Interaction Properties of the Procollagen C-proteinase Enhancer Protein Shed Light on the Mechanism of Stimulation of BMP-1

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150–400 nm. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nm. Finally, pN-collagen (i.e.procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases. Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150–400 nm. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nm. Finally, pN-collagen (i.e.procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases. bone morphogenetic protein-1 procollagen C-proteinase PCP enhancer short gastrulation twisted gastrulation module found in complement subcomponents C1r/C1s,Uegf, and BMP-1 netrin-like tissue inhibitor of metalloproteinases isolated C-terminal propeptide trimer from the procollagen I molecule isolated C-terminal propeptide trimer from the procollagen III molecule procollagen molecule lacking the C-terminal propeptide domain surface plasmon resonance N-succinimidyl 6-[4′-azido-2′-nitrophenylamino]hexanoate monoclonal antibody secreted protein, acidic and rich in cysteine cartilage oligomeric matrix protein mannan binding lectin Bone morphogenetic protein-1 (BMP-1)1 and other tolloid-related metalloproteinases (1Kessler E. 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Biochem. 2001; 268: 2991-2996Crossref PubMed Scopus (34) Google Scholar), which lacks intrinsic proteinase activity. Similarly, in the case of chordin and SOG, the protein TSG or its homologues stimulates cleavage by tolloid proteinases (20Scott I.C. Blitz I.L. Pappano W.N. Maas S.A. Cho K.W. Greenspan D.S. Nature. 2001; 410: 475-478Crossref PubMed Scopus (159) Google Scholar, 21Yu K. Srinivasan S. Shimmi O. Biehs B. Rashka K.E. Kimelman D. O'Connor M.B. Bier E. Development. 2000; 127: 2143-2154Crossref PubMed Google Scholar), thus raising the possibility that PCP processing of different substrates might be specifically regulated by distinct enhancer proteins. Both tolloid proteinases and PCPE are multidomain glycoproteins containing multiple copies of the so-called CUB domain (22Bork P. Beckmann G. J. Mol. Biol. 1993; 231: 539-545Crossref PubMed Scopus (521) Google Scholar), a protein module common to several extracellular and plasma membrane-associated proteins (23Thielens N.M. 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Biochemistry. 2000; 39: 3231-3239Crossref PubMed Scopus (53) Google Scholar). In PCPE and the recently identified PCPE2 (29Xu H. Acott T.S. Wirtz M.K. Genomics. 2000; 66: 264-273Crossref PubMed Scopus (36) Google Scholar) the N-terminal region consists of two CUB domains, whereas the C-terminal domain is homologous to the NTR domain (30Bányai L. Patthy L. Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (147) Google Scholar), which is also found in netrins, complement proteins, and TIMPs. PCP-enhancing activity is a property of the CUB domain region of PCPE (18Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Janisait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 31Kessler E. Adar R. Eur. J. Biochem. 1989; 186: 115-121Crossref PubMed Scopus (83) Google Scholar, 32Hulmes D.J.S. Mould A.P. Kessler E. Matrix Biol. 1997; 16: 41-45Crossref PubMed Scopus (49) Google Scholar). In contrast, the NTR domain, which is released relatively easily from the rest of the molecule by proteolytic attack (17Kessler E. Mould A.P. Hulmes D.J. Biochem. Biophys. Res. Commun. 1990; 173: 81-86Crossref PubMed Scopus (38) Google Scholar), appears to have a moderate TIMP-like inhibitory activity against matrix metalloproteinases (33Mott J.D. Thomas C.L. Rosenbach M.T. Takahara K. Greenspan D.S. Banda M.J. J. Biol. Chem. 2000; 275: 1384-1390Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Thus, different regions of PCPE may show either stimulatory or inhibitory activities to different subfamilies of metzincin metalloproteinases in the extracellular matrix. In addition, PCPE expression is implicated in the control of cell growth (34Masuda M. Igarashi H. Kano M. Yoshikura H. Cell Growth & Differ. 1998; 9: 381-391PubMed Google Scholar, 35Kanaki T. Morisaki N. Bujo H. Takahashi K. Ishii I. Saito Y. Biochem. Biophys. Res. Commun. 2000; 270: 1049-1054Crossref PubMed Scopus (15) Google Scholar), as has also been reported for TIMPs (36Hoegy S.E., Oh, H.R. Corcoran M.L. Stetler-Stevenson W.G. J. Biol. Chem. 2001; 276: 3203-3214Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The mechanism of PCP stimulation by PCPE is unknown. From kinetic studies (1Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (457) Google Scholar, 16Adar R. Kessler E. Goldberg B. Collagen Relat. Res. 1986; 6: 267-277Crossref PubMed Scopus (54) Google Scholar), PCPE increases both the Km andVmax for PCP/BMP-1 cleavage of the C-propeptide region from procollagen I. Furthermore, maximum enhancement is an of PCPE to that enhancement the of the and not the the interactions of PCPE with the procollagen I molecule as well as with the C-propeptide that are released from procollagens I and III by PCP show by surface plasmon resonance that C-propeptide binding to PCPE is on divalent metal binding to the cross-linking that PCPE binds to the C-propeptide trimer in a of Finally, PCPE binding to intact procollagen molecules appears to be to the isolated C-propeptide suggesting binding sites in other regions of the procollagen a by the binding of PCPE to procollagen molecules devoid of the C-propeptide PCPE as well as the C-terminal propeptide trimer from procollagen III were using L. Bernocco S. Font B. Rivkin H. Eichenberger D. Chejanovsky N. Hulmes D.J.S. Kessler E. Eur. J. Biochem. 2001; 268: 2991-2996Crossref PubMed Scopus (34) Google Scholar, S. S. C. Eichenberger D. M. Farjanel J. Hulmes D.J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, K. E.M. H. G. Sieron A.L. A. D.J. Matrix Biol. 1997; 16: PubMed Scopus Google Scholar). Procollagen I and its C-propeptide trimer domain were from the of R. Farjanel J. Eichenberger D. Hulmes D.J.S. Biochem. 2000; 277: PubMed Scopus Google Scholar) or Hulmes D.J.S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar), the of pN-collagen procollagen I was with recombinant BMP-1 (1Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (457) Google Scholar) in the of PCPE and from both and BMP-1 by on a of with and K.E. Y. D.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and was as L. Bernocco S. Font B. Rivkin H. Eichenberger D. Chejanovsky N. Hulmes D.J.S. Kessler E. Eur. J. Biochem. 2001; 268: 2991-2996Crossref PubMed Scopus (34) Google Scholar). The binding of the monoclonal and to sites and of the as M. P. J. W. M. W. M. Matrix Biol. 1998; PubMed Scopus Google Scholar). Binding was using a PCPE was to The surface was by the of a of and PCPE was in PCPE and were to the of The were by the of 1 were a of 5 The of was that the was cells were by the in the presence of were to control binding to the surface as well as from in the of the were from with immobilized ligand to binding Binding were in containing and procollagen and pN-collagen I were against and several and different immobilized PCPE. The surface was with a of kinetic constants were by to the and dissociation to the equilibrium dissociation constants were as the were from the equilibrium resonance as a of D.J. D.J. Biochem. 1996; PubMed Scopus Google Scholar), by to using of resonance as a of the of D.R. D.J. Biochem. 1997; PubMed Scopus Google Scholar). were by to the expression is the maximum binding of the surface and is the using binding was studied by ligand and K. T. S. J. Biochem. PubMed Scopus Google Scholar, T. K. PubMed Scopus Google Scholar) using and of proteins were using a well was with containing and 5 were with a of for with for 5 and to in a with an from A. was as a control M. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and collagen was as a control T. K. PubMed Scopus Google Scholar). These were in two PCPE in was in the with a of the for of was and the was for the of the was by through a with with was for in the with an of in the with 5 was for with a a of from the using a in proteins were for were by blotting, using and the other using monoclonal antibody against M. P. J. W. M. W. M. Matrix Biol. 1998; PubMed Scopus Google Scholar). were in the of the cross-linking were studied using surface plasmon resonance (BIAcore) technology. the surface of the was found to bind both specifically and in a to immobilized PCPE the PCPE molecule was immobilized and is that PCPE was immobilized the NTR domain the two acidic CUB domains and for and to with the is by in which PCPE lacking the NTR domain was immobilized interaction with was not PCPE enhancement of PCP/BMP-1 is known to be a property of the CUB domain region of PCPE (18Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Janisait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 31Kessler E. Adar R. Eur. J. Biochem. 1989; 186: 115-121Crossref PubMed Scopus (83) Google Scholar, 32Hulmes D.J.S. Mould A.P. Kessler E. Matrix Biol. 1997; 16: 41-45Crossref PubMed Scopus (49) Google Scholar), is that in the of the NTR domain with binding sites in the CUB The binding of to immobilized PCPE was but saturation equilibrium of was rapid. These of a kinetic to the and the of and binding not well to the different in the with or a the use of a with a the of the Furthermore, equilibrium was not during the the use of to the apparent equilibrium dissociation constant was not to the equilibrium dissociation were to using in to the equilibrium binding D.J. D.J. Biochem. 1996; PubMed Scopus Google Scholar, D.R. D.J. Biochem. 1997; PubMed Scopus Google Scholar). to the expression and also shown in an apparent of and a of resonance for the interaction of with PCPE. binding of PCPE to also be in the by the PCPE molecule immobilized the with immobilized PCPE were with relatively of immobilized a that kinetic and for the relatively saturation of immobilized binding shown in the kinetics for the binding of to immobilized PCPE were to for the dissociation was of the constant from the dissociation of but for using the 1:1 dissociation In this found to be the the dissociation was was different and for different and or on different of immobilized PCPE and resonance that the interaction was not by and not following binding or binding of to a of the was not with the were from equilibrium as for also shown in an apparent of and of resonance for the interaction of with PCPE. In the show that binding of the procollagen C-propeptide I or to PCPE is of moderate in the range 150–400 nm. to immobilized PCPE the surface was in the presence or of 5 Furthermore, binding was by of the PCPE surface with containing by in In contrast, was of divalent metal cations by against containing and against PCPE, the binding of to immobilized PCPE was The of or the binding These that divalent metal binding to is for binding to immobilized PCPE. To the using the binding of calcium to by the ligand K. T. S. J. Biochem. PubMed Scopus Google Scholar). Procollagen and a distinct for PCPE, which not the T. K. PubMed Scopus Google Scholar), collagen not bind whereas a calcium binding protein as a control M. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), The of binding of to immobilized PCPE was in the presence of and Furthermore, PCPE to a in the or presence of up to not Thus, binding to PCPE not to sites in either To PCPE binding sites the C-propeptide domain, monoclonal directed against different regions of M. P. J. W. M. W. M. Matrix Biol. 1998; PubMed Scopus Google Scholar). monoclonal were for with and the was immobilized PCPE. directed against an the N-terminal of the to bind to immobilized PCPE, but the was with antibody binding was in the of These that the to PCPE, thus an and suggested that the N-terminal region of not in PCPE using directed against other regions of were not To the of molecules of PCPE interactions the two in the presence of a of PCPE were by shown in in the presence of the an of was by the whereas the trimer in the was The was also by the and was the molecular to be by both The apparent molecular of the by cross-linking to the of the apparent molecular of trimer molecule of PCPE the binding stoichiometry to be that immobilized PCPE not with and not suggesting that the C-propeptide be in its to with PCPE. procollagen I molecules were also found to bind specifically to immobilized PCPE in a In to the with isolated C-propeptide the and in the dissociation were slow, suggesting that the the procollagen I molecule and PCPE was Association and dissociation were for using a 1:1 and were and corresponding to an of and of resonance The of not the a was also using the with To another of was a which a suggesting that not to a the In addition, a or in the and a procollagen not the of the and that not with the of the the binding of the intact procollagen molecule to PCPE 1 to be the binding of isolated C-propeptide 150–400 is that might be sites for PCPE binding in the procollagen To this the binding of pN-collagen I procollagen lacking the C-propeptide to immobilized PCPE. binding of pN-collagen to PCPE was and the of the corresponding to with the procollagen I molecule with a dissociation The of the pN-collagen I binding studies a range of These that PCPE binds to sites in the procollagen molecule in to in the C-propeptide that the C-propeptide of procollagens I and III bind binding has been for the C-propeptide trimer of procollagen also known as A. 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Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Similarly, the and and also the protein with the region of N.M. S. S. T. Arlaud G.J. J. 2001; PubMed Scopus Google Scholar, R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The that PCPE might with the region of the procollagen molecule that CUB domains might be well for with collagen are to determine the binding sites for PCPE on the procollagen molecule in the C-propeptide region and and also to the interactions of PCPE with procollagen C-proteinases and R. and T. for A. for the of and C. for with the