Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate
2010; Elsevier BV; Volume: 285; Issue: 44 Linguagem: Inglês
10.1074/jbc.m110.141366
ISSN1083-351X
AutoresTali Weiss, Sylvie Ricard‐Blum, Laura Moschcovich, Eitan Wineman, Shlomit Mesilaty, Efrat Kessler,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoProcollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action. Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action. IntroductionFibrillar procollagen precursors contain N- and C-terminal propeptide extensions at both ends of their pro α chains. These propeptides must be removed for proper collagen fibril assembly to occur (1Ricard-Blum S. Ruggiero F. van der Rest M. Top. Curr. Chem. 2005; 247: 35-84Crossref Scopus (62) Google Scholar). The C-propeptide is removed by procollagen C-proteinases (PCPs), 2The abbreviations used are: PCPprocollagen C-proteinaseBAPTAcalcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidBMP-1bone morphogenetic protein-1CUBmodule found in complement subcomponents C1r/C1s, Uegf, and BMP-1ECMextracellular matrixHPheparinHSheparan sulfateHEKhuman embryonic kidneyNTRnetrin-likePEGpolyethylene glycolPCPEprocollagen C-proteinase enhancerRUresonance unitSPRsurface plasmon resonanceTLCKNα-tosyl-l-lysine chloromethyl ketoneTPCKN-p-tosyl-l-phenylalanine chloromethyl ketone. a number of closely related tolloid family metalloproteinases that play important regulatory roles in developmental processes and extracellular matrix assembly. This functional versatility results from their ability to process a variety of matrix components in addition to fibrillar procollagens, including non-fibrillar procollagens, small leucine-rich proteoglycans, growth factors (e.g. growth differentiation factors 8 and 11), and associated regulatory proteins (chordin, latent TGF-β-binding protein) and lysyl oxidases, enzymes responsible for elastin and collagen cross-linking (reviews in Refs. 2Kessler E. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes, Vol. 1. 2nd Ed. Elsevier, Academic Press2004: 609-617Google Scholar, 3Hopkins D.R. Keles S. Greenspan D.S. Matrix Biol. 2007; 26: 508-523Crossref PubMed Scopus (198) Google Scholar). Procollagen processing by bone morphogenetic protein-1 (BMP-1), the prototype and apparently most active PCP (3Hopkins D.R. Keles S. Greenspan D.S. Matrix Biol. 2007; 26: 508-523Crossref PubMed Scopus (198) Google Scholar, 4Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (450) Google Scholar), can be stimulated by procollagen C-proteinase enhancers-1 and 2 (PCPE-1 and -2), two extracellular matrix glycoproteins lacking proteolytic activity of their own (5Adar R. Kessler E. Goldberg B. Coll. Relat. Res. 1986; 6: 267-277Crossref PubMed Scopus (53) Google Scholar, 6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 7Xu H. Acott T.S. Wirtz M.K. Genomics. 2000; 66: 264-273Crossref PubMed Scopus (35) Google Scholar, 8Steiglitz B.M. Keene D.R. Greenspan D.S. J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Enhancement of BMP-1 activity by PCPE-1 appears to be restricted to fibrillar procollagen precursors because PCPE-1 does not affect BMP-1 activity on other tolloid substrates (9Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. François V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).PCPE-1 is abundant in connective tissues rich in collagen I and in fibrotic tissues where it functions as a positive regulator of collagen deposition (6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 10Kessler E. Mould A.P. Hulmes D.J. Biochem. Biophys. Res. Commun. 1990; 173: 81-86Crossref PubMed Scopus (38) Google Scholar). Increased expression of PCPE-1 in both liver (11Ogata I. Auster A.S. Matsui A. Greenwel P. Geerts A. D'Amico T. Fujiwar K. Kessler E. Rojkind M. Hepatology. 1997; 26: 611-617Crossref PubMed Scopus (36) Google Scholar) and cardiac fibrosis (12Shalitin N. Schlesinger H. Levy M.J. Kessler E. Kessler-Icekson G. J. Cell. Biochem. 2003; 90: 397-407Crossref PubMed Scopus (41) Google Scholar, 13Kessler-Icekson G. Schlesinger H. Freimann S. Kessler E. Int. J. Biochem. Cell Biol. 2006; 38: 358-365Crossref PubMed Scopus (32) Google Scholar) points at PCPE-1 as a potential therapeutic target in fibrosis. PCPE-1 expression is also increased in cultured smooth muscle cells and in intimal thickening induced by arterial injury. It may thus play a role in the proliferation of smooth muscle cells and extracellular matrix production during atheroma formation (14Kanaki T. Morisaki N. Bujo H. Takahashi K. Ishii I. Saito Y. Biochem. Biophys. Res. Commun. 2000; 270: 1049-1054Crossref PubMed Scopus (15) Google Scholar). The other procollagen C-proteinase enhancer, PCPE-2, has a much more limited distribution of expression than PCPE-1 (8Steiglitz B.M. Keene D.R. Greenspan D.S. J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar).PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptide and are required for enhancing activity (6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 15Kessler E. Adar R. Eur. J. Biochem. 1989; 186: 115-121Crossref PubMed Scopus (83) Google Scholar) followed by a C-terminal NTR (netrin-like) domain (16Bányai L. Patthy L. Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (146) Google Scholar) that binds to heparin (17Moschcovich L. Bernocco S. Font B. Rivkin H. Eichenberger D. Chejanovsky N. Hulmes D.J. Kessler E. Eur. J. Biochem. 2001; 268: 2991-2996Crossref PubMed Scopus (34) Google Scholar) and as was recently demonstrated (18Bekhouche M. Kronenberg D. Vadon-Le Goff S. Bijakowski C. Lim N.H. Font B. Kessler E. Colige A. Nagase H. Murphy G. Hulmes D.J. Moali C. J. Biol. Chem. 2010; 285: 15950-15959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), also interacts with BMP-1. The NTR domain of PCPE-1 also binds to β2-microglobulin, which may help initiate β2-microglobulin amyloid fibril formation in connective tissues (19Morimoto H. Wada J. Font B. Mott J.D. Hulmes D.J. Ookoshi T. Naiki H. Yasuhara A. Nakatsuka A. Fukuoka K. Takatori Y. Ichikawa H. Akagi S. Nakao K. Makino H. Matrix. Biol. 2008; 27: 211-219Crossref PubMed Scopus (10) Google Scholar). The CUB and NTR domains in PCPE-1 are separated by two linkers: a short linker (9 amino acids in human PCPE-1) between the two CUB domains and a long linker (44 amino acids in human PCPE-1) that is sensitive to proteolysis between the second CUB and the NTR domain (6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 19Morimoto H. Wada J. Font B. Mott J.D. Hulmes D.J. Ookoshi T. Naiki H. Yasuhara A. Nakatsuka A. Fukuoka K. Takatori Y. Ichikawa H. Akagi S. Nakao K. Makino H. Matrix. Biol. 2008; 27: 211-219Crossref PubMed Scopus (10) Google Scholar, 20Kronenberg D. Vadon-Le Goff S. Bourhis J.M. Font B. Eichenberger D. Hulmes D.J. Moali C. J. Biol. Chem. 2009; 284: 33437-33446Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The NTR domain has homology with the N-terminal domain of tissue inhibitors of metalloproteinases and with the C-terminal domain of netrins, complement components C3, C4, and C5, and secreted frizzled-related proteins (16Bányai L. Patthy L. Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (146) Google Scholar). The NTR fragment of PCPE-1 has been reported to act as a weak inhibitor of matrix metalloproteinases (21Mott 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). However, no inhibitory activity was detected by other investigators against a range of metalloproteinases, including MMPs-1, -2, -3, and -9, BMP-1 and different ADAMTS proteinases (18Bekhouche M. Kronenberg D. Vadon-Le Goff S. Bijakowski C. Lim N.H. Font B. Kessler E. Colige A. Nagase H. Murphy G. Hulmes D.J. Moali C. J. Biol. Chem. 2010; 285: 15950-15959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22Liepinsh E. Banyai L. Pintacuda G. Trexler M. Patthy L. Otting G. J. Biol. Chem. 2003; 278: 25982-25989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The PCPE-1 molecule has been shown to be a rod-like molecule, with a length of ∼15 nm (23Bernocco S. Steiglitz B.M. Svergun D.I. Petoukhov M.V. Ruggiero F. Ricard-Blum S. Ebel C. Geourjon C. Deleage G. Font B. Eichenberger D. Greenspan D.S. Hulmes D.J. J. Biol. Chem. 2003; 278: 7199-7205Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The short linker between the CUB domains of PCPE-1 provides a flexible tether linking two binding sites within the individual CUB domains that act cooperatively in the binding of PCPE-1 to the procollagen substrate (20Kronenberg D. Vadon-Le Goff S. Bourhis J.M. Font B. Eichenberger D. Hulmes D.J. Moali C. J. Biol. Chem. 2009; 284: 33437-33446Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).PCPE-1 binding to heparin suggests that PCPE-1 may also interact with heparan sulfate proteoglycans (HSPGs). HSPGs carry out distinct biological functions, from maintenance of basement membrane homeostasis to modulation of growth factor activity and angiogenesis (24Iozzo R.V. Zoeller J.J. Nyström A. Mol. Cells. 2009; 27: 503-513Crossref PubMed Scopus (172) Google Scholar). They are abundant at cell surfaces and within the extracellular matrix and heparan sulfate (HS) is a complex and highly active biopolymer, which binds to a wide variety of growth factors, morphogens, chemokines, and extracellular matrix proteins (25Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1524) Google Scholar, 26Whitelock J.M. Iozzo R.V. Chem. Rev. 2005; 105: 2745-2764Crossref PubMed Scopus (342) Google Scholar, 27Gallagher J.T. Biochem. Soc. Trans. 2006; 4: 438-441Crossref Scopus (56) Google Scholar). HSPGs act as co-receptors for several tyrosine kinase receptors and have been shown to be important in controlling the biological activities of the bound factor (26Whitelock J.M. Iozzo R.V. Chem. Rev. 2005; 105: 2745-2764Crossref PubMed Scopus (342) Google Scholar). In this study, using surface plasmon resonance (SPR) binding assays, we characterized at the molecular level the binding of PCPE-1 to heparin and heparan sulfate, which is likely to be the physiological partner of PCPE-1. This included determination of the kinetics and affinity constants and demonstration of the role of divalent cations in this interaction. We have also investigated the role of this interaction in mediating cell adhesion to PCPE-1 and in anchoring PCPE-1 to the cell membrane, revealing that cell-bound PCPE-1 can augment procollagen processing by BMP-1 at the cell surface. The binding of PCPE-1 to heparan sulfate might play a major role in the assembly of the molecular machines comprised of bone morphogenetic protein-1, PCPE-1, and procollagens, which ensure the processing of procollagen molecules at the cell surface.DISCUSSIONIn the present report, we found that PCPE-1 binds to heparin/heparan sulfate with an affinity in the high nanomolar range (KD = 63.2 and 122 nm for heparin and heparan sulfate, respectively). This is lower than the affinity we have calculated for collagen I (KD = 3.4 and 3.5 nm for heparin and heparan sulfate, respectively) and for collagen V (KD = 5.62 and 2.0 nm for heparin and heparan sulfate, respectively (31Ricard-Blum S. Beraud M. Raynal N. Farndale R.W. Ruggiero F. J. Biol. Chem. 2006; 281: 25195-25204Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), but much higher than the affinity of endostatin, a soluble fragment of collagen XVIII inhibiting angiogenesis (KD ∼ 2 μm for both glycosaminoglycans) (30Ricard-Blum S. Féraud O. Lortat-Jacob H. Rencurosi A. Fukai N. Dkhissi F. Vittet D. Imberty A. Olsen B.R. van der Rest M. J. Biol. Chem. 2004; 279: 2927-2936Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) or of the α5β1 integrin (1.14 μm and 2.04 μm for heparin and heparan sulfate, respectively (33Faye C. Moreau C. Chautard E. Jetne R. Fukai N. Ruggiero F. Humphries M.J. Olsen B.R. Ricard-Blum S. J. Biol. Chem. 2009; 284: 22029-22040Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).The strength of PCPE-1 binding to heparin has been previously evaluated by affinity chromatography on heparin-Sepharose. PCPE-1 eluted from the column between 0.4 and 0.5 m NaCl (17Moschcovich L. Bernocco S. Font B. Rivkin H. Eichenberger D. Chejanovsky N. Hulmes D.J. Kessler E. Eur. J. Biochem. 2001; 268: 2991-2996Crossref PubMed Scopus (34) Google Scholar) or between 0.3 and 0.4 m NaCl (8Steiglitz B.M. Keene D.R. Greenspan D.S. J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). However, heparin-Sepharose may act as a cation exchanger because of the highly negative charge of heparin. The amount of salt required for elution of a protein from the affinity column is a quantitative measure of the ionic component of the binding, but fails to measure the hydrophobic and hydrogen bonding contributions to binding (25Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1524) Google Scholar), whereas the SPR assays take into account all of the possible bonding mechanisms. Deviations between the relative ionic strength elution of proteins from heparin-Sepharose and direct affinity measurements have been observed (34Powell A.K. Yates E.A. Fernig D.G. Turnbull J.E. Glycobiology. 2004; 14: 17R-30RCrossref PubMed Scopus (223) Google Scholar), suggesting that differential ionic strength elution of proteins may not reflect their true relative affinities. The presence of two possible binding sites identified by fitting SPR kinetic data to the heterogeneous ligand model is in agreement with the fact that significant quantities of PCPE-1 eluted at NaCl concentrations lower than 0.3 m (8Steiglitz B.M. Keene D.R. Greenspan D.S. J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar).The NTR domain does not contain any of the three linear consensus sequences proposed for heparin binding (BBXBX, BBXXBX, and XBBBXXBBBXXBBX, where B is a basic and X a hydropathic, neutral and hydrophobic, amino acid residue (25Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1524) Google Scholar). The precise binding site of heparin/heparan sulfate inside the NTR domain remains to be mapped. The three-dimensional structure of the NTR domain has been elucidated by NMR (22Liepinsh E. Banyai L. Pintacuda G. Trexler M. Patthy L. Otting G. J. Biol. Chem. 2003; 278: 25982-25989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). It includes a patch with uncharged residues in the center and positive charges at the rim, including Lys32, Arg36, Arg120, Arg134, Arg147 (numbering of amino acid positions is as used in the NTR construct studied by Liepinsh et al. (22Liepinsh E. Banyai L. Pintacuda G. Trexler M. Patthy L. Otting G. J. Biol. Chem. 2003; 278: 25982-25989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), in which position 30 corresponds to Cys318 in full-length PCPE-1). Electrostatic surface representation of the NTR domain shows that several basic amino acids of the NTR domain are clustered on one face of the molecule, forming two other basic clusters that may bind heparin/heparan sulfate. The first one comprises Arg35, Arg36, and Arg147 and the second one Lys105, Lys106, Lys56, Lys99, and Arg60. These basic clusters might also bind heparan sulfate.The binding of PCPE-1/NTR to heparin/heparan sulfate requires divalent cations, and most likely calcium, as reported previously for other extracellular proteins such as endostatin (30Ricard-Blum S. Féraud O. Lortat-Jacob H. Rencurosi A. Fukai N. Dkhissi F. Vittet D. Imberty A. Olsen B.R. van der Rest M. J. Biol. Chem. 2004; 279: 2927-2936Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), a fragment of collagen V (31Ricard-Blum S. Beraud M. Raynal N. Farndale R.W. Ruggiero F. J. Biol. Chem. 2006; 281: 25195-25204Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), the α5β1 integrin (33Faye C. Moreau C. Chautard E. Jetne R. Fukai N. Ruggiero F. Humphries M.J. Olsen B.R. Ricard-Blum S. J. Biol. Chem. 2009; 284: 22029-22040Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and certain annexins (35Shao C. Zhang F. Kemp M.M. Linhardt R.J. Waisman D.M. Head J.F. Seaton B.A. J. Biol. Chem. 2006; 281: 31689-31695Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The role of calcium ions in the binding of the NTR domain of PCPE-1 to heparin is unclear. Based on its three-dimensional structure (22Liepinsh E. Banyai L. Pintacuda G. Trexler M. Patthy L. Otting G. J. Biol. Chem. 2003; 278: 25982-25989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), this protein domain does not contain calcium ions. It is possible however that, as was shown for annexin A2 (35Shao C. Zhang F. Kemp M.M. Linhardt R.J. Waisman D.M. Head J.F. Seaton B.A. J. Biol. Chem. 2006; 281: 31689-31695Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), some heparin binding sites may rely on calcium binding and not on arrays of basic residues. In this instance, main-chain and side chain nitrogen atoms and two calcium ions play an important role in the binding. Of special interest in this regard is the finding that a new calcium-binding site is formed in annexin A2 upon initial binding to heparin, in which two sugar residues within a small heparin derivative used as a model for heparin, provided oxygen ligands for this calcium ion. It is conceivable that a similar mechanism operates in the binding of the NTR domain to heparin, where initial binding to clusters of basic residues in the NTR domain may trigger the formation of a functionally important calcium binding site(s) for heparin.Our data on the interaction of PCPE-1 with cells, including inhibition by heparin, a NTR-specific monoclonal antibody and the NTR fragment itself, strongly suggest that the NTR domain of PCPE-1 mediates the binding of PCPE-1 to cell surface HSPGs. This binding was observed with both HEK-293 cells that do not express collagen type I but express HSPGs and 3T3 mouse fibroblasts that produce and process relatively large amounts of procollagen I and also express HSPGs. The binding patterns and patterns of inhibition of cell attachment to PCPE-1 by heparin seen with these cell types were however different (Fig. 2). Higher cell binding and a lower degree of inhibition by heparin were seen with the fibroblasts as compared with the HEK-293 cells. This suggests that the interaction of PCPE-1 with fibroblasts may involve another receptor in addition to HSPGs, while the interaction of PCPE-1 with the HEK-293 cells was mainly mediated by HSPGs. Nonetheless, the interaction of the NTR domain with HSPGs seems to be cardinal for PCPE-1 binding to cells, as prolonged incubation of 3T3 mouse fibroblasts with heparin alone was sufficient to achieve complete displacement of PCPE-1 Flag from the cell surface. The finding that cell-bound PCPE-1 can be fully displaced by heparin (which does not penetrate intact cells) indicates that cell-bound PCPE-1 is not internalized, and suggests that PCPE-1 may function on the cell surface, accelerating procollagen processing at this location.The biological significance of PCPE-1 interaction with heparan sulfate is not fully elucidated. To gain an insight on this question, we examined whether cell-bound PCPE-1 can stimulate PCP activity pericellularly. We used radioactively labeled procollagen and Flag-tagged PCPE-1 to facilitate their localization, and both were localized to the cell surface/cell layer during enzymatic processing by BMP-1. The results of the processing assays, which were performed with and without addition of PCPE-Flag, document for the first time cell-associated enhancing activity of PCPE-1. This suggests the cell surface as a physiological site of PCPE-1 enhancing activity, and is in agreement with previous reports indicating that in tendon, fibrillogenesis is initiated in a series of extracellular compartments defined by the fibroblasts and close to their surface (36Zhang G. Young B.B. Ezura Y. Favata M. Soslowsky L.J. Chakravarti S. Birk D.E. J Musculoskelet Neuronal Interact. 2005; 5: 5-21PubMed Google Scholar).The highest degree of enhancement (3.5-fold) was however lower than that obtained in the standard assay of PCP activity (∼10-fold) that is performed in solution at optimal (1:1) molar ratios of PCPE-1 and procollagen (29Ricard-Blum S. Bernocco S. Font B. Moali C. Eichenberger D. Farjanel J. Burchardt E.R. van der Rest M. Kessler E. Hulmes D.J. J. Biol. Chem. 2002; 277: 33864-33869Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This could be due to the fact that in the cell-based assay, we only measured an increment in enhancing activity, above that provided by endogenous PCPE-1, which also binds to the cell membrane and can be displaced from it by heparin (37Wineman E. Weiss T. Farzam N. Savion N. Smorodinsky I.N. Kessler E. Federation of the European Connective Tissue Societies, Marseille, France, July 9–13. 2008; (2008, Federation of the European Connective Tissue Societies)Google Scholar).The interaction of the NTR domain with heparan sulfate seems to play a role in anchoring PCPE-1 to the cell membrane where it can take part in the processing of a number of extracellular matrix proteins by BMP-1, including fibrillar procollagen precursors. Assembly of functional multiprotein complexes has been reported on the heparan sulfate polymer chain (27Gallagher J.T. Biochem. Soc. Trans. 2006; 4: 438-441Crossref Scopus (56) Google Scholar). Heparan sulfate may act as a guiding scaffold to bring BMP-1, PCPE-1, and procollagen molecules together in areas of collagen fibrillogenesis, playing a pivotal role in the building of the molecular machines involved in procollagen processing. Indeed, heparan sulfate chains bind with moderate affinity to PCPE-1, which is required to enhance BMP-1 activity, to procollagen I with high affinity in the nanomolar range (31Ricard-Blum S. Beraud M. Raynal N. Farndale R.W. Ruggiero F. J. Biol. Chem. 2006; 281: 25195-25204Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and transiently to BMP-1 with low affinity in the micromolar range (0.5–2 μm) (18Bekhouche M. Kronenberg D. Vadon-Le Goff S. Bijakowski C. Lim N.H. Font B. Kessler E. Colige A. Nagase H. Murphy G. Hulmes D.J. Moali C. J. Biol. Chem. 2010; 285: 15950-15959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Once the complexes are anchored at the cell surface via several proteins, rearrangements could occur, and some PCPE-1 molecules could bind to BMP-1 via their NTR domain (18Bekhouche M. Kronenberg D. Vadon-Le Goff S. Bijakowski C. Lim N.H. Font B. Kessler E. Colige A. Nagase H. Murphy G. Hulmes D.J. Moali C. J. Biol. Chem. 2010; 285: 15950-15959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The functional importance of complexes assembled by heparan sulfate is highlighted by the recent demonstration that heparin/heparan sulfate can increase the enhancing activity of PCPE-1 further (about 2-fold) in the processing of (mini) procollagen III by BMP-1, an effect described as "superstimulation" of PCPE-1 (18Bekhouche M. Kronenberg D. Vadon-Le Goff S. Bijakowski C. Lim N.H. Font B. Kessler E. Colige A. Nagase H. Murphy G. Hulmes D.J. Moali C. J. Biol. Chem. 2010; 285: 15950-15959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Similar complexes can be formed during the processing of pro-α1(V) N-propeptides and pro-α2(V) C-propeptides by BMP-1-like enzymes (38Unsöld C. Pappano W.N. Imamura Y. Steiglitz B.M. Greenspan D.S. J. Biol. Chem. 2002; 277: 5596-5602Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Finally, PCPE-2 may also function as a component of such a multimolecular complex because, similarly to PCPE-1, it binds to heparin and there is evidence indicating that much of the PCPE-2 antigen is found pericellularly (8Steiglitz B.M. Keene D.R. Greenspan D.S. J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). IntroductionFibrillar procollagen precursors contain N- and C-terminal propeptide extensions at both ends of their pro α chains. These propeptides must be removed for proper collagen fibril assembly to occur (1Ricard-Blum S. Ruggiero F. van der Rest M. Top. Curr. Chem. 2005; 247: 35-84Crossref Scopus (62) Google Scholar). The C-propeptide is removed by procollagen C-proteinases (PCPs), 2The abbreviations used are: PCPprocollagen C-proteinaseBAPTAcalcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidBMP-1bone morphogenetic protein-1CUBmodule found in complement subcomponents C1r/C1s, Uegf, and BMP-1ECMextracellular matrixHPheparinHSheparan sulfateHEKhuman embryonic kidneyNTRnetrin-likePEGpolyethylene glycolPCPEprocollagen C-proteinase enhancerRUresonance unitSPRsurface plasmon resonanceTLCKNα-tosyl-l-lysine chloromethyl ketoneTPCKN-p-tosyl-l-phenylalanine chloromethyl ketone. a number of closely related tolloid family metalloproteinases that play important regulatory roles in developmental processes and extracellular matrix assembly. This functional versatility results from their ability to process a variety of matrix components in addition to fibrillar procollagens, including non-fibrillar procollagens, small leucine-rich proteoglycans, growth factors (e.g. growth differentiation factors 8 and 11), and associated regulatory proteins (chordin, latent TGF-β-binding protein) and lysyl oxidases, enzymes responsible for elastin and collagen cross-linking (reviews in Refs. 2Kessler E. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes, Vol. 1. 2nd Ed. Elsevier, Academic Press2004: 609-617Google Scholar, 3Hopkins D.R. Keles S. Greenspan D.S. Matrix Biol. 2007; 26: 508-523Crossref PubMed Scopus (198) Google Scholar). Procollagen processing by bone morphogenetic protein-1 (BMP-1), the prototype and apparently most active PCP (3Hopkins D.R. Keles S. Greenspan D.S. Matrix Biol. 2007; 26: 508-523Crossref PubMed Scopus (198) Google Scholar, 4Kessler E. Takahara K. Biniaminov L. Brusel M. Greenspan D.S. Science. 1996; 271: 360-362Crossref PubMed Scopus (450) Google Scholar), can be stimulated by procollagen C-proteinase enhancers-1 and 2 (PCPE-1 and -2), two extracellular matrix glycoproteins lacking proteolytic activity of their own (5Adar R. Kessler E. Goldberg B. Coll. Relat. Res. 1986; 6: 267-277Crossref PubMed Scopus (53) Google Scholar, 6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 7Xu H. Acott T.S. Wirtz M.K. Genomics. 2000; 66: 264-273Crossref PubMed Scopus (35) Google Scholar, 8Steiglitz B.M. Keene D.R. Greenspan D.S. J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Enhancement of BMP-1 activity by PCPE-1 appears to be restricted to fibrillar procollagen precursors because PCPE-1 does not affect BMP-1 activity on other tolloid substrates (9Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. François V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).PCPE-1 is abundant in connective tissues rich in collagen I and in fibrotic tissues where it functions as a positive regulator of collagen deposition (6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 10Kessler E. Mould A.P. Hulmes D.J. Biochem. Biophys. Res. Commun. 1990; 173: 81-86Crossref PubMed Scopus (38) Google Scholar). Increased expression of PCPE-1 in both liver (11Ogata I. Auster A.S. Matsui A. Greenwel P. Geerts A. D'Amico T. Fujiwar K. Kessler E. Rojkind M. Hepatology. 1997; 26: 611-617Crossref PubMed Scopus (36) Google Scholar) and cardiac fibrosis (12Shalitin N. Schlesinger H. Levy M.J. Kessler E. Kessler-Icekson G. J. Cell. Biochem. 2003; 90: 397-407Crossref PubMed Scopus (41) Google Scholar, 13Kessler-Icekson G. Schlesinger H. Freimann S. Kessler E. Int. J. Biochem. Cell Biol. 2006; 38: 358-365Crossref PubMed Scopus (32) Google Scholar) points at PCPE-1 as a potential therapeutic target in fibrosis. PCPE-1 expression is also increased in cultured smooth muscle cells and in intimal thickening induced by arterial injury. It may thus play a role in the proliferation of smooth muscle cells and extracellular matrix production during atheroma formation (14Kanaki T. Morisaki N. Bujo H. Takahashi K. Ishii I. Saito Y. Biochem. Biophys. Res. Commun. 2000; 270: 1049-1054Crossref PubMed Scopus (15) Google Scholar). The other procollagen C-proteinase enhancer, PCPE-2, has a much more limited distribution of expression than PCPE-1 (8Steiglitz B.M. Keene D.R. Greenspan D.S. J. Biol. Chem. 2002; 277: 49820-49830Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar).PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptide and are required for enhancing activity (6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 15Kessler E. Adar R. Eur. J. Biochem. 1989; 186: 115-121Crossref PubMed Scopus (83) Google Scholar) followed by a C-terminal NTR (netrin-like) domain (16Bányai L. Patthy L. Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (146) Google Scholar) that binds to heparin (17Moschcovich L. Bernocco S. Font B. Rivkin H. Eichenberger D. Chejanovsky N. Hulmes D.J. Kessler E. Eur. J. Biochem. 2001; 268: 2991-2996Crossref PubMed Scopus (34) Google Scholar) and as was recently demonstrated (18Bekhouche M. Kronenberg D. Vadon-Le Goff S. Bijakowski C. Lim N.H. Font B. Kessler E. Colige A. Nagase H. Murphy G. Hulmes D.J. Moali C. J. Biol. Chem. 2010; 285: 15950-15959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), also interacts with BMP-1. The NTR domain of PCPE-1 also binds to β2-microglobulin, which may help initiate β2-microglobulin amyloid fibril formation in connective tissues (19Morimoto H. Wada J. Font B. Mott J.D. Hulmes D.J. Ookoshi T. Naiki H. Yasuhara A. Nakatsuka A. Fukuoka K. Takatori Y. Ichikawa H. Akagi S. Nakao K. Makino H. Matrix. Biol. 2008; 27: 211-219Crossref PubMed Scopus (10) Google Scholar). The CUB and NTR domains in PCPE-1 are separated by two linkers: a short linker (9 amino acids in human PCPE-1) between the two CUB domains and a long linker (44 amino acids in human PCPE-1) that is sensitive to proteolysis between the second CUB and the NTR domain (6Takahara K. Kessler E. Biniaminov L. Brusel M. Eddy R.L. Jani-Sait S. Shows T.B. Greenspan D.S. J. Biol. Chem. 1994; 269: 26280-26285Abstract Full Text PDF PubMed Google Scholar, 19Morimoto H. Wada J. Font B. Mott J.D. Hulmes D.J. Ookoshi T. Naiki H. Yasuhara A. Nakatsuka A. Fukuoka K. Takatori Y. Ichikawa H. Akagi S. Nakao K. Makino H. Matrix. Biol. 2008; 27: 211-219Crossref PubMed Scopus (10) Google Scholar, 20Kronenberg D. Vadon-Le Goff S. Bourhis J.M. Font B. Eichenberger D. Hulmes D.J. Moali C. J. Biol. Chem. 2009; 284: 33437-33446Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The NTR domain has homology with the N-terminal domain of tissue inhibitors of metalloproteinases and with the C-terminal domain of netrins, complement components C3, C4, and C5, and secreted frizzled-related proteins (16Bányai L. Patthy L. Protein Sci. 1999; 8: 1636-1642Crossref PubMed Scopus (146) Google Scholar). The NTR fragment of PCPE-1 has been reported to act as a weak inhibitor of matrix metalloproteinases (21Mott 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). However, no inhibitory activity was detected by other investigators against a range of metalloproteinases, including MMPs-1, -2, -3, and -9, BMP-1 and different ADAMTS proteinases (18Bekhouche M. Kronenberg D. Vadon-Le Goff S. Bijakowski C. Lim N.H. Font B. Kessler E. Colige A. Nagase H. Murphy G. Hulmes D.J. Moali C. J. Biol. Chem. 2010; 285: 15950-15959Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22Liepinsh E. Banyai L. Pintacuda G. Trexler M. Patthy L. Otting G. J. Biol. Chem. 2003; 278: 25982-25989Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The PCPE-1 molecule has been shown to be a rod-like molecule, with a length of ∼15 nm (23Bernocco S. Steiglitz B.M. Svergun D.I. Petoukhov M.V. Ruggiero F. Ricard-Blum S. Ebel C. Geourjon C. Deleage G. Font B. Eichenberger D. Greenspan D.S. Hulmes D.J. J. Biol. Chem. 2003; 278: 7199-7205Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The short linker between the CUB domains of PCPE-1 provides a flexible tether linking two binding sites within the individual CUB domains that act cooperatively in the binding of PCPE-1 to the procollagen substrate (20Kronenberg D. Vadon-Le Goff S. Bourhis J.M. Font B. Eichenberger D. Hulmes D.J. Moali C. J. Biol. Chem. 2009; 284: 33437-33446Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).PCPE-1 binding to heparin suggests that PCPE-1 may also interact with heparan sulfate proteoglycans (HSPGs). HSPGs carry out distinct biological functions, from maintenance of basement membrane homeostasis to modulation of growth factor activity and angiogenesis (24Iozzo R.V. Zoeller J.J. Nyström A. Mol. Cells. 2009; 27: 503-513Crossref PubMed Scopus (172) Google Scholar). They are abundant at cell surfaces and within the extracellular matrix and heparan sulfate (HS) is a complex and highly active biopolymer, which binds to a wide variety of growth factors, morphogens, chemokines, and extracellular matrix proteins (25Capila I. Linhardt R.J. Angew. Chem. Int. Ed. Engl. 2002; 41: 391-412Crossref PubMed Scopus (1524) Google Scholar, 26Whitelock J.M. Iozzo R.V. Chem. Rev. 2005; 105: 2745-2764Crossref PubMed Scopus (342) Google Scholar, 27Gallagher J.T. Biochem. Soc. Trans. 2006; 4: 438-441Crossref Scopus (56) Google Scholar). HSPGs act as co-receptors for several tyrosine kinase receptors and have been shown to be important in controlling the biological activities of the bound factor (26Whitelock J.M. Iozzo R.V. Chem. Rev. 2005; 105: 2745-2764Crossref PubMed Scopus (342) Google Scholar). In this study, using surface plasmon resonance (SPR) binding assays, we characterized at the molecular level the binding of PCPE-1 to heparin and heparan sulfate, which is likely to be the physiological partner of PCPE-1. This included determination of the kinetics and affinity constants and demonstration of the role of divalent cations in this interaction. We have also investigated the role of this interaction in mediating cell adhesion to PCPE-1 and in anchoring PCPE-1 to the cell membrane, revealing that cell-bound PCPE-1 can augment procollagen processing by BMP-1 at the cell surface. The binding of PCPE-1 to heparan sulfate might play a major role in the assembly of the molecular machines comprised of bone morphogenetic protein-1, PCPE-1, and procollagens, which ensure the processing of procollagen molecules at the cell surface.
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