at alkaline pH (4; 5; 13; 14). Alternatively, the plasmin disulfide bonds can be cleaved enzymatically by a plasmin reductase such as phosphoglycerate kinase (15; 16).  
           [0048]    In co-pending patent application PCT/US01/44515 (published as WO0244328 A and reference 14), which is incorporated herein by reference, it was shown that the primary AAPF present in mouse and human blood has a molecular weight of 61 kDa. This AAPF, called A 61 , was produced in a cell-free system consisting of uPA and plasminogen. A, was shown to be a novel four-kringle containing plasminogen fragment consisting of the amino acid sequence, Lys 78 -Lys 468  (SEQ ID NO:1) (14). The release of A 61  from plasmin required cleavage of the Lys 468 -Gly 469  (SEQ ID NO:1) bond by plasmin autoproteolysis and also cleavage of the Cys 462 -Cys 541  (SEQ ID NO:1) disulfide. Since A 61  was generated in a cell-free system from plasmin at alkaline pH in the absence of sulfhydryl donors, it was concluded that cleavage of the Cys 462 -Cys 541  disulfide was catalyzed by hydroxyl ions in vitro. In contrast, at physiological pH, it was observed that the conversion of plasminogen to Al was very slow. These results contrasted with the observation that at physiological pH, HT1080 fibrosarcoma and bovine capillary endothelial (BCE) cells stimulated the rapid formation of A 61 . Heretofore, the mechanism by which these cells stimulated plasmin reduction and the release of A 61  from plasmin was unclear.  
         SUMMARY OF THE INVENTION  
         [0049]    According to the present invention, it was discovered that an annexin II heterotetramer or its subunits stimulates the conversion of plasminogen to A 61  in vitro. It was also discovered that an annexin II heterotetramer or its subunits (p36, p11) possesses an intrinsic plasmin reductase activity, and that the cysteinyl residues of both subunits of the annexin II heterotetramer (i.e., p36 and p11) participate in the reduction of plasmin. Preferred Annexins include annexin II (p36) and AIIt. The invention is also drawn to inhibitors of AIIt activity, which diminish A 61  production by cells. Preferred inhibitors of AIIt activity include antisense nucleotides, which down-regulate expression of AIIt on the surface of cells.  
           [0050]    The present invention is drawn to a method of producing an anti-angiogenesis plasmin fragment comprising contacting a plasminogen polypeptide with a plasminogen activator and a plasmin reductase, wherein a reduced plasmin protein is produced. The anti-angiogenesis plasmin fragment, which has anti-angiogenesis activity, is released from the reduced plasmin protein. Preferably but not exclusively, the plasminogen activator is a urokinase-type plasminogen activator, a plasmin reductase is an annexin II heterotetramer, annexin II p36 subunit, or p11 and the anti-angiogenesis plasmin fragment is an A 61  corresponding to SEQ ID NO:7 or SEQ ID NO:8.  
           [0051]    In another embodiment, the present invention is drawn to a method of producing an anti-angiogenesis plasmin fragment comprising contacting a plasmin protein with a plasmin reductase, wherein the anti-angiogenesis plasmin fragment, which has anti-angiogenesis activity, is released from a reduced plasmin protein. Preferably but not exclusively, the plasmin reductase is an annexin II heterotetramer, annexin II p36 subunit, or p111, and the anti-angiogenesis plasmin fragment is an A 61  corresponding to SEQ ID NO:7 or SEQ ID NO:8.  
           [0052]    In another embodiment, the present invention is drawn to a method of inhibiting the formation of an A 61  anti-angiogenesis fragment comprising contacting a cell with a polynucleotide, wherein (a) the polynucleotide encodes a p11 antisense polynucleotide, which enters the cell inhibits the expression of p11 on the surface of the cell, and (b) expression of p11 is requited for efficient production of the A 61  anti-angiogenesis fragment.  
           [0053]    In another embodiment, he present invention is drawn to a method of increasing the formation of an A 61  anti-angiogenesis fragment comprising contacting a cell with a polynucleotide, wherein (a) the polynucleotide encodes a p11 sense polynucleotide, which enters the cell increases the expression of p11 subunits on the surface of the cell, and (b) expression of p11 is required for efficient production of the A 61  anti-angiogenesis fragment. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0054]    [0054]FIG. 1 illustrates the stimulation of conversion of plasminogen to A 61  by AIIt. (A,B) AIIt dose-dependent generation of A 61 . [Glu]-plasrrinogen (4 μM) was incubated with u-PA (0.075 μM) and various concentrations of AIIt, then subjected to non-reduced SDS-PAGE followed by Coomassie blue staining (A). Portions of the reaction mixtures were incubated with MPB (100 μM), reduced glutathione (200 μM), iodoacetamide (400 μM), L-lysine-Sepharose, and then subjected to non-reduced SDS-PAGE followed by Western blot with streptavidin-HRP (B). (C,D) Time-course generation of A 61 . [Glu]-plasminogen (4 μM) was incubated with uPA (0.075 μM) and AIIt (4 μM) for various times at 37°. The results are shown as Coomassie blue staining (C) and Western blot with streptavidin-HRP (D).  
         [0055]    [0055]FIG. 2 Illustrates the role of p11 and p36 subunits in the plasmin reductase activity of AIIt. [Glu]-plasminogen (4 μM) was incubated with uPA (0.075 μM) and various subunits of AIIt or AIIt (4 μM). The results are shown as Coomassie blue staining (I) and Western blot with streptavidin-HRP (B).  
         [0056]    [0056]FIG. 3 illustrates the thiol reactivity of AIIt and its subunits. AIIt or its various subunits (2 μM) were incubated with MPB (100 μM), reduced glutathione and iodoacetamide, sequentially. The reaction mixtures were subjected to reduced SDS-PAGE followed by either Coomassie blue staining (A) or Western blot with streptavidin-HRP (B). (C) AIIt (18 μM) was incubated with iodoacetic acid (IAA, 10 mM) or MPB (200 μM). [Glu]-plasminogen (4 μM) was incubated with u-PA (0.075 μM) and the indicated AIIt (4 μM). The result shown is a Western blot with streptavidin-HRP.  
         [0057]    [0057]FIG. 4 illustrates the specificity of the plasmin reductase activity of AIIt. [Glu]-plasminogen (4 μM) was incubated with uPA (0.075 μM) and the indicated annexin proteins (4 μM). The result shown is a Western blot with streptavidin-HRP.  
         [0058]    [0058]FIG. 5 illustrates the binding of A 61  to AIIt. The wells of Removawell™ strips were coated with phospholipid, blocked with bovine serum albumin, incubated with AIIt, and extensively washed. Various concentrations of iodinated A 61  were added into the AIIt-coated wells in the absence filled circles) or presence of 30-fold molar excess of bovine serum albumin (filled triangles) or cold A 61  (open circles). Individual wells were detached and measured for radioactivity with y-counter. The data shown (n=6) were the average of two separate experiments.  
         [0059]    [0059]FIG. 6 illustrates a comparison of AIIt with other disulfide reductases. [Glu]-plasminogen (4 μM) was incubated with uPA (0.075 FM) and the various proteins as indicated. The result shown is a Western blot with streptavidin-HRP.  
         [0060]    [0060]FIG. 7 illustrates that the down-regulation of AIIt blocks A, generation by HT1080 cells. (A) Flow cytometric analysis of transduced HT1080 cells. (B,C) Comparison of the generation of A 61  by transduced HT1080 cells. Transduced HT1080 cells were incubated with DMEM containing 2 μM [Glu]-plasminogen (B) or plasmin (C). After the indicated time of incubation, the medium was analyzed by reduced SDS-PAGE (B) or non-reduced SDS-PAGE (C) followed by Western blot with monoclonal anti-human plasminogen kringle 1-3 antibody.  
         [0061]    [0061]FIG. 8 diagrammatically illustrates the mechanism of A 61  formation. K represents the kringle domain of plasminogen. S—S indicates the disulfide bond and SH represents the free thiol generated. Plasmin catalyzes the cleavage of the Lys 77 -Lys 78  and Lys469-Gly 469  bonds of plasmninogen. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0062]    The inventors have discovered that an annexin II heterotetramer or its subunits (p36, p11), contains an intrinsic plasmin reductase activity and is useful in the generation of the antiangiogenic plasminogen fragment, A 61 , which is a four-kringle containing plasminogen fragment comprising the amino acid sequence Lys 78 -Lys 468 , wherein the numbering of Lys 78 -Lys 468  is based upon the numbering of SEQ ID NO:1. It is also disclosed that cells transduced with a vector encoding a p11 antisense RNA (“antisense p11”) show reduced extracellular AIIt and A 61  production, demonstrating the utility of AIIt as an antiangiogenic agent and in the formation of other antiangiogenic agents.  
         [0063]    While not intending to be bound by theory, the inventors postulate that plasminogen is converted to A 61  in a three-step process FIG. 8). First, u-PA cleaves the Arg561-Val 562  of plasminogen (SEQ ID NO:1) resulting in the formation of plasmin. Second, plasmin autoproteolysis results in the cleavage of the Lys 77 -Lys 78  and Lys 468 -Gly 469  bond. A minor cleavage site at Arg 471 -Gly 472  has also been shown to be present (minor A 61  fragment depicted in SEQ ID NO:8). However, the presence of a Cys 462 -Cys 541  disulfide prevents release of A 61  (Lys 78 -Lys 468 ). Third, AIIt catalyzes the reduction of the Cys 462 -Cys 54 1 disulfide which allows the release of A 61  (SEQ ID NO:7, major form) from the rest of the molecule. Since AIIt does not reduce catalytically inactive plasmin, one skilled in the art may conclude that plasmin autoproteolysis must occur before plasmin reduction, suggesting that autoproteolyzed plasmin is the substrate for AIIt&#39;s plasmin reductase activity.  
         [0064]    It is herein disclosed that plasmin autoproteolysis precedes plasmin reduction and that plasmin reduction is accelerated by AIIt. The AIIt present at the surface of certain cells participates in the conversion of plasminogen to A 61  and loss of extracellular AIIt results in the inhibition of cell-generated A 61 . AIIt likely functions as a catalyst, since large amounts of plasminogen are converted to A 61  by AIIt expressing cells. This suggests that in order for AIIt to continually reduce plasmin it must proceed through cycles of oxidation by plasmin and reduction by unknown reducing equivalents. The mechanism by which oxidized extracellular AIIt is reduced is unknown at this time.  
         [0065]    It is further disclosed that both the p36 and p11 subunits of AIIt possess plasmin reductase activity. In the case of the p36 subunit the Cys 334  residue (the cysteine at position number 334 according to SEQ ID NO:2) is essential for plasmin reductase activity. In the case of the p11 subunit both Cys 61  and Cys 82  (according to SEQ ID NO:3) are capable of participating in plasmin reduction. Although speculative, the simplest explanation for these observations is that plasmin and autoproteolyzed plasmin can bind to AIIt but the unique conformational change induced by the binding of autoproteolyzed plasmin to AIIt may result in an increased accessibility of cysteinyl residues of AIIt, which participate in reduction of autoproteolyzed plasmin. Since, according to the examples presented below, Cys 334  of p36 was not labeled with the thiol specific reagent MPB, Cys 334  of p36 is likely shielded from the solvent. Furthermore, a p36 mutant, which comprises a Cys to Ser substitution at position 334, is inactive in terms of plasmin reductase activity. It is therefore reasonable for one skilled in the art to suspect that Cys 334  may be shielded and only accessible for reduction of plasmin upon binding of autoproteolyzed plasmin to AIIt.  
         [0066]    Protein disulfide reductases typically contain the Cys-X-X-Cys motif in their active sites. Members of this family of proteins include thioredoxin, protein disulfide isomerase, fibronectin, von Willebrand factor and platelet integrin α IIb β 3  (28; 29; 32-36). Typically these proteins share the general property of catalyzing the reduction of insulin disulfides. In contrast, AIIt does not contain the Cys-X-X-Cys motif and does not catalyze the reduction of insulin disulfides. This suggests that the intrinsic plasmin reductase activity of AIIt is due to a novel mechanism of disulfide reduction.  
         [0067]    The phrase “anti-angiogenesis plasmin fragment” (“AAPF”), as used herein, means a polypeptide fragment of plasminogen or plasmin, which inhibits the recruitment or growth of blood vessels, or the recruitment or growth of endothelial cells, wherein the plasminogen or plasmin may be from any species of metazoan. For example, AAPFs include, p22 and A 61  (WO0244328 A and reference 14), a 38 kDa AAPF isolated from the conditioned media of tumor-infiltrating macrophages (2), a 43 kDa and 38 kDa AAPF identified in the conditioned media of Chinese hamster ovary and HT1080 fibrosarcoma cells, a 48 kDa AAPF present in macrophage conditioned media (3), a 43 kDa and a 38 kDa AAPF isolated from the conditioned media of human prostrate carcinoma PC-3 cells (4;5) and AAPFs of 66, 60 and 57 kDa detected in the conditioned media of HT1080 and Chinese hamster ovary cells (6). Preferred AAPFs include A 61 .  
         [0068]    The term “plasmin protein” means any plasmin protein, which includes active plasmin polypeptide, proteolyzed plasmin, and reduced plasmin, from any species. A preferred plasmin protein has an N-terminal lysine, which corresponds to Lys 78  of plasminogen.  
         [0069]    The phrase “plasminogen activator” means an enzyme that catalyzes the proteolysis of a plasminogen polypeptide to produce an active plasmin protein. Preferred plasminogen activators include urokinase-type plasminogen activators (uPA), streptokinase and tissue-type plasminogen activators (tPA).  
         [0070]    The phrase “plasmin reductase” means an agent, preferably a protein, which is capable of catalyzing the reduction of disulfide bridges of a plasminogen polypeptide or plasmin protein. Preferred plasmin reductases include annexin II heterotetramer (used interchangeably with “annexin II tetramer” or “AIIt”), annexin II p36 subunit (“p36”), S100A10 subunit (“p11”), thioredoxin and protein disulfide isomerase.  
         [0071]    The phrase “anti-angiogenesis activity” means (a) the ability of a substance to inhibit endothelial cell proliferation or migration, (b) kill proliferating endothelial cells, or (c) the ability of a substance to inhibit the formation of new blood vessels in a tissue. Preferred substances are peptides such as A 61 . Preferred A 61  polypeptides have a sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8. The term “angiogenesis” means the formation of new blood vessels in a tissue, the stimulation of endothelial cells to proliferate, or the promotion of survival of proliferating endothelial cells.  
         [0072]    The phrase “p11 antisense polynucleotide” means a single stranded RNA molecule, which is complementary to a p11 RNA that can be translated to produce a p11 polypeptide, or a fragment thereof. Functionally, a p11 antisense polynucleotide is capable of decreasing the expression of p11 protein in a cell. A preferred p11 antisense polynucleotide may be a DNA or RNA as set forth in SEQ ID NO:5. The inventors also envision that a siRNA (small interfering RNA) comprising a p11 sequence, which in preliminary experiments suggests that it is effective in decreasing the expression of p11 in a cell, may be useful in stimulating angiogenesis in a tissue, such as in myopathological heart tissue.  
         [0073]    The phrase “p11 sense polynucleotide” means a single stranded RNA molecule, which is can be translated to produce a p11 polypeptide, or a fragment thereof. Functionally, a p11 antisense polynucleotide is capable of increasing the expression of p11 protein in a cell. A preferred p11 sense polynucleotide may be a DNA or RNA as set forth in SEQ ID NO:6. The terms “p11”, “p11 polypeptide”, “p11 protein”, “p11 subunit”, “annexin II p11”, “annexin II p11 subunit”, “S100A10”, and “S100A10 subunit” are equivalent and are used interchangeably throughout the instant specification and claims.  
         [0074]    The term “vector” refers to a polynucleotide that enables the expression of a constituent polynucleotide in a cell, wherein expression means the transcription of DNA into a RNA. Preferred vectors include retroviral vectors, such as pLin.  
       EXAMPLE 1  
     Stimulation of A 61  Production  
       [0075]    A 61  is an internal fragment of plasminogen that encompasses the sequence Lys 78 -Lys 468  (SEQ ID NO: 1). The release of A 61  from plasmin is facilitated by the reduction of the Cys 462 -Cys 541  disulfide bond of plasmin. Therefore, the release of Al generates a free sulfhydryl residue at Cys 462 . Since plasminogen and plasmin contain only disulfides, A 61  can be discriminated from these proteins on the basis of its reactivity with free sulfhydryl-reactive reagents such as 3-(N-raleidylpropionyl)biocytin (MPB). The reaction of free-sulfhydryl-containing proteins with MPB results in the biotinylation of the protein which allows easy detection with streptavadin-HRP.  
         [0076]    As shown in FIG. 1A (lane 2), the incubation of uPA with plasminogen resulted in the generation of plasmin. As expected, the plasmin generated by this reaction did not contain a free cysteinyl residue and therefore did not react with MPB (FIG. 1B, lane 2). However, the addition of AIIt to the u-PA-plasminogen reaction resulted in the appearance of A6, (which displays a single major band and two minor bands of about M r  50 K on non-reduced SDS-PAGE) and disappearance of plasmin (FIG. 1A). Furthermore, the A 61  generated in these reactions reacted with MPB, confirming the presence of a free sulfhydryl in A 61  (FIG. 1B).  
         [0077]    AIIt stimulated the dose- and time-dependent conversion of plasminogen to A 61  (FIGS.  1 A-D). The maximal conversion of plasminogen to A 61  occurred at approximately equimolar concentrations of AIIt and plasminogen (FIGS. 1A,B). At equimolar concentrations the half-maximal conversion of plasminogen to A 61  occurred between 30-60 min (FIGS. 1C,D). Since AIIt stimulated the generation of A 61  in the absence of sulfhydryl donors, the data reasonably suggest that AIIt promoted the cleavage of a plasmin disulfide, presumably the Cys 462 -Cys 541  disulfide, resulting in the release of A 61  from plasmin and the generation of a free cysteine (Cys 462 ) in A 61 .  
         [0078]    AIIt is composed of two copies of a p36 polypeptide subunit and two copies of a p11 polypeptide subunit. As shown in FIGS. 2A,B the incubation of either the p36 or p11 subunit with u-PA and plasminogen stimulated the formation of A 61 . However, AIIt appeared to be a more potent plasmin reductase than either subunit, suggesting that the interaction of the subunits potentiated the plasmin reductase activity of either subunit.  
         [0079]    Theoretically, either the disulfides or cysteinyl residues of AIIt could participate in the reduction of plasmin. It is possible that upon plasminogen binding the disulfide of annexin II is reduced by the thiols of annexin II and the newly formed thiols participate in reduction of plasmin. Since the p11 subunit does not contain disulfides, it is reasonable to suspect that the thiols of this subunit were important for its plasmin reductase activity. p11 contains two cysteinyl residues: Cys 61  which plays a critical role in the binding of p36 and Cys 82  which is a free thiol (25). Two recombinant forms of this subunit were prepared, in which individual cysteinyl residues were mutated to serine. This conservative mutation results in the substitution of a thiol group for a hydroxyl group. As shown in FIG. 2B, substitution of either of these cysteine residues resulted in a loss of plasmin reductase activity of the p11 subunit. This suggests that both cysteinyl residues of p11 are required to sustain the plasmin reductase activity of the protein.  
         [0080]    Human p36 contains two thiol-containing cysteines, Cys 8  and Cys 334 . As shown in FIG. 2B, substitution of Cys 334 , but not Cys 8 , with serine blocked the plasmin reductase activity of p36. This result suggests that the Cys 334  thiol is critical for plasmin reductase activity of the p36 subunit.  
         [0081]    To identify the reactive thiols of AIIt and its subunits, AIIt, p36 and p11 were incubated with MPB and resolved with SDS-PAGE followed by Coomassie blue staining (FIG. 3A) or Western blot with streptavidin-HRP (FIG. 3B). As shown in FIG. 3B, the p11 subunit and both Cys 61 Ser and Cys 82 Ser p11 mutants were labeled with MBP. In contrast, although p36 and the Cys 334 Ser p36 mutant were labeled with MPB, the Cys 8 Ser mutant was not labeled. This suggests that the thiols of the isolated p11 are accessible to MPB whereas the Cys 334  of p36 is not accessible. Interestingly, both the p36 and p11 subunits within AIIt were labeled with MPB. However, preincubation of AIIt with iodoacetic acid or MPB resulted in only a small decrease in the plasmin reductase activity of AIIt (FIG. 3C). This suggests that the Cys 334  thiol of the p36 subunit of AIIt is inaccessible to the solvent.  
         [0082]    Since the Cys 334  residue is highly conserved among many of the annexins, seven other annexins were examined for plasmin reductase activity. As shown in FIG. 4, only annexin II p36 subunit and AIIt possessed plasmin reductase activity. This result establishes that plasmin reductase activity is not a common feature of the annexins.  
         [0083]    It is generally accepted in the art that AIIt binds both plasminogen and plasmin and is present at discrete regions of the extracellular surface (17; 20; 26). This suggested that AIIt could act as a scaffolding protein and focus the proteolytic activity of plasmin to the plasma membrane. It was unclear if the A 61  generated by cells could remain bound to AIIt at the cell surface or be immediately released into the media. However, as shown in FIG. 5, A 61  bound to AIIt with a k d  of 1.0±0.05 μM (n-6). According to the observation made by the inventors that A 61  did not block the stimulation of tPA-catalyzed conversion of plasminogen to plasmin, one skilled in the art may reasonably expect that the binding sites on AIIt for A 61  are distinct from those for plasminogen or plasmin. This suggests that some of the A 61  produced by the AIIt&#39;s plasmin reductase reaction may remain bound to AIIt at the cell surface.  
       EXAMPLE 2  
     Stimulation of A 61  Production by Other Disulfide Reductases  
       [0084]    Protein disulfide isomerase, thioredoxin and phosphoglycerate kinase are three protein disulfide reductases that are secreted by cultured cells (16;27-29). The three reductases have been shown to act as plasmin reductases (15;16). As shown in FIG. 6, under the assay conditions AIIt was a more potent plasmin reductase than the other reductases in vitro.  
         [0085]    Thioredoxin and protein disulfide isomerase share a common sequence, Trp-Cys-Gly-Pro-Cys-Lys (SEQ ID NO:4), which participates in the cleavage, formation and reshuffling of disulfide bonds. This sequence is not present in phosphoglycerate kinase or AIIt, suggesting that these reductases have distinct catalytic mechanisms. Typically, the disulfide reductase activity of thioredoxin or protein disulfide isomerase is measured by determination of their rates of reduction of insulin disulfide (30;31). Interestingly, although protein disulfide isomerase and thioredoxin exhibit potent insulin reductase activity, AIIt failed to exhibit insulin reductase activity. This further confirms that the catalytic mechanism of AIIt is distinct from that of some other protein reductases.  
       EXAMPLE 3  
     Down-Regulation of AIIT Blocks A6, Generation  
       [0086]    HT1080 fibrosarcoma cells were stably transfected (transduced) with a pLin retroviral vector encoding a p11 gene in the sense (pLin-p11S) or antisense (pLin-p11AS) orientation, or an empty pLin vector (pLin-V). The pLin-p11AS transduced cells showed a decrease in both p11 and p36 subunits on the cell surface whereas pLin-plS transduced cells showed an increase in both p11 and p36 subunits (FIG. 7A). As shown in FIG. 7B, incubation of the pLin-p11S transduced cells with plasminogen resulted in enhanced A 61  formation compared to the pLin-V control cells. In contrast, the pLin-p11AS transduced cells failed to produce A 61 . Additionally, HeLa cells transfected with a p11 antisense expressing vector also failed to convert plasminogen to A 61 .  
         [0087]    The data in FIG. 7B establishes a role for AIIt in A 61  formation in HT1080 fibrosarcoma cells. However, it is unclear if plasmin could be directly reduced or if plasmin autoproteolysis preceded plasmin reduction. As shown in FIG. 7C, the incubation of plasmin with the pLin-p11S cells also resulted in the accelerated disappearance of plasmin and concomitant enhanced appearance of A 61  compared with pLin-p11V cells. In contrast, the pLin-p11AS cells showed reduced plasmin loss and concomitant A 61  formation compared to the pLin-p11V cells. In another series of experiments, plasmin was inactivated by prior treatment of the serine protease inhibitor, DIFP and then incubated with the HT1080 cells. However, A 61  production by the cells was not observed. Furthermore, the catalytically inactive plasmin that was incubated with the cells was not reduced since it did not react with MPB. This result may be interpreted by the skilled artisan to mean that plasmin autoproteolysis is required before plasmin reduction can occur. Collectively, the experimental observations herein disclosed suggest that the mechanism of A 61  formation involves the u-PA-dependent conversion of plasminogen to plasmin followed by plasmin autoproteolysis and reduction of cleaved plasmin (FIG. 8).  
       EXAMPLE 4  
     Materials and Procedures Employed  
       [0088]    Two-chain urokinase-type plasminogen activator (EPA) was a generous gift from Dr. H. Stack (Abbott Laboratories). [Glu]-plasminogen and plasmin were purchased from American Diagnostica. Antiangiogenic plasminogen fragment (A 61 ) was purified as outlined in copending patent application PCT/US01/44515. Annexin II heterotetramer (AIIt) and other annexins were purified from bovine lung as described in reference 21. Monoclonal anti-human plasminogen kringle 1-3 antibody was purchased from Enzyme Research Laboratories Inc. Monoclonal anti-annexin II and anti-annexin II fight chain antibodies were purchased from Transduction Laboratories. Anti-mouse horseradish peroxidase-conjugated secondary antibody was purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated streptavidin and protein disulfide isomerase were purchased from Calbiochem. Anti-mouse R-phycoerytbrin-conjugated secondary antibody was purchased from Caltag Laboratories Inc. N α -(3-maleimidylpropionyl)biocytin (MPB) was purchased from Molecular Probes. Reduced glutathione, iodoacetamide, lodoacetic acid, diisopropylfluorophosphate (DIFP), and thioredoxin were purchased from Sigma. L-lysine-Sepharose was purchased from Amersham Phannacia Biotech. lodobeads were purchased from Pierce. Phosphoglycerate kinase (PGK) was a generous gift from Dr. P. J. Hogg (Center for Thrombosis and Vascular Research, University of New South Wales, Sydney, Australia). Stably transfected HeLa cells expressing p11 antisense or sense mRNA were a generous gift from Dr. J. H. Shelhamer (Critical Care Medicine Department, National Institutes of Health, USA). HT1080 fibrosarcoma cells were obtained from American Type Culture Collection. Dulbecco&#39;s modified eagle medium (DMEM) was purchased from Life Technologies.  
         [0089]    Mutagenesis of Annexin II and p11—Bacterial expression vectors containing the wild-type sequence for annexin II (pAED4.91-annexin II) and p11 (pAED4.91-p11) were mutated using the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene). Briefly, mutagenic primers were synthesized that introduced Cys→Ser mutations at positions 8 and 334 of annexin II, as well as positions 61 and 82 of p11. All of the mutations introduced were verified by DNA sequence analysis. These various plasrids were then transformed into  E. coli  BL21 (DE3) and grown as previously described (22).  
         [0090]    Purification of Wild-type and Mutant Annexin II—After 4 hours of induction with IPTG, bacteria were collected by low speed centrifugation. The cells were subsequently sonicated in lysis buffer (10 mM irmidazole, pH 7.5, 150 mM NaCI, 2 mM EGTA, 1 mM DTT+protease inhibitors) and centrifuged at 100,000×g for 1 hour at 4° C. Both mutant annexin H proteins were purified in the same manner as wild-type annexin II via hydroxyapatite, heparin-Sepharose affinity and gel penneation chromatography as reported previously (22). The elution profiles of the recombinant wild-type and mutant annexin II on hydroxyapatite, heparin affinity and gel permeation chromatography were indistinguishable. In addition, the circular dichroism spectra of each of the proteins were very similar, indicating little secondary structure perturbation.  
         [0091]    Purification of Wild-type and Mutant p11—After 4 hours of induction with IPTG, bacteria were collected by low speed centrifugation. The cells were subsequently sonicated in lysis buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM MgCk, 2 mM DTT+protease inhibitors) and centrifuged at 100,000×g for 1 h at 4° C. Both p11 mutants were purified in the same manner as wild-type p11 (23). Briefly, the cell lysis supematant was precipitated with 50% (NH 4 ) 2 SO 4 , and the supernatant was applied to a Butyl-Sepharose column equilibrated in lysis buffer containing 50% 4)2SO 4 . The p11 was eluted with a linear gradient of (NH 4 ) 2 SO 4  from 50% to 0%, and peak fractions containing p11 were pooled and dialyzed against 10 mM imidazole, pH 7.4, 1 mM EGTA, 0.5 mM DTT, and 0.1 mM EDTA. The dialyzed fractions were subsequently applied to a DEAE-Sepharose column equilibrated in the same buffer. The p11 was eluted with a linear NaCl gradient, concentrated to 4 mL, and applied to a Sephacryl S-100 column equilibrated in 40 mM Tris-HCl, pH 7.4, 0.1 mM EGTA, and 0.1 mM DTT. A single protein peak was recovered at the expected molecular weight based on gel filtration standards.  
         [0092]    Plasmid Construction and Transfection of HT1080 Cells—Sense and antisense p11 expression vector were produced by cloning the full-length human p11 cDNA into the pLin retroviral vector in the sense (pLin-p11S) or antisense orientation (pLin-p11AS) as reported (24). Control cells transduced with the vector alone (pLin-V) were also established. The pLin vector carries the Moloney murine leukemia virus 5′ LTR enhancer/promoter region to promote strong, constitutive expression of the cloned p11 inserts and neomycin phosphotransferase gene in mammalian cells. The pLin constructs were propagated in a PA317 retroviral packaging cell line. Packaging cells were selected in 300 μg/ml neomycin and conditioned media that contained high titers of the virus were used to transduce the HT1080 fibrosarcoma cells. After viral transduction the neomycin resistant HT1080 fibrosarcoma cells were cloned and permanent cell lines established (Choi, K-S et al., in press at FASB J.).  
         [0093]    Dialysis of Candidate Plasmin Reductase Proteins—After purification or reconstitution, AIIt, p11, p36 (annexin II), other annexins, thioredoxin, protein disulfide isomerase, and phosphoglycerate kinase were dialyzed against 20 mM Tris (pH 7.5) and 140 mM NaCI under argon gas to prevent possible oxidation.  
         [0094]    Plasmin Reductase Assay—[Glu]-plasminogen (4 μM) was incubated with 0.075 μM u-PA and a candidate plasmin reductase protein (4 μM, unless described) in a buffer containing 20 mM Tris (pH 7.5) and 140 mM NaCl at 37° C. for 2 h. A portion of reaction mixture was diluted with SDS-PAGE sample buffer and subjected to non-reduced SDS-PAGE followed by Coomassie blue staining. To label any free thiol groups of produced protein(s), the reaction mixture was incubated with 100 μM MPB at room temperature for 30 min. The reaction mixture was then treated with 200 μM reduced glutathione at room temperature for 10 min to quench the unreacted MPB. The unreacted glutathione and other free thiols in the reaction mixture were blocked with 400 μM of iodoacetamide at room temperature for 10 min. Then the reaction mixture was incubated with L-lysine-Sepharose at room temperature for 30 min to purify the kringle-containing, plasminogenderived proteins. The matrix was extensively washed with PBS and the bound proteins were eluted by boiling the resin with SDS-PAGE sample buffer. Each sample was subjected to non-reduced SDS-PAGE followed by Western blot with horseradish peroxidase-conjugated streptavidin (streptavidin-HRP) as indicated below.  
         [0095]    Detection offree thiols in AIIt—2 μM AIIt, p11, or p36 was incubated with 100 μM MPB in a buffer containing 20 mM Tris (pH 7.5) and 140 mM NaCl at room temperature for 30 min. After incubation, 200 μM reduced glutathione and 400 μM iodoacetamide were added sequentially, and the reaction mixture was subjected to reduced SDS-PAGE followed by either Coomassie blue staining or Western blot with streptavidin-HRP as indicated below.  
         [0096]    Cell-Mediated Generation of A 61 —Transduced HT1080 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 units/ml penicillin G, 10 μM streptomycin sulfate, and 300 μg/ml neomycin. Approximately 1×10 5  cells were added to each well of 24-well tissue culture plates and incubated at 37° C. for 24 h. The cell monolayers were then washed three times with DMEM and 2 μM [Glu]-plasminogen, plasmin, or diisopropylfluorophosphate (DIFP)-treated plasmin in DMEM was added to each well. The conditioned medium was removed at indicated times, diluted with SDS-PAGE sample buffer with or without β-mercaptoethanol and subjected to SDS-PAGE followed by Western blot with monoclonal anti-human plasminogen kringle 1-3 antibody (anti-K1-3) as indicated below.  
         [0097]    Electrophoresis and Western Blot—Samples were diluted with SDS-PAGE sample buffer and subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose membrane (0.45 μm pore size) at 4° C. for 1 h. The membrane was blocked in TPBS (phosphate buffered saline containing 0.1% Tween-20) with 5% skim milk at room temperature for 1 h and incubated at 4° C. overnight with a 0.4 μg/mnl monoclonal antihuman plasminogen ktingle 1-3 antibody in TPBS with 5% skim milk. The blot was extensively washed with TPBS at room temperature and then incubated at room temperature for 1 h with a 0.16 μg/ml horseradish peroxidase-conjugated goat anti-mouse secondary antibody in TPBS with 5% skim milk. In the case of MPB-reacted protein samples, the membrane was blocked and incubated at room temperature for 1 h with a 0.1 μg/in streptavidin-HRP in TPBS with 5% skim milk. The membrane was extensively washed with TPBS and visualized by enhanced chemiluminescence (Pierce).  
         [0098]    Binding of A 61  to AIIt—The purified A 61  was iodinated according to manufacturer&#39;s procedures. Iodinated A 61  retained biological activity as determined by the endothelial cell proliferation assay (14). 96-well Immulon-1 Removawell strips (Dynex Technologies) were coated with phospholipid mixture containing 3:1 ratio of phosphatidylserine to phosphatidylcholine and air-dried. The coated strips were blocked with 1% fatty acid-free bovine serum albumin (BSA) in a buffer containing 20 mM Hepes (pH 7.4), 140 mM NaCl, and 2 mM CaCl 2  (buffer A) at room temperature for 2 h. The strips were washed with buffer A and incubated with 1 μM AIIt in buffer A at room temperature for 4 h. The strips were then washed and incubated with 0.008-5 μM iodinated Al with or without 30-fold molar excess of cold Ai or BSA at 4° C. After overnight incubation, the strips were washed five times with PBS, and individual wells were detached and measured for radioactivity with a 7-counter. The data shown (n=6) are the average of two separate experiments.  
         [0099]    Flow Cytometric Analysis of Transduced HT 1080 Cells—Transduced HT 1080 cells were harvested and 1×10 6  cells in PBS were divided into each tube. The cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and washed twice with PBS. The cells were then incubated with 1 μg of monoclonal anti-annexin II or anti-annexin II light chain antibody at room temperature for 30 min. For the control staining, 1 μg of mouse Ig G was used. The cells were washed and incubated with 2 μg/ml anti-mouse R-phycoerythrin-conjugated secondary antibody at room temperature for 30 min. The cells were washed and subjected to flow cytometric analysis using FACScan™ (Beckton Dickinson) and analyzed by the FlowJo™ program. The data shown are a representative of three separate experiments.  
         [0100]    As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and sequence listing, and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.  
         [0101]    All references cited in this specification are hereby incorporated by reference. The discussion of references herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.  
     
       
       
         1 
         
           
             8  
           
           
             1  
             791  
             PRT  
             mammalian  
           
            1 

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser 
  1               5                  10                  15 

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala 
             20                  25                  30 

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His 
         35                  40                  45 

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser 
     50                  55                  60 

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr 
 65                  70                  75                  80 

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met 
                 85                  90                  95 

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser 
            100                 105                 110 

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu 
        115                 120                 125 

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp 
    130                 135                 140 

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu 
145                 150                 155                 160 

Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly 
                165                 170                 175 

Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser 
            180                 185                 190 

Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys 
        195                 200                 205 

Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro 
    210                 215                 220 

Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile 
225                 230                 235                 240 

Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys 
                245                 250                 255 

Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val 
            260                 265                 270 

Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His 
        275                 280                 285 

Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr 
    290                 295                 300 

Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn 
305                 310                 315                 320 

Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser 
                325                 330                 335 

Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr 
            340                 345                 350 

Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly 
        355                 360                 365 

Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser 
    370                 375                 380 

Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala 
385                 390                 395                 400 

Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro 
                405                 410                 415 

Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu 
            420                 425                 430 

Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val 
        435                 440                 445 

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe 
    450                 455                 460 

Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly 
465                 470                 475                 480 

Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile 
                485                 490                 495 

Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys 
            500                 505                 510 

Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn 
        515                 520                 525 

Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro 
    530                 535                 540 

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly 
545                 550                 555                 560 

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln 
                565                 570                 575 

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu 
            580                 585                 590 

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser 
        595                 600                 605 

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val 
    610                 615                 620 

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu 
625                 630                 635                 640 

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala 
                645                 650                 655 

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr 
            660                 665                 670 

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr 
        675                 680                 685 

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val 
    690                 695                 700 

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val 
705                 710                 715                 720 

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser 
                725                 730                 735 

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys 
            740                 745                 750 

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro 
        755                 760                 765 

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile 
    770                 775                 780 

Glu Gly Val Met Arg Asn Asn 
785                 790 

 
           
             2  
             319  
             PRT  
             mammalian  
           
            2 

Met Ser Thr Val His Glu Ile Leu Cys Lys Leu Ser Leu Glu Gly Asp 
  1               5                  10                  15 

His Ser Thr Pro Pro Ser Ala Tyr Gly Ser Val Lys Ala Tyr Thr Asn 
             20                  25                  30 

Phe Asp Ala Glu Arg Asp Ala Leu Asn Ile Glu Thr Ala Ile Lys Thr 
         35                  40                  45 

Lys Gly Val Asp Glu Val Thr Ile Val Asn Ile Leu Thr Asn Arg Ser 
     50                  55                  60 

Asn Ala Gln Arg Gln Asp Ile Ala Phe Ala Tyr Gln Arg Arg Thr Lys 
 65                  70                  75                  80 

Lys Glu Leu Ala Ser Ala Leu Lys Ser Ala Leu Ser Gly His Leu Glu 
                 85                  90                  95 

Thr Val Ile Leu Gly Leu Leu Lys Thr Pro Ala Gln Tyr Asp Ala Ser 
            100                 105                 110 

Glu Leu Lys Ala Ser Met Lys Gly Leu Gly Thr Asp Glu Asp Ser Leu 
        115                 120                 125 

Ile Glu Ile Ile Cys Ser Arg Thr Asn Gln Glu Leu Gln Glu Ile Asn 
    130                 135                 140 

Arg Val Tyr Lys Glu Met Tyr Lys Thr Asp Leu Glu Lys Asp Ile Ile 
145                 150                 155                 160 

Glu Asp Gly Ser Val Ile Asp Tyr Glu Leu Ile Asp Gln Asp Ala Arg 
                165                 170                 175 

Asp Leu Tyr Asp Ala Gly Val Lys Arg Lys Gly Thr Asp Val Pro Lys 
            180                 185                 190 

Trp Ile Ser Ile Met Thr Glu Arg Ser Val Pro His Leu Gln Lys Val 
        195                 200                 205 

Phe Asp Arg Tyr Lys Ser Tyr Ser Pro Tyr Asp Met Leu Glu Ser Ile 
    210                 215                 220 

Arg Lys Glu Val Lys Gly Asp Leu Glu Asn Ala Phe Leu Asn Leu Val 
225                 230                 235                 240 

Gln Cys Ile Gln Asn Lys Pro Leu Tyr Phe Ala Asp Arg Leu Tyr Asp 
                245                 250                 255 

Ser Met Lys Gly Lys Gly Thr Arg Asp Lys Val Leu Ile Arg Ile Met 
            260                 265                 270 

Val Ser Arg Ser Glu Val Asp Met Leu Lys Ile Arg Ser Glu Phe Lys 
        275                 280                 285 

Arg Lys Tyr Gly Lys Ser Leu Tyr Tyr Tyr Ile Gln Gln Asp Thr Lys 
    290                 295                 300 

Gly Asp Tyr Gln Lys Ala Leu Leu Tyr Leu Cys Gly Gly Asp Asp 
305                 310                 315 

 
           
             3  
             97  
             PRT  
             mammalian  
           
            3 

Met Pro Ser Gln Met Glu His Ala Met Glu Thr Met Met Phe Thr Phe 
  1               5                  10                  15 

His Lys Phe Ala Gly Asp Lys Gly Tyr Leu Thr Lys Glu Asp Leu Arg 
             20                  25                  30 

Val Leu Met Glu Lys Glu Phe Pro Gly Phe Leu Glu Asn Gln Lys Asp 
         35                  40                  45 

Pro Leu Ala Val Asp Lys Ile Met Lys Asp Leu Asp Gln Cys Arg Asp 
     50                  55                  60 

Gly Lys Val Gly Phe Gln Ser Phe Phe Ser Leu Ile Ala Gly Leu Thr 
 65                  70                  75                  80 

Ile Ala Cys Asn Asp Tyr Phe Val Val His Met Lys Gln Lys Gly Lys 
                 85                  90                  95 

Lys 
 97 

 
           
             4  
             6  
             PRT  
             mammalian  
           
            4 

Trp Cys Gly Pro Cys Lys 
  1               5 

 
           
             5  
             291  
             DNA  
             mammalian  
           
            5 

cttctttccc ttctgcttca tgtgtactac aaaatagtca ttgcatgcaa tggtgaggcc     60 

cgcaattagg gaaaagaagc tctggaagcc cactttgcca tctctacact ggtccaggtc    120 

cttcattatt ttgtccacag ccagagggtc tttttgattt tccaaaaatc cagggaactc    180 

cttttccatg agtactctca ggtcctcctt tgttaagtag cctttatccc cagcgaattt    240 

gtgaaatgta aacatcatgg tttccatggc gtgttccatt tgagatggca t             291 

 
           
             6  
             291  
             DNA  
             mammalian  
           
            6 

atgccatctc aaatggaaca cgccatggaa accatgatgt ttacatttca caaattcgct     60 

ggggataaag gctacttaac aaaggaggac ctgagagtac tcatggaaaa ggagttccct    120 

ggatttttgg aaaatcaaaa agaccctctg gctgtggaca aaataatgaa ggacctggac    180 

cagtgtagag atggcaaagt gggcttccag agcttctttt ccctaattgc gggcctcacc    240 

attgcatgca atgactattt tgtagtacac atgaagcaga agggaaagaa g             291 

 
           
             7  
             391  
             PRT  
             mammalian  
           
            7 

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg 
  1               5                  10                  15 

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser 
             20                  25                  30 

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser 
         35                  40                  45 

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln 
     50                  55                  60 

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys 
 65                  70                  75                  80 

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn 
                 85                  90                  95 

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala 
            100                 105                 110 

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe 
        115                 120                 125 

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu 
    130                 135                 140 

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu 
145                 150                 155                 160 

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr 
                165                 170                 175 

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala 
            180                 185                 190 

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro 
        195                 200                 205 

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp 
    210                 215                 220 

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His 
225                 230                 235                 240 

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys 
                245                 250                 255 

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro 
            260                 265                 270 

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser 
        275                 280                 285 

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser 
    290                 295                 300 

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr 
305                 310                 315                 320 

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp 
                325                 330                 335 

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr 
            340                 345                 350 

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro 
        355                 360                 365 

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp 
    370                 375                 380 

Cys Met Phe Gly Asn Gly Lys 
385                 390 

 
           
             8  
             394  
             PRT  
             mammalian  
           
            8 

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg 
  1               5                  10                  15 

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser 
             20                  25                  30 

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser 
         35                  40                  45 

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln 
     50                  55                  60 

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys 
 65                  70                  75                  80 

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn 
                 85                  90                  95 

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala 
            100                 105                 110 

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe 
        115                 120                 125 

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu 
    130                 135                 140 

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu 
145                 150                 155                 160 

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr 
                165                 170                 175 

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala 
            180                 185                 190 

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro 
        195                 200                 205 

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp 
    210                 215                 220 

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His 
225                 230                 235                 240 

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys 
                245                 250                 255 

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro 
            260                 265                 270 

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser 
        275                 280                 285 

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser 
    290                 295                 300 

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr 
305                 310                 315                 320 

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp 
                325                 330                 335 

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr 
            340                 345                 350 

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro 
        355                 360                 365 

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp 
    370                 375                 380 

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg 
385                 390