Inhibition of Bone Morphogenetic Protein 1 by Native and Altered Forms of α2-Macroglobulin

Abstract

The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein α2-macroglobulin is unable to inhibit the astacin-like proteinases meprin α and meprin β, we herein demonstrate α2-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the α2-macroglobulin “bait” region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I–III. α2-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified α2-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced ∼24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of α2-macroglobulin in the treatment of fibrotic conditions. The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein α2-macroglobulin is unable to inhibit the astacin-like proteinases meprin α and meprin β, we herein demonstrate α2-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the α2-macroglobulin “bait” region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I–III. α2-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified α2-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced ∼24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of α2-macroglobulin in the treatment of fibrotic conditions. Bone morphogenetic protein 1 (BMP1) 3The abbreviations used are: BMP, bone morphogenetic protein; α2M, α2-macroglobulin; ECM, extracellular matrix; EGF, epidermal growth factor; pCP, procollagen C-proteinase; C-propeptide, COOH-terminal propeptide; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline. is the prototype of a subgroup of structurally similar, secreted metalloproteinases, each member of which has an astacin-like protease domain, complement-uegf-BMP1 protein-protein interaction domains, and epidermal growth factor motifs (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (356) Google Scholar, 2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar). Mammalian members of this subgroup proteolytically convert a variety of precursor molecules into mature, functional proteins involved in formation of the extracellular matrix (ECM) (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (356) Google Scholar, 2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar). Members of this class of proteinases also activate a subset of the transforming growth factor-β superfamily of proteins in a broad range of species, through cleavage of extracellular protein antagonists (2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar). Thus, the four mammalian BMP1-like proteinases are likely to be key regulators and orchestrators of ECM formation and signaling by certain transforming growth factor-β-like molecules, in morphogenetic events and homeostasis. Surprisingly, studies regarding regulation of the expression and activities of these key proteinases have been limited. Nevertheless, one such study showed that, whereas transcription of the BMP1 gene and secretion of BMP1 are both up-regulated ∼8-fold by treatment of cells with transforming growth factor-β1, induction of detectable procollagen C-proteinase activity is only ∼2-fold, suggesting the existence of an endogenous inhibitor(s) (3Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In fact, a number of studies have detected unexpectedly low levels of procollagen C-proteinase (pCP) activity in tissues and cell cultures, also leading to suggestions of endogenous inhibitors (4Hojima Y. van der Rest M. Prockop D.J. J. Biol. Chem. 1985; 260: 15996-16003Abstract Full Text PDF PubMed Google Scholar). α2-Macroglobulin (α2M) is a member of the α-macroglobulin family of proteins found in the circulation and egg whites of a broad range of species (5Sottrup-Jensen L. J. Biol. Chem. 1989; 264: 11539-11542Abstract Full Text PDF PubMed Google Scholar). Human α2M is found at relatively high levels (2–4 mg/ml) in plasma and is produced by hepatocytes, but is also produced by a number of other cell types that include lung fibroblasts, macrophages, astrocytes, and tumor cells (6Borth W. FASEB J. 1992; 6: 3345-3353Crossref PubMed Scopus (413) Google Scholar, 7Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (977) Google Scholar). Human α2M is a tetramer of four identical 185-kDa subunits, each of which has an exposed 39-amino acid “bait region” that contains cleavage sites for a variety of proteinases (6Borth W. FASEB J. 1992; 6: 3345-3353Crossref PubMed Scopus (413) Google Scholar, 8Feinman R.D. Ann. N. Y. Acad. Sci. 1994; 737: 245-266Crossref PubMed Scopus (33) Google Scholar). Cleavage within the bait region results in exposure of a highly reactive α2M thioester that can covalently bind the cleaving proteinase (6Borth W. FASEB J. 1992; 6: 3345-3353Crossref PubMed Scopus (413) Google Scholar, 8Feinman R.D. Ann. N. Y. Acad. Sci. 1994; 737: 245-266Crossref PubMed Scopus (33) Google Scholar). Cleavage within the bait region also results in a conformational change that entraps the proteinase within the interior of the α2M molecule, thus inhibiting further proteinase activity by steric hindrance (6Borth W. FASEB J. 1992; 6: 3345-3353Crossref PubMed Scopus (413) Google Scholar, 8Feinman R.D. Ann. N. Y. Acad. Sci. 1994; 737: 245-266Crossref PubMed Scopus (33) Google Scholar). The conformational change also gives rise to what can be considered an “activated” form of α2M, with exposed sites for binding of α2Mto its cognate cell surface receptor, the low-density lipoprotein receptor-related protein, and for binding to a number of cytokines, including transforming growth factor-β, platelet-derived growth factor, interleukin-1β, basic fibroblast growth factor, and nerve growth factor (6Borth W. FASEB J. 1992; 6: 3345-3353Crossref PubMed Scopus (413) Google Scholar). Binding to lipoprotein receptor-related protein results in rapid clearance of α2M-proteinase complexes from the extracellular space and catabolism, although activated α2M appears able to bind cytokines in a reversible manner that allows it to serve as a carrier and targeting protein involved in modulating the biological responses of various cell types (6Borth W. FASEB J. 1992; 6: 3345-3353Crossref PubMed Scopus (413) Google Scholar). BMP1-like proteinases have astacin-like protease domains (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (356) Google Scholar, 2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar). It has previously been reported that α2M does not inhibit vertebrate proteinases with astacin-like protease domains (7Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (977) Google Scholar, 9Kruse M.N. Becker C. Lottaz D. Köhler D. Yiallouros I. Krell H.W. Sterchi E.E. Stöcker W. Biochem. J. 2004; 378: 383-389Crossref PubMed Scopus (143) Google Scholar), such as α meprin and β meprin (9Kruse M.N. Becker C. Lottaz D. Köhler D. Yiallouros I. Krell H.W. Sterchi E.E. Stöcker W. Biochem. J. 2004; 378: 383-389Crossref PubMed Scopus (143) Google Scholar), although bovine α2M has been shown to have inhibitory activity toward the crayfish digestive protease astacin (10Stöcker W. Breit S. Sottrup-Jensen L. Zwilling R. Comp. Biochem. Physiol. B. 1991; 98: 501-509Crossref PubMed Scopus (36) Google Scholar). Here we identify a potential site for cleavage of α2M by BMP1-like proteinases. We demonstrate cleavage at this site by BMP1 and the potent inhibition of BMP1 proteolytic activities via the formation of covalent complexes with cleaved α2M. A genetically altered version of α2M, in which the bait region has been replaced by the BMP1 cleavage site of the small leucine-rich proteoglycan precursor probiglycan is shown to have a greatly enhanced ability to inhibit BMP1, and is able to inhibit procollagen I processing by cells. Implications of the data are discussed. Cleavage of α2M by BMP1—200 nm FLAG-tagged recombinant BMP1, prepared, and purified as previously described (11Scott I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar), was incubated overnight at 37 °C with 200 nm human α2M (Sigma) in 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 10 mm CaCl2. Subsequently, reaction proteins were analyzed by SDS-PAGE under reducing conditions on a 7.5% gel and staining with Coomassie Brilliant Blue R-250. For Western blot analysis, 320 nm BMP1 was incubated with 200 nm α2M at 37 °C overnight, and samples were loaded on 7.5% gels and separated by SDS-PAGE, under both nonreducing and reducing conditions. Separated proteins were transferred to polyvinylidene difluoride membranes and probed with a 1:5000 dilution of anti-FLAG antibody (Sigma). Subsequently, membranes were incubated with a 1:25000 dilution of goat anti-mouse IgG as the of BMP1 by the α2M was with BMP1 for at 37 The study of cleavage of α2M by BMP1 that cleavage of α2M by BMP1 is a under the conditions Thus, the of procollagen processing is to the of BMP1 to α2M. processing of procollagen was α2M and was to of BMP1 Cleavage of by of BMP1 was the of α2M in 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 10 mm at 37 Subsequently, of as previously described I.C. Imamura Y. Pappano W.N. Greenspan D.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), was to a of and incubated Cleavage were by of of 10 of of mm Tris-HCl, pH mm mm and 10 of mm by at 37 °C for were to SDS-PAGE on a gel and Western blot was antibody 1995; PubMed Scopus Google of W. at a 1:5000 and a 1:25000 dilution of goat IgG as the of α2M by α2M was incubated with 50 mm Tris-HCl, pH in the of mm for by 50 mm Tris-HCl, pH 7.5, 100 mm and α2M samples were for the ability to be cleaved by BMP1, and ability to inhibit BMP1 cleavage of of α2M sequences were from human in used 1 and and and and were into the α2M in and of via an site the and sites of the expression The α2M, that the native is replaced by the for of In cleavage of the a at the of α2M α2M, an site was into the of α2M bait region the protein, via used and of 1 and and and were used as for 1 and The was and sites in the α2M sequences were with and The was with and and and sites in the modified α2M In the α2M the sequences flanking the human probiglycan BMP1 cleavage site for the bait region cells were in Dulbecco's modified Eagle's with and bovine at were with 1 of expression cells were in the of with 200 of secreted α2M, induction with 1 was detected via Western cells were with phosphate-buffered and incubated in at 37 were with PBS, by of 1 to protein and was and protease inhibitors were to of mm 1 mm 1 mm and 1 mm was to and were at FLAG-tagged α2M and were from an anti-FLAG the nm recombinant was incubated with mm (Sigma) in at was by to a of were separated by SDS-PAGE on gels and to of and for cells recombinant BMP1 were with PBS, and incubated in at 37 were with PBS, by of 1 to protein and was and protease inhibitors were to of 1 mm 1 mm and 1 mm BMP1 was purified from an anti-FLAG the nm BMP1 was incubated with and nm plasma α2M, recombinant α2M with and nm 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, and 10 mm CaCl2. were at and α2M and recombinant at and by SDS-PAGE and at Protein samples were separated on 7.5% SDS-PAGE which were with and exposed to of BMP1 into high complexes were by and were via the of G. K. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). of by cells were in a to overnight, and with 50 in DMEM, for were with PBS, and incubated in at 37 were with PBS, by of 50 and nm α2M an of mm Tris-HCl, pH 7.5, mm were as described Cell were with PBS, and into and cell samples were to SDS-PAGE on transferred to and probed with antibody 1995; PubMed Scopus Google from as BMP1 of the acid of the α2M bait region found a site and that the of BMP1 cleavage sites The in the of and and to with are found in and an is found in the of previously of proteinases (2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google the of previously cleavage sites of BMP1-like proteinases have with small in the 2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google and such that the of α2M in to and is also of a BMP1 cleavage α2M can be cleaved by BMP1-like the proteins were and α2M for Although α2M incubated at 37 °C overnight was 185-kDa α2M at 37 °C overnight with BMP1 was cleaved to of and acid of the the to the of secreted α2M. acid of the the thus cleavage of α2M by BMP1 at the site and in the bait α2M a with a in the inhibition of proteinases by α2M is formation of a covalent α2M and the we to the of α2M to covalently bind BMP1 is detected on SDS-PAGE gels as a (3Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, I.C. Blitz I.L. Pappano W.N. Imamura Y. Clark T.G. Steiglitz B.M. Thomas C.L. Maas S.A. Takahara K. Cho K.W. Greenspan D.S. Dev. Biol. 1999; 213: 283-300Crossref PubMed Scopus (233) Google Scholar), but it can also be detected as a form on a to with α2M conditions BMP1 can be detected as The and COOH-terminal of cleaved α2M can covalently via and this form can be to other and 185-kDa via R.D. Ann. N. Y. Acad. Sci. 1994; 737: 245-266Crossref PubMed Scopus (33) Google Scholar). Thus, the high under conditions likely BMP1 covalently via reaction with the to and although the of each on the gel The are with Western probed with which α2M to to the high as BMP1 under both reducing and conditions. The of covalent binding of BMP1 to the α2M cleavage is with the α2M has been found to covalently bind other proteinases that it R.D. Ann. N. Y. Acad. Sci. 1994; 737: 245-266Crossref PubMed Scopus (33) Google Scholar). α2M the of the and activity of BMP1 is as a E. Takahara K. L. M. Greenspan D.S. PubMed Scopus Google Scholar), we to this activity is in the of α2M. demonstrated that BMP1 cleavage of plasma α2M by Thus, to the of interaction on BMP1 a of BMP1 was with of α2M to overnight with I were to SDS-PAGE and cleavage of procollagen was by of can be with α2M to potent inhibition of BMP1 with a of α2M the Cleavage of by α2M can inhibit BMP1 cleavage of other we the ability of α2Mto inhibit BMP1 cleavage of can be BMP1 was able to convert probiglycan to under whereas with a excess of α2M for to with probiglycan in inhibition of a of probiglycan Thus, α2M appears to be a inhibitor of BMP1 activity various of α2M of and inhibition of proteinases by α2M is to cleavage of the bait The results in of a highly reactive α2M thioester which can form an lysyl of the proteinase and the of the thioester R.D. Ann. N. Y. Acad. Sci. 1994; 737: 245-266Crossref PubMed Scopus (33) Google Scholar). In cleavage of the bait region a conformational change in α2M, such that it the thus it and inhibiting its ability to with protein via steric hindrance R.D. Ann. N. Y. Acad. Sci. 1994; 737: 245-266Crossref PubMed Scopus (33) Google Scholar). of α2M with which with the also conformational in α2M, and its ability to bind and inhibit proteinases by the described binding and inhibition of BMP1 by α2M is likely to be via the described α2M was with to with BMP1 was unable to cleave α2M α2M with not form complexes with BMP1 and α2M with was unable to inhibit BMP1 processing of probiglycan to of BMP1 to cleave α2M was the of the conformational change in α2M by interaction with the α2M to this conformational the bait region is not for results thus the that binding of BMP1 by α2M can formation of an BMP1 lysyl and the of the α2M and that inhibition α2M conformational to cleavage of the bait α2M to have previously in the with which are cleaved by G. and D. S. of the by BMP1 is probiglycan I.C. Imamura Y. Pappano W.N. Greenspan D.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We to we the ability of α2M to inhibit BMP1 by the native bait region with sequences the probiglycan bone It was found that the recombinant α2M complexes with and is cleaved by BMP1 plasma α2M recombinant α2M, under the conditions as the inhibitory activities of of recombinant α2M and were and to of and The of nm for recombinant α2M in BMP1 inhibition the for plasma α2M whereas the of is with inhibitory In fact, nm BMP1 be to be by nm α2M under one of BMP1 is by one of α2M, the of nm that molecules of are capable of inhibiting one of BMP1, with an of molecules of BMP1 by one of in that is cleaved by BMP1, only in such are molecules of α2M to inhibit at a (5Sottrup-Jensen L. J. Biol. Chem. 1989; 264: 11539-11542Abstract Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). Although cross-linking demonstrated both recombinant α2M and to form small in the activity of the recombinant protein the of the overnight that of the protein from is shown to have in inhibiting BMP1 with α2M under identical with plasma α2M. The of interaction of with BMP1, with α2M, is further by the with which is cleaved by BMP1 with cleavage of recombinant plasma α2M a of the of interaction of BMP1 with the various and of α2M, we the of G. K. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar), which of covalent to of proteinase with α2M. In it can be that, to a of nm BMP1 with nm α2M, BMP1 is into complexes with of with recombinant of plasma of α2M. with a of BMP1 incubated with of each form as in G. K. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar), of and nm for and recombinant and plasma α2M, from the data showed in with α2M was recombinant α2M under identical conditions and α2M from plasma BMP1 inhibitory of plasma and recombinant α2M in a α2M of by α2M is capable of inhibiting the activity of BMP1, we to it be able to inhibit the processing of procollagen by cells. this cells were incubated in the of recombinant α2M and levels of processing of procollagen and into the cell were can be processing of procollagen was by both and α2M. in the of from detectable collagenous was in the form of processing which the but not the is whereas in from cells detectable collagenous was in the form of A and These results inhibition of BMP1-like proteins by and inhibition by The of in the and procollagen in the that both are able to inhibit cleavage in cell by the proteinase is with a that α2M is capable of inhibiting in I. J. J. L. B. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the ability to inhibit to a potential cleavage site at the of the native bait region that is in the was not detected in the of cells in and was only detected exposure of the Western blot of processing and of collagen into the cell cell only whereas cell of with α2M both and which the but not the is were detected only in the of and were not found in the cell of of the cultures, are not into ECM under (3Lee S. Solow-Cordero D.E. Kessler E. Takahara K. Greenspan D.S. J. Biol. Chem. 1997; 272: 19059-19066Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). of cells with plasma α2M not on procollagen processing similar to from treatment of cells with recombinant α2M. BMP1-like proteinases are regulators of ECM in In are of to the formation of collagen fibrils types procollagen C-propeptides to the major fibrous components of cleave a to lysyl oxidase, the enzyme that the formation of covalent in collagen and the and in of procollagen of the collagen types and (2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar). The are into fibrils of collagen types I and and to the of the fibrils D.E. J. Cell Sci. PubMed Google Scholar, Takahara K. D.E. Greenspan D.S. PubMed Scopus Google Scholar). Cleavage of procollagen C-propeptides appears to be the that not collagen and it has been demonstrated in that, whereas I collagen that are into collagen fibrils as as which C-propeptides are from D.J. of from and of and Scholar). of of but not in ECM with cell is with the in The inhibitory of C-propeptides on be via steric hindrance by the relatively of the highly of necessary for and C-propeptides to collagen which be to with D.J. of from and of and Scholar). of with results in fibrils with in results in the of and Scholar). In has been with to cleave such be with and thus with BMP1-like proteinases have been demonstrated to not procollagen C-proteinase activity in E. Takahara K. L. M. Greenspan D.S. PubMed Scopus Google Scholar, W.N. Steiglitz B.M. I.C. D.R. Greenspan D.S. Biol. PubMed Scopus (105) Google Scholar), and of the is for collagen the BMP1-like proteinases are attractive targets for therapeutic interventions in inhibition of collagen is Although the formation of collagen fibrils is to and to the of and bone in the formation of of fibrous collagenous ECM is the of much in the These conditions range from of the to the formation of to of such as the and The are as the of by of fibrous collagenous ECM, that α2M does not inhibit astacin-like proteinases (7Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (977) Google Scholar, 9Kruse M.N. Becker C. Lottaz D. Köhler D. Yiallouros I. Krell H.W. Sterchi E.E. Stöcker W. Biochem. J. 2004; 378: 383-389Crossref PubMed Scopus (143) Google Scholar), we herein demonstrate α2M to be an inhibitor of the BMP1-like a subgroup of the astacin-like proteinases (1Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (356) Google Scholar). Thus, α2M the endogenous inhibitor of BMP1-like proteinases to be as this was prepared, and B. Full Text Full Text PDF PubMed Scopus Google demonstrated the secreted protein to be an inhibitor of such proteinases. The that α2M is a potent inhibitor of BMP1-like proteinases toward therapeutic interventions in the These in of such as and via for and ectopic expression of α2M via gene expression via gene in to targeting of high levels of α2M to specific also in of recombinant α2M with endogenous BMP1-like proteinases in the cells. The inhibitory as it has been that of the by BMP1-like proteinases the cell surface (2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar, Y. J. Cell Biol. 2004; PubMed Scopus (233) Google Scholar), and it has also been previously demonstrated that recombinant α2M is capable of inhibiting both and with endogenous proteinases in the cells L. W. Biochem. J. 1997; PubMed Scopus Google Scholar). Although the be with α2M the also of the bait region of α2M, such that the for inhibition of BMP1-like proteinases is The of the has been in the in which of the native α2M bait region with sequences flanking the probiglycan BMP1 cleavage site the The of the in of inhibiting of a collagenous ECM, is also demonstrated in it is shown that the was in inhibiting procollagen processing by whereas α2M was much Although is for the cleavage sites of BMP1-like we can to identify acid at each flanking such on of the sites of of BMP1-like proteinases (2Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar). In the modified versions of α2M be further for cleavage by BMP1-like proteinases by into flanking cleavage sites for BMP1-like proteinases in bait In of sites for cleavage of the bait region by other proteinases the recombinant α2M specific for inhibition of BMP1-like the of from inhibition of other proteinases. In to the treatment of it be to versions of α2M that inhibit BMP1 which are involved in collagenous ECM not inhibiting matrix involved in the of collagenous to the described are We for