MXPA98000891A - Mocarhagina, a protease from the cobra poison and therapeutic uses of the mi - Google Patents

Abstract

The present invention relates to mochahagina, a protease of cobra venom. Pharmaceutical compositions and therapeutic uses of the protease are also provided

Description

MOCARHAGINA. A P OTEASE OF THE COBRA VENOM AND THERAPEUTIC USES IN THE SAME DESCRIPTION OF THE INVENTION In response to the inflammatory stimulus, the neutrophils in the adjacent vascular system initially coiled in the wall of the blood vessel, then in the form of a cane, and finally transmigrated to the site. of injury. The initial coiling event involves a class of addition proteins called selectins (P-, E-, and L-selectin) which mediate the interactions between leukocytes and endothelial cells by their recognition against specific carbohydrate structures, including Lewis x of sialyl The sequence structure / primary motif of each of the selectins is similar. Each contains a calcium-dependent lectin domain at amino acid 118 terminated at N, an EGF motif, a variable number of respective random motifs related to motifs found in complementary regulatory domains, a transmembrane domain and a short cytoplasmic tail. . P-selectin is an integral granular membrane glycoprotein of 140 kDa located in the granules of the platelet and Weibel-Palade bodies of endothelial cells and is rapidly expressed in both cell types in cell activation. This suggests that endothelial P-selectin is a critical molecule that measured the initial adhesion events in water inflammation, an observation recently reported by an in vivo number of inflammatory models that include acute neutrophil-dependent lung damage (Uulligan et al. (1992) J. Clin. Invest. 90, 1960), endotoxin-induced neutrophrenia (Coughlan et al (1994) J. Exp. Med. 179, 329), reperfusion injury (Asako et al. (1994) J. Clin. Invest 93, 1508) and curl of leukocytes induced by histamine in venules (Eyrich et al (1993) J. Clin Invest. 91, 2620). P-selectin binds 10,000-20,000 copies of a single class of binding site in neutrophils and HL60 cells. Sako et al. ((1993) Cell 75, 1179) has cloned the ligand for P-selectin, named glycoprotein P-selectin ligand 1 (PSGL-1) (see also copending application Serial No. 08 / 316,305). PSGL-1 is a 220 kDa disulfide-linked homodimeric sialomucin which, when expressed in COS cells with the appropriate fucosyltransferase, binds P-selectin in a similar way to calcium-dependent receptor in neutrophils. PSGL-1 has a signal peptide sequence of 17 amino acids followed by a 24 amino acid PACE propeptide sequence. The mature N-terminus of PSGL-1 contains an unusual twenty-amino acid extension which is rich in negatively charged aspartate and glutamate residues and which contains three tyrosine residues which meets the consensus sequence for O-sulfonation by a sulfotransferase of Golgi. At least one of these tyrosine residues is sulfated as assessed by site-directed mutagenesis. In addition to linking P-selectin, PSGL-1 also binds E-selectin. In contrast to P-selectin, however, the requirements for recognition of E-selectin are much less rigid. E-selectin binds a wide variety of sialomucin structures if they co-express the Le is x structure of sialyl. L-selectin binds to a number of different counter-receptors, GLYCAM-1, MadCAM-1 and CD34, which similarly to PSGL-1, are also sialomucins. A major issue currently unresolved is what determines the specificity of selectin in the recognition of structures against specific receptors. P-, E-, and L-selectin are 60-70 & homologs in their lectin motifs of amino acid 118 terminated in N and each similarly recognizes the Lewis x structures of sialyl and Lewis sialyl carbohydrates. In addition, the binding of P-selectin to its receptor on neutrophils is four to five orders of magnitude more avid than Lewis x binding of sialyl. While the differences in specificity and avidity may be partly taken into account by either the presentation of multiple Lewis sialyl carbohydrate structures on the mucin receptor core or by subtle differences in the carbohydrate structure, it is likely that the component of the sialomucin protein also determines the interaction of selectin. Although the inflammatory response mediated by the interaction of P-selectin / PSGL-1 is a part of the body's normal defense system, those on aggressive inflammatory responses may also result in the development of several inflammatory disease states. It may, therefore, be desirable to provide agents to interfere with or block the interaction of P-selectin / PSGL-1 in order to treat the inflammatory disease. The present invention provides compositions comprising the mochahagin protein substantially free of other cobra proteins. In the preferred embodiments, the mocarhagin protein is full-length mocarhagin (as described below). In other embodiments, the protein mochahagin is a fragment of full-length mochahagin that has proteolytic activity of mochahagin. Preferably, the mochahagin protein is characterized by at least one characteristic selected from the group consisting of: (a) A molecular weight of about 55 kDa under reducing conditions. (b) A molecular weight of approximately 55 kDa under non-reducing conditions. (c) A N-terminus amino acid sequence comprising Treo-Xaa-Cis-Pro-Glu-Leu-Xaa-Pro-Tir-Leu-Gln-Xaa-Lis-Cis-Tir-Ile-Glu-Fen-Tir- Val- Val-Val-Asp-Asn (Sequence ID No.l), where the Xaa in the second position is Lis or Asn, the Xaa in the seventh position is lie or Lis, and the Xaa in the twelve position is Lis or Ala; (d) Proteolytic activity of mocarhagin; (e) The ability to inhibit the binding of the platelet to vWF; (f) Requirement of the calcium ion for activity; (g) Requirement of zinc ion for activity; (h) An activity substantially inhibited by excess EDTA; and (i) An activity substantially inhibited by high concentrations of DFP. In particularly preferred embodiments, the mochahagin protein is capable of cleaving PSGL-1. Also provided are compositions comprising a therapeutically effective amount of the mochahagin protein and a pharmaceutically acceptable carrier. Methods for treating an inflammatory disease and inhibiting the selectin-mediated binding comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the mocarhagin protein to a mammalian subject are described. The invention also provides a method for isolating mochahagin from venom, the method comprising: (a) subjecting a composition comprising cobra venom to a heparin affinity chromatography column; and (b) eluting the mochahagin from the column by affinity to heparin. Other methods for mochahagin purification encompassed by the present invention further comprise: (c) subjecting the eluted solution of the column by affinity to heparin to a gel filtration column; and (d) eluting the mochahagin from the gel filtration column. Compositions comprising a protein isolated according to the methods (and optionally further comprising a pharmaceutically acceptable carrier) are also encompassed by the claimed invention. Such compositions can also be used in methods for treating an inflammatory disease and inhibiting the selectin-mediated binding which comprise administering a therapeutically effective amount of such compositions to a mammalian subject. Also provided are compositions comprising an antibody which specifically reacts with mochahagin or a fragment thereof having mochahagin proteolytic activity.

Figure 1: Effect of mocarhagin on the binding of P-selectin to neutrophils. Neutrophils are pretreated for 30 min. at room temperature with increases in concentrations of mocarhagin (circles), or with mocarhagin which has been treated with DFP (diisopropylfluorophosphate) (Triangles). Figure 2: Protease cleavage sites in PSGL-1. The sequence of the N-terminated protein of PSGL-1 indicating the cleavage sites for the signal peptidase, PACE and mocarhagin. the peptide that is used to increase the polyclonal antibody Rb3443 has a superior stripe. The cleavage site of mocarhagine for PSGL-1 contrasts with the cleavage site on GP iba. Figure 3: Mocarhagin digestion of the ligand of the soluble P-selectin glycoprotein. A medium conditioned with COS containing PSGL-1.T7 labeled with [35S] methionine is treated (bands 1,3,5,6 and 8) or digested with 5μg / mg of mochahagina (bands 2,4,7 and 9). The samples are taken by direct electrophoresis (bands 1 and 2), or precipitated with LEC, IgG chimera of P-selectin (bands 3 and 4), or precipitated with LEC? Which is pretreated with mochahagin (LEC? + ni, band 5), or immunoprecipitates with Rb3026 (bands 6 and 7) or with Rb3443 (bands 8 and 9). Figure 4. Effect of IgG anti PSGL-1 on the binding of P-selectin in neutrophils. Dose response curves for inhibition of specific binding of [125 I] P-selectin to neutrophils by polyclonal IgG against synthetic peptide sequences from Gln-1 to Glu-15 (circles) and Asp-9 to Arg-23 (frames). Figure 5: Polyacrylamide gel electrophoresis by 5-20% SDS exponential gradient of purified mocarhagin under non-reducing (NR) and reducing (R) conditions stained with Coomassie blue. The molecular weight standards are myosin (200 kDa), β-galactosidase (130 kDa), phosphorylase B (94 kDa), BSA (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and soybean trypsin inhibitor (21 kDa). Figure 6: Comparison of the N-terminus amino acid sequence determined for mocarhagin with metalloproteases-disintegrins of viper venom. Jararhagina is a 52 kDa protease purified from Bothrops jararaca (Paine et al (1992) J. Biol. Chem. 267, 22869-22876), HR1B and Hr2A are homorragins derived from the venom Trimeresurus flavoviridis (Habu) (Takeya et al. (1989) J. Biochem. (Tokyo) 106, 151-157; Takeya et al. (1990) J. Biol. Chem. 265, 16068-16073); the sequences for protrigramin and pro-rhodostomine are inferred from the cDNA sequences of the disintegrin trigramins (from the poison of T. Gra ineus) (Neeper et al. (1990) Nucleic Acids Res. 18, 4255)) and rhodostomine (from Calloselasma rhodosto a venom (Malayan python viper) (Au et al. (1991) Biochem. Biophys. Res. Commun 181, 585-593)). The identical or conserved waste is framed.

The present invention provides a highly specific metalloproteinase, mochahagin, which has been purified from the venom of the Mozambique spitting cobra, Naja mocambique mocambique. Mocarhagin cuts a peptide of ten amino acids from the mature N-terminus of PSGL-1 and suppresses the ability of PSGL-1 to bind to P-selectin. These results according to the negatively charged sulphated tyrosine group at the N-terminus of PSGL-1 are an important determinant of P-selectin recognition in addition to recognition of the carbohydrate structure. The mochahagina can be purified from the cobra venom according to the method described in the following examples. Other methods for purifying mochahagina from cobra venom will also be apparent to those skilled in the art. The progress of any purification scheme for mochahagin can be monitored based on the biochemical characteristics of the mochahagin described here and the assays for digestion of PSGL-1 and neutrophil cell binding / HL60 described below. The main step in the preferred method of purification is heparin affinity chromatography, based on the observation by Ogilvie and Gartner (1984) that the cobra lectin-dependent hemagglutination is inhibited by heparin. Mochahagina is further purified by gel filtration on Sepharose CL-6B, and has an apparent molecular weight by electrophoresis in 55 kDa polyacrylamide gel under non-reducing and reducing conditions (Figure 5). Typically, 1-2 mg of purified mochahagin is obtained from 0.5 g of lyophilized venom. Mochahagina faintly stained with Schiff's acid reagent is consistent with the presence of glycosylated residues. The N-termination sequencing of purified mocarhagin is achieved with up to 26 residues (Figure 6), although the initial circles show multiple peaks that may be suggestive of a variable N-terminus. The proteinase requires either calcium ion or zinc ion for activity and is substantially inhibited (currently in total form) by excess EDTA and by high concentrations of DFP. The pretreatment of platelets with mocarhagin suppresses their ability to bind the adhesive ligand, von Willebrand Factor (vWF). This is due to the protolysis between Glu-282 and Asp-283 in the chain a of the vWF receptor of the platelet, the complex GP lb-V-IX, which appears as the only detectable excision on the surface of the platelet intact In the course of studies with vWF, it is observed that mochahagina is also a potent inhibitor of the binding of P-selectin to its myeloid receptor in neutrophils. Pretreatment of either neutrophils or HL60 cells with mocarhagin profoundly and reproducibly affect the subsequent binding of P-selectin to these cells treated with an apparent IC50 (inhibitor concentration giving 50% inhibition) of 0.1 μg / ml. An inhibition curve representative of multiple studies performed with both HL60 and neutrophil cells is shown in figure 1. Equivalent data are obtained independently whether the cells treated with mocarhagin are washed or not before the addition of P-selective, ie if the Linkage assay is actually performed in the absence or presence of etaloproteinase. In addition, the inhibition is not reversed by incubation of cells treated with fresh medium for up to three hours. Finally, mochahagin has no effect on the molecular size of P-selectin or on its inherent ability to bind ieloid cells (no data shown and see Figure 2). The treatment of mocarhagin with DFP completely blocks its ability to inhibit P-selective binding even if the myeloid cells are incubated with concentrations of mocarhagina treated with DFP up to 100 μg / ml (Figure 1), a result according to the proteolysis of the P-selectin receptor. Consistent with this point of view, the ability of mocarhagina to inhibit subsequent P-selective binding is dependent on a divalent cation since the incubation of neutrophils with excess EDTA before the addition of mochahagina prevents its inhibitory effect. In addition, in the course of time the effect of mocarhagina is also consistent with a proteolytic event. If the cells are incubated with 12 μg / ml of mocarhagin for ten seconds before the addition of EDTA and the cells are washed, the binding of P-selectin is reduced, even with this brief treatment, to 40% of normal. The concentrated supernatant of the cells treated with mocarhagin, after the removal of mochahagin by absorption with Sepharose CL-6B, does not inhibit the binding of P-selectin to HL60 cells indicating that a functional fragment of the P-selectin receptor is not released by the treatment with mocarhagina. Although the data suggest that mocarhagin suppresses the binding of P-selectin to neutrophils by a proteolytic effect on the P-selectin receptor, the cell-labeled studies fail to identify a major substrate for mocarhagin on either neutrophils or HL60 cells. Both neutrophils and HL60 cells labeled on the surfaces with radioiodinated lactoperoxidase and periodate / tritium are treated with mocarhagin and the resulting cells and supernatants are compared with controls by a one- and two-dimensional SDS polyacrylamide gel analysis. With neutrophils and HL60 cells with radioiodinated lactoperoxidase, no substrate or product consistent with the specificity of exquisite substrate of mocarhagina suggested by platelet studies is identified. With cells marked with periodate, however, analyzes under reducing conditions reveal the partial loss of a 40 kDa band and several barely visible bands are evident in the supernatant. There are also increased amounts of a 140 kDa protein in the supernatant of treated cells, although this band of glycoprotein is also present in supernatants of controls and probably corresponds to the spontaneous release of small amounts of leukosialin. P-selectin has previously been established not to bind leukosialin. Since blotting assays of the P-selectin ligand have not identified a 40 kDa molecular weight binding protein and since the 40 kDa glycoprotein is only partially cut at a much higher concentration of mochahagin than required to cancel the P-selectin binding (Figure 1), it is likely that none of these minor events represents the cleavage of the P-selectin receptor. Glycoprotein 1 P-selectin (PSGL-1) has recently been identified as a functional ligand for P-selectin on HL60 cells. Form . soluble of PSGL-1, designated SPSGL-1.T7 (comprising amino acids 18-295 of PSGL-1), when expressed in COS cells with 1,3 / 1,4 fucosyltransferase, also measured the P-selectin binding in a calcium-dependent form (Sako et al.) One of the salient features of PSGL-1 is its similarity to platelet chain I GP Ib. Both are sialomucins and each has an N-terminus immediately for the mucin nucleus, a sequence rich in negatively charged amino acids with three potential tyrosine residues (Sako et al.; López et al. (1987) Proc. Nati Acad. Sci. USA 78, 3403; Titani et al. (1987) Proc. Nati Acad. Sci. USA 84, 5610). Since mochahagin breaks the I chain of GP ib within this sulphated / negatively charged tyrosine group (Figure 2), it is speculated that mochahagin can cancel the binding of P-selectin to neutrophils and HL60 cells breaking near the N-terminus. of PSGL-1, result which could explain the failure to identify a main substrate for mocarhagin on myeloid cells. It is confirmed that this is indeed the case for the data of Figure 3. Figure 3 shows that the digestion of mochahagin from cut PACE, fucosylated sPSGL-1 (comprising amino acids 42-295 of PSGL-1) results in only a minor change, if any, in the electrophoretic mobility of the protein on an SDS polyacrylamide gel (lanes 1 and 2), but completely abolish the binding of SPSGL-1.T7 to the IgG chimera of P-selectin, LEC? l (Sako et al.), coupled to protein A of Sepharose (lanes 3 and 4). To exclude the possibility that the protease treatment interferes with the LEC binding by destroying the LEC complex and protein A of Sepharose, the following control experiment is performed. The beads of LEC? Sepharose protein A are incubated with mochahagin and then washed repeatedly to remove any residual protease. The treated protease beads are not affected in their ability to bind sPSGL-l.T7 (band 5). Figure 3 also shows the reactivity of sPSGL-1 digested with mocarhagin and untreated with two polyclonal antibodies Rb3026 (Sako et al.) Which increases against COS yields SPSGL-1.T7, precipitates sPSGL-1 independently of digestion with mochahagina (lanes 6 and 7), whereas Rb3443, which is increased against the N-terminated peptide of cut PACE PSGL-1 (QATEYEYLDYDFLPE, SEQUENCE ID NO: 2), only precipitates untreated SPSGL-1.T7 (bands 8 and 9), indicating that the terminating epitope N for Rb3443 is lost after digestion with mochahagin. The exact cleavage or cleavage site of mocarhagin in PSGL-1 is determined by microsequencing of N-termini of the SPSGL-1T7 protein cut with mochahagin (Figure 2). The N-terminus sequence of PSGL-1 cut with mochahagin is determined to be DFLPETEPPEML (SEQUENCE ID NO: 6). Mochahagin removes the first ten amino acids of Spsgl-l.T7 cut with PACE. This N-terminated peptide comprises three tyrosine residues, at least one of which is sulphated as determined by NMR. The mochahagin cleavage site is confirmed to be between Tir-10 and Asp-11 using the synthetic peptide, TEYEYLDYDFLPETE (SEQUENCE ID NO: 3), corresponding to residues 3-17 of mature N-terminus PSGL-1. The cleavage sites of mocarhagin on PSGL-1 and the chain a of GP Ib are similar. Each occurs on the site of the N terminus of an aspartate residue and the C terminal side of three potential sulphated tyrosine residues and within a negatively charged group (Figure 2). Since the proteolytic activity of mocarhagin is inhibited by heparin and polyanions (Ward et al., In manuscript in the preparation), this preference for group of negative charges can in part explain the specificity to the remarkable substrate of mocarhagin. Confirmation of the critical importance of the sequence of the N-terminus of PSGL-1 at the P-selectin linkage is obtained using antipeptide antibodies. The binding of P-selectin to neutrophils by 80-90% is inhibited by an affinity purified polyclonal antibody against Gln-1 to Glu-15 residues of mature PSGL-1 (QATEYEYLDYDFLPE, SEQUENCE ID NO: 2), but not with a affinity purified polyclonal antibody against Asp-9 to Arg-23 (DYDFLPETEPPEMLR, SEQUENCE ID NO: 4) (Figure 4) or non-immune rabbit IgG (not shown). This result, together with the observed sensitivity of the myeloid P-selectin receptor for mocarhagin, is consistent with PSGL-1 which is the predominant and main receptor for P-selectin on myeloid cells. Although P-, E-, and L-selectin recognize similar sialylated carbohydrate structures such as Lewis x from sialyl, and many glycoproteins on the surface of myeloid cells containing Lewis x from sialyl, P-selectin appears to be highly specific in its recognition from PSGL-1 (Sako et al.). The present data suggest that a cause for this specificity is the sulphated tyrosine / negative charge group at the N terminus of mature PSGL-l. The proteolytic removal of a N-terminated ten amino acid peptide by mochahagin cancels the binding of P-selectin to PSGL-1 even though this sequence (QATEYEYLDY, SEQUENCE ID NO: 5) is not glycosylated. One explanation for this phenomenon is that the removal of this sequence alters the conformational integrity of PSGL-1 such that P-selectin can no longer interact with critical carbohydrate structures associated with the mucin core of PSGL-1. This is probably for two reasons. First, an affinity purified polyclonal antibody against the fifteen N-terminus amino acids of mature PSGL-l also strongly inhibits the binding of P-selectin to neutrophils. Second, E-selective as well as P-selectin also binds to PSGL-1, but, unlike P-selectin, E-selectin binds equally well to PSGL-1 cut by mochahagin suggesting that carbohydrate recognition structures about PSGL-l are still inherently accessible. An alternative explanation of the present observations is that the binding of P-selectin to PSGL-1 is bimodal since the P-selectin binding not only involves the recognition of the carbohydrate but also the sulphated / negatively charged tyrosine group. The approximately order of magnitude difference in avidity for the binding of P-selectin to Lewis x of sialyl against the receptor strongly suggests that additional structural determinants are involved in the binding of P-selectin to its myeloid receptor. This is further supported by the observation that the binding of P-selectin to myeloid cells is not only dependent on the N-terminus lectin domain, but also on the adjacent EGF-like motif (Kansas et al. (1994) J. Cell Biol. 124, 609). For the purposes of the present invention, it is defined that a protein has "mocarhagin proteolytic activity" when (1) it digests PSGL-1, such as the PSGL-1 digestion assay described below, and / or (2) inhibits the binding of P-selectin to neutrophils or HL60 cells, such as in the binding inhibition assay described below. Preferably, complete cleavage of "[S] -sPSGL-1.T7 in 20 minutes using 10 μg / ml mocarhagin protein is achieved in the PSGL-1 digestion assay.; more preferably in 20 min. using less than 1 μg / ml of the mocarhagin protein; preferably, in the neutrophil binding / HL60 inhibition assay, the mochahagin protein exhibits an IC50 of less than about 100 μg / ml; more preferably less than about 1 μg / ml. Mochahagin fragments having mochahagin proteolytic activity are also encompassed by the present invention. Mochahagin fragments having mochahagin proteolytic activity can be identified by the PSGL-1 digestion assay and neutrophil-binding / HL60 inhibition assay described below. The mochahagin fragments may be in a linear form or may be cyclized using known methods, for example, as described in H: U: Saragovi, et al., Bio / Technology 1Q_, 773-778 (1992) and in R.S. Me Dowell, et al., J. Amer. Chem. Soc. 114. 9245-9253 (1992), which are incorporated herein by reference. For purposes of the present invention, all references to "mocarhagin protein" herein include mochahagin and fragments having mochahagin proteolytic activity. The isolated mochahagin protein may be useful for treating conditions characterized by medium intercellular adhesion by E- or P-selectin. Such conditions include, without limitation, myocardial infarction, bacterial or viral infection, metastatic conditions, inflammatory disorders such as arthritis, acute respiratory distress syndrome, asthma, emphysema, delayed-type hypersensitivity reaction, systematic lupus erythematosus, thermal damage such as burns or freezing, autoimmune thyroiditis, experimental allergic encephalomyelitis, multiple sclerosis, multiple organ damage syndrome secondary to trauma, diabetes, Reynaud's syndrome, neutrophilic dermatosis (Sweet's syndrome), inflammatory bowel disease, Grave's disease, glomerulonephritis, gingivitis, periodontitis, haemolytic uraemic syndrome, ulcerative colitis, Crhon's disease, necrotizing enterocolitis, granulocyte transfusion associated syndrome, cytosine-induced toxicity, and the like. The mochahagin protein can also be useful in organ transplantation, both to prepare the organs for transplantation and stop the rejection of organ transplantation. The mochahagin protein can be used to treat patients with hemodialysis and leukophoresis. The mocarhagin protein may be useful on its own as an intercellular adhesion inhibitor mediated by P- or E-selectin or for designing intercellular adhesion inhibitors mediated by E-selectin. The present invention encompasses both pharmaceutical compositions containing the mocarhagin protein and therapeutic methods of treatment or use which employ the mocarhagin protein. The mochahagin protein can be used as a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition can comprise, in addition to the mochahagin protein and the carrier, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of or of the active ingredients. The characteristics of the carrier will depend on the administration route. The pharmaceutical composition of the invention may also contain cytosines, lymphosines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6. , IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, G-CSF, Meg-CSF, germ cell factor, and erythropoietin. The pharmaceutical composition may contain thrombolytic or antithrombotic factors such as plasminogen activator and Factor VIII. The pharmaceutical composition may also contain other anti-inflammatory agents. Such additional factors and / or agents can be included in the pharmaceutical composition to produce a synergistic effect with the mochahagin protein, or minimize the side effects caused by the protein mocarhagina. Conversely, the mocarhagin protein can be included in the formulations of cytosine, lymphosin, another hematopoietic factor, thrombolytic or antithrombotic factor, or anti-inflammatory agent to minimize the side effects of cytosine, lymphosin, another hematopoietic factor, thrombolytic or antithrombotic factor, or anti-inflammatory agent. The pharmaceutical composition of the invention may be in the form of a liposome in which the mochahagin protein is combined, in addition to other pharmaceutically acceptable carriers, with antipathy agents such as these. as lipids which exist in aggregate form as micelles, insoluble monolayers, liquid crystals, or laminar layers which are in aqueous solution. Suitable lipids for a liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfates, lysolecithin, phospholipids, saponin, bile acids, and the like. The preparation of such liposomal formulations is within the level of skill in the art, as described, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; U.S. Patent No. 4,737,323; all of which are incorporated here for reference. As used herein, the term "therapeutically effective amount" means the total amount of each of the active components of the pharmaceutical composition or method that is sufficient to show a significant benefit to the patient, ie, cure of chronic conditions characterized by cell adhesion mediated by P-selectin or E-selectin or increase in the rate of cure of such conditions. When applied for the individual active ingredient, administered alone, the term refers to that single ingredient. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, a therapeutically effective amount of the mocarhagin protein is administered to a mammal having a disease state mediated by P-selectin. The mochahagin protein can be administered according to the method of the invention either alone or administered with other therapies such as treatments employing cytosines, lymphosines, or other hematopoietic factors. When administered with one or more cytosines, lymphosines or other hematopoietic factors, the isolated mocarhagin protein can be administered either simultaneously with cytosines, lymphosines, other hematopoietic factors, thrombolytic or antithrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administration of the isolated mocarhagin protein in combination with cytosines, lymphosines, other hematopoietic factors, thrombolytic or antithrombotic factors. The administration of the mochahagin protein used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional forms, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous injection to the patient is preferred. When a therapeutically effective amount of the mochahagin protein is administered orally, the mochahagin protein will be in the form of a tablet, capsule, powder, solution or elixir. When administered in the tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% mochahagin protein, and preferably from about 25 to 90% mochahagin protein. When administered in liquid form, a liquid carrier such as water, petroleum, animal or vegetable oils such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oil can be added. The liquid form of the pharmaceutical composition may further contain physiological saline, dextrose, or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or propylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the mochahagin protein and preferably from about 1 to 50% of the mochahagin protein. When a therapeutically effective amount of the mocarhagin protein is administered by intravenous injection, cutaneous or subcutaneous, the mocarhagin protein will be in the form of a parental, aqueous, pyrogen-free, acceptable solution. The preparation of such parentally acceptable protein solutions, taking into account pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous or subcutaneous injection must contain, in addition to the mocarhagin protein an isotonic vehicle such as a sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, Ringer's injection lactase, or other vehicle as is known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The amount of mocarhagin protein in the pharmaceutical composition of the present invention will depend on the nature and severity of the condition being treated, and on the nature of the prior treatments to which the patient has been subjected. Finally, the attending physician will decide the amount of mocarhagin protein with which each individual patient will treat. Initially, the attending physician will administer low doses of the mocarhagin protein and observe the patient's response. Large doses of the mocarhagin protein can be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dose is not further increased. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.1 μg to about 100 mg of the mocarhagin protein per kg body weight. The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the potential idiosyncratic condition and response of each individual patient. It is contemplated that the duration of each application of the mocarhagin protein will be in the range of 12 to 24 hours of continuous intravenous administration. Finally, the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention. The mochahagin protein of the invention can also be used to immunize animals to obtain polyclonal and monoclonal antibodies which react specifically with the mochahagin protein and which can inhibit cell adhesion mediated by P-selectin. Such antibodies can be obtained using the complete mochahagin protein as an immunogen, or using fragments of the mochahagin protein such as the soluble mature mochahagin protein. The peptide immunogens may additionally contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Additional peptide immunogens can be generated by replacing tyrosine residues with sulphated tyrosine residues. Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Examples The following examples are presented to illustrate, not limit, the present invention. Materials Naja mocambique mocambique venom, diisopropyl fluorophosphate (DFP), aprotonin and pepstatin from Sigma, St. Louis, MO; Triton X-100 from BDH, Kilsyth, Victoria, Australia; RPMI tissue culture medium and mono-poly resolution medium (Ficoll-Hipaque) from Flow Laboratories, Irvine, Scotland; fetal bovine serum from Cytosystems, Castle Hill, N.S.W., Australia; Heparine-Sepharose CL-6B, Protein A from Sepharose and Sepharose CL-6B from Pharmaci, Uppsala, Sweden; leupeptin from Auspep Pty. Ltd, Melhourne, Victoria, Australia, [125] iodine and [3H] sodium borohydride from New England Nuclear, Wilmington, DE. The peptide TEYEYLDYDFLPETE, corresponding to residues 3-17 of mature PSGL-l is synthesized by Chiron Mimotopes Pty., Melbourne, Australia. Antipeptide antibodies Synthetic peptides based on the amino acid sequences PSGL-1 Gln-1 to Glu-5 and Asp-9 to Arg-23 containing an N-terminus cysteine residue to facilitate coupling are purified by reverse-phase HPLC and characterized by mass spectroscopy (Chiron). The peptides are coupled to the keyhole limpet hemocyanin (Sigma) with m-maleimidobenzoyl-N-hydroxysuccinimide (Pierce, Rockfork, IL) essentially as previously described (Lerner et al (1981) Proc. Nati. Acad. Sci. USA 78, 3403) and rabbit antiserum is obtained by a standard protocol (Chong et al (1994) Blood 83, 1535). Anti-peptide IgG is purified by affinity on the peptide coupled to a 1: 1 mixture of Affi-gel 10 and 15 (0.5 mg of peptide / 5 ml of resin and 0.5 mg of peptide conjugated to BSA / 5 ml of resin) according to the manufacturer's instructions (Bio Rad, Richmond, CA). After washing with 0.01 M Tris, 0.15 M sodium chloride, pH 7.4 (TS buffer), IgG bound with 0.1 M glycine, pH 2.8 is eluted, the maximum fractions are immediately reneutralized by the addition of one fifth volume of Tris 0.1, pH 8, and the antibody is dialyzed in the TS buffer. The non-immune rabbit IgG is prepared as previously described (Skinner et al (1991) J. Biol. Chem. 266, 5371). Purification of mochahagina The purification of mochahagina from the Naja mocambique mocambique venom is based on the heparin binding properties of cobra lectins (Ogilvie &Gartner, 1984). The lyophilized venom was purified without purification (0.5 g) in 10 ml of water and loaded at 25 ml / h on a column with Sepharose CL-6B of 1.5 x 40 cm heparin in TS buffer (Tris 0.01 M, sodium chloride 0.15 M, pH 7.4) at 22 ° C, and washed thoroughly with TS buffer. The bound protein is eluted with 250 ml of a linear 0.15-1.0 M sodium chloride gradient in 0.01 M Tris, pH 7.4. Fractions containing mochahagin as evaluated by SDS polyacrylamide gel electrophoresis are pooled and concentrated using an Amicon ultrafiltration device fixed with a YM30 membrane, and applied at 25 ml / h on a 1.5 x 70 Sepharose CL-6B column. cm equilibrated with 0.01 M Tris, 0.5 M sodium chloride, pH 7.4. The maximum mochahagin fractions are pooled and dialyzed against the TS buffer. The mocarhagina treated with DFP was prepared by incubating 250 μg of mochahagina in 1 ml of TS buffer with 8 mM DFP (final concentration) for 1 hour at 22 ° C, followed by dialysis against the TS buffer. Sequencing of N-termini of mochahagin Protein samples for N-termination sequencing are dialyzed against 0.02% SDS and concentrated by vacuum centrifugation (Speedvac concentrator, Savant), the N-terminator sequence is determined using a Model 470A protein sequencer. Applied Biosystems equipped with a continuous Model 120A phenylthiohydantoin analyzer. Polybrene is used as a carrier.

Linkage of P-selectin to neutrophils and HL60 cells (Neutrophil binding inhibition assay / HL60). Neutrophils are isolated from poisonous blood coagulated with heparin (20 units / ml, final concentration) according to the method of Bignold and Ferrante ((1987) J. Immunol.Meth.96.29). Neutrophils are > 95% pure as assessed by flow cytometry and > 98% viable by exclusion of trypan blue. HL60 cells are cultured in RPMI medium supplemented with 10% fetal calf serum. Immediately before use, the cells are washed twice with phosphate buffered saline (0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4). The neutrophils and cultured cells are finally resuspended at 2 x 10 7 / ml in RPMI medium supplemented with 1% fetal calf serum. The binding of P-selectin labeled with 125 I (Skinner et al.) To neutrophils or HL60 cells is evaluated by incubating 125 I-labeled P-selectin (0.5 μg / ml, final concentration) with cells (1 x 107 / ml, final concentration) at 22 ° C in a final volume of 200 μg of 17% sucrose (weight / volume) in RPMI medium containing 1% fetal calf serum. Neutrophils are formed in granules at 8.750 x g for 2 min. After careful suctioning of the supernatant, the radiolabel associated with the cell granules is measured in a counter. The non-specific binding of P-selectin labeled with 125 I is calculated using a 50-fold excess of P-selectin (Skinner et al.).

To examine the effect of pretreatment of neutrophils or HL60 cells with mochahagin on the p-selectin binding, the washed cells (2 x 10 7 / ml) in RPMI made in 1% fetal calf serum are incubated in the presence or absence of 10 mM EDTA followed by mochahagin (0.025-100 μg / ml, final concentrations) for 30 min. at 22 ° C. The P-selectin binding is then evaluated either directly or is evaluated after centrifugation of the cells, which are then washed twice and finally resuspended in RPMI with 1% fetal calf serum. In some experiments, mocarhagina treated with DFP is used instead of mocarhagin. To evaluate the effect of the supernatant of cells treated with mocarhagin on the P-selectin bond, HL60 cells are incubated at 10a / ml in 0.01 M Tris, 0.015 M sodium chloride, 0.001M calcium chloride, pH 7.4, with mochahagina (12 μg / ml) for 10 min at 22 ° C. The supernatant collected after centrifugation at 1000 x g for 10 min is brought to 0.1% in BSA and loaded onto the Sepharose CL-6B heparin column (0.5 x 5 cm) to remove the mochahagin. The flow through is then tested for its effect on the binding of P-selectin to HL60 cells. Effect of mochahagin on neutrophils and marked HL60 cells on the surface. Neutrophils or washed HL60 cells are labeled on the surface with either lactoperoxidase-catalysed radioiodination or with sodium periodate / [3] sodium borohydride (Berndt et al (1981) J. Biol. Chem. 256, 59; Booth et al. al. (1984) J. Clin Invest. 73.291). The cells labeled in 0.01 M Tris, 0.15 M sodium chloride, 0.001 M calcium chloride, pH 7.4, are incubated with mocarhagina (12 μg / ml, final concentration) or buffer for 10 min at 22 ° C. Cells are centrifuged at 150 xg for 10 minutes, and washed twice with 0.01 M Hepes, 0.15 M sodium chloride, 0.001 M EDTA, pH 7.4. Cells are then lysed with 1% Triton X-100 (volume / volume) at 4 ° C for 1 hour in the presence of the following protease inhibitors: diisopropyl difluorophosphate (DFP) (0.5 mM), aprotonin (10 μg / ml), pepstatin (1 M), leupeptin (100 μg / ml). U benzamidine (10 M). The soluble fractions in Triton X-100 separated by centrifugation at 1,000 xg for 10 min. and control supernatants and cells treated with mochahagin are mixed with the buffer of the SDS sample and then electrophoresed on a 5-15% SDS polyacrylamide gel under reducing and nonreducing conditions, or on a non-reducing / reducing gel of one to two dimensions as described by Phillips and Agin ((1977) J. Biol. Chem. 252, 2121). The gels are then stained with Coomassie bright blue R and either prepared for fluorography according to Bonner and Lasky ((1974) Eur. J. Biochem., 46, 83) or for autoradiography. Digestion with Mocarhagina from PSGL-1 (PSGL-1 digestion assay) COS cells are cotransferred with three plasmids encoding soluble PSGL-1 (pED, sPSGL-1.T7; Sako et al.), Alpha 1,3 / 1,4 fucosyltransferase (pEA.3 / 4FT) and soluble PACE (pEA-PACE SOL, Wasley et al (1993) J. Biol. Chem. 268, 8458-8465). The conditioned medium with [35 S] methionine-labeled COS containing SPSGL-1.T7 is digested with 5 μg / ml of mocarhagin in TBS, 2 mM calcium chloride; 1 mg / ml of BSA for 20 minutes at 37 ° C. The ability of sPSGL-1 to bind P-selectin is evaluated by precipitation with LEC? P-selectin IgG chimera (Sako et al.) Preabsorbed on Sepharose protein A counts in TBS, 2 M calcium chloride , 1 mg / ml of BSA for 4 hours at 4C. A control experiment is also performed where the sepharose protein A counts with LEC? L are pretreated with mochahagin and then thoroughly washed prior to the presentation of SPSGL-1.T7 for immunoprecipitation analysis of untreated sPSGL-1 and treated with mochahagin, the protease is deactivated by the addition of 5 mM EDTA. PSGLS-1T7 is then precipitated with anti-PSGL-1 polyclonal antibodies Rb3026 (obtained against sPSGL-1 which produces COS, Sako et al.) Or Rb3443 (originated against the N-termination peptide of PSGL-1 cleaved with PACE; QATEYEYLDYDFLPE). Purification of SPSGL-1.T7. The conditioned medium with COS cells transfected with the three pED peptides. sPSGL-1. T7, pEA.3 / 4FT and pEA-PACE Sol, is diluted twice with 50 mM MOPS, 150 mM NaCl, 0.5 mM CaCl2 and 0.5 mM MnCl2, pH 7.2, and applied to a Sepharose 4B lectin column equilibrated with the same shock absorber. After loading, the column is washed with the same buffer until the optical absorbance at 280 nm decreases to a stable baseline. The column is then eluted with the same buffer which has been adjusted for 0.5 M a-methyl mannoside and 0.3 M NaCl. Recombinant SPSGL-1.T7 is collected on 5-15 column volumes of this elution buffer, the The lentilectin eluate is then subjected to a precipitation of 0-70% ammonium sulfate by adding 472 g of ammonium sulfate per liter of column eluate at 4 ° C. After stirring for 30 minutes, the precipitate is suspended in a minimum volume of TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) and applied to a TSK G4000SWXL gel filtration column equilibrated with TBS. The flow velocity on the column is 0.5 ml / min and a protection column is used. In aliquots of < 250 μl, the resuspended ammonium sulfate granule is injected into the column and the fractions are analyzed by SDA-PAGE and Western analysis.

Identification of the cleavage site for mocarhagin on PSGL-1. 20 μg of purified sPSGL-1 are digested with 1 μg of mochahagina in a total volume of 100 μl of TBS containing 2 mM CaCl 2 for 1 hour at 37 ° C. The protease is inactivated by the addition of 10 mM EDTA. The sample is concentrated directly on Pro-spin (Applied Biosystems, Foster City, CA) and subjected to N-termination sequencing over an ABI m476 gas-phase protein sequencer.

SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: Berndt, Michael C. Dunlop, Lindsay Andrews, Robert Deluca, Mariagrazia (ii) TITLE OF THE INVENTION: MOCARHAGINA, A NEW PROTEASE OF THE COBRA VENOM, AND THERAPEUTIC USES OF THE SAME. (iii) NUMBER OF SEQUENCES: 6 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Genetics Institute, Inc. (B) STREET: 87 CambridgePark Drive (C) CITY: Cambridge (D) STATE: Massachusetts (E) COUNTRY: USA (F) POSTAL CODE: 02140 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM Compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARES: Patentein Reeléase # 1.0, version # 1.25. (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) INFORMATION OF THE APPORTER / AGENT (A) NAME: Brown, Scott A. (B) NUMBER OF REGISTRATION: 32,724 (C) REFERENCE NUMBER / CERTIFICATE: GI5239 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (617) 498-8224 (B) TELEFAX: (617) 876-5851 (2) INFORMATION FOR SEQUENCE ID NO : 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) CHAINS: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 1: iv Tre Xaa Cis Pro Glu Leu Xaa Pro Tir Leu Gln Xaa Lis 1 5 10 Cis Tir He Glu Fe Tir Val Val Val Asp Ans 15 (2) INFORMATION FOR SEQUENCE ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) CHAINS: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 2: Gln Ala Treo Glu Tir Glu Tir Leu Asp Tir Asp Fen 1 5 10 Leu Pro Glu 15 (2) INFORMATION FOR SEQUENCE ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) CHAINS: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : peptide (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 4: Asp Tir Asp Fen Leu Pro Glu Treo Glu Pro Pro Glu Me 1 5 10 Met Leu Arg 15 (2) INFORMATION FOR SEQUENCE ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHAINS: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCING: SEQUENCE ID NO: 5: Glu Ala Treo Glu Tir Glu Tir Leu Asp Tir 1 5 10 (2) INFORMATION FOR SEQUENCE ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid ( C) CHAINS: one (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (xi) SEQUENCE DESCRIPTION: SEQUENCE ID NO: 6: Asp Fen Leu Pro Glu Tre Glu Pro Pro Glu Met Leu 1 50 10

Claims (17)

1. CLAIMS 1. A composition characterized in that it comprises the mochahagin protein substantially free of other proteins of the cobra.
2. 2. The composition according to claim 1, characterized in that the mocarhagin protein is full-length mochahagin.
3. The composition according to claim 1 characterized in that the mochahagin protein is a fragment of the full-length mochahagin having the proteolytic length of the mochahagin.
4. 4. The composition according to claim 1 characterized in that the mochahagin protein is exhibits an IC50 of less than about 100 μl / ml in a neutrophil binding / HL60 inhibition assay.
5. The composition according to claim 1 wherein the mochahagin protein is characterized by at least one characteristic selected from the group consisting of: (a) A molecular weight of about 55 kDa under reducing conditions. (b) A molecular weight of approximately 55 kDa under non-reducing conditions. (c) A N-terminus amino acid sequence comprising Treo-Xaa-Cis-Pro-Glu-Leu-Xaa-Pro-Tir-Leu-Gln-Xaa-Lis-Cis-Tir-Ile-Glu-Fen-Tir- Val- Val-Val-Asp-Asn, where the Xaa in the second position is Lis or Asn, the Xaa in the seventh position is He or Lis, and the Xaa in the twelve position is Lis or Ala; (d) Proteolytic activity of mocarhagin; (e) The ability to inhibit the binding of the platelet to vWF; (f) Requirement of the calcium ion for activity; (g) Requirement of zinc ion for activity; (h) An activity substantially inhibited by excess EDTA; and (i) An activity substantially inhibited by high concentrations of DFP.
6. 6. The composition in accordance with the claim 1 characterized in that the mocarhagin protein is capable of cleaving PSGL-1.
7. 7. A composition characterized in that it comprises a therapeutically effective amount of a composition according to claim 1 and a pharmaceutically effective carrier.
8. 8. A method for treating an inflammatory disease characterized in that it comprises administering a therapeutically effective amount of a composition according to claim 7 to a mammalian subject.
9. 9. A method for inhibiting the selectin-mediated binding characterized in that it comprises administering a therapeutically effective amount of a composition according to claim 7 to a mammalian subject.
10. 10. A method for isolating mocarhagina from venose, the method is characterized in that it comprises: (a) subjecting a composition comprising cobra venom to a heparin affinity chromatography column; and (b) eluting the mochahagin from the column by affinity to heparin.
11. 11. The method according to claim 10, characterized in that it further comprises: (c) subjecting the solution eluted from the column by affinity to heparin to a gel filtration column; and (d) eluting the mochahagin from the gel filtration column.
12. 12. The composition characterized in that it comprises an isolated protein according to the method of claim 10.
13. 13. The composition according to claim 12, characterized in that it also comprises a pharmaceutically acceptable carrier.
14. A method for treating an inflammatory disease characterized in that it comprises administering a therapeutically effective amount of a composition according to claim 13 to a mammalian subject.
15. 15. A method for inhibiting the selectin-mediated binding characterized in that it comprises administering a therapeutically effective amount of a composition according to claim 13 to a mammalian subject.
16. 16. A composition characterized in that it comprises administering an antibody which specifically reacts with mochahagin or a fragment thereof having proteolytic activity of mochahagin.
17. 17. The composition according to claim 4 characterized in that the mocarhagin protein exhibits an IC50 of less than about 1 μl / ml in a neutrophil binding assay / HL60.