Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold
2007; Elsevier BV; Volume: 282; Issue: 23 Linguagem: Inglês
10.1074/jbc.m701610200
ISSN1083-351X
AutoresGuillaume Blanc, B. Font, D. Eichenberger, Christophe Moreau, Sylvie Ricard‐Blum, David Hulmes, Catherine Moali,
Tópico(s)Peptidase Inhibition and Analysis
ResumoProcollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies. Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies. CUB 3The abbreviations used are: CUB, module found in complement subcomponents C1r/C1s, Uegf, and BMP-1; BMP-1, bone morphogenetic protein-1; mTld, mammalian tolloid; mTLL-1, mammalian tolloid-like 1; mTLL-2, mammalian tolloid-like 2; PCP, procollagen C-proteinase; PCPE, PCP enhancer. 3The abbreviations used are: CUB, module found in complement subcomponents C1r/C1s, Uegf, and BMP-1; BMP-1, bone morphogenetic protein-1; mTld, mammalian tolloid; mTLL-1, mammalian tolloid-like 1; mTLL-2, mammalian tolloid-like 2; PCP, procollagen C-proteinase; PCPE, PCP enhancer. domains are widely occurring structural motifs, found almost exclusively in extracellular and plasma membrane-associated proteins. These proteins are involved in a wide range of biological functions, including complement activation (1Gaboriaud C. Thielens N.M. Gregory L.A. Rossi V. Fontecilla-Camps J.C. Arlaud G.J. Trends Immunol. 2004; 25: 368-373Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 2Sorensen R. 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Pediatr. Nephrol. 2004; 19: 714-721Crossref PubMed Scopus (123) Google Scholar), and tumor suppression (15Hooi C.F. Blancher C. Qiu W. Revet I.M. Williams L.H. Ciavarella M.L. Anderson R.L. Thompson E.W. Connor A. Phillips W.A. Campbell I.G. Oncogene. 2006; 25: 3924-3933Crossref PubMed Scopus (18) Google Scholar, 16Kang W. Reid K.B. FEBS Lett. 2003; 540: 21-25Crossref PubMed Scopus (75) Google Scholar). Many CUB domain-containing proteins are proteases (1Gaboriaud C. Thielens N.M. Gregory L.A. Rossi V. Fontecilla-Camps J.C. Arlaud G.J. Trends Immunol. 2004; 25: 368-373Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 2Sorensen R. Thiel S. Jensenius J.C. Springer Semin. Immunopathol. 2005; 27: 299-319Crossref PubMed Scopus (108) Google Scholar, 5Greenspan D.S. Top. Curr. Chem. 2005; 247: 149-183Crossref Scopus (54) Google Scholar, 10Tao Z. Peng Y. Nolasco L. Cal S. Lopez-Otin C. Li R. Moake J.L. Lopez J.A. Dong J.F. Blood. 2005; 106: 4139-4145Crossref PubMed Scopus (86) Google Scholar, 17Duke-Cohan J.S. Tang W. Schlossman S.F. Adv. Exp. Med. Biol. 2000; 477: 173-185Crossref PubMed Google Scholar, 18Ge W. Hu H. Ding K. Sun L. Zheng S. J. Biol. Chem. 2006; 281: 7406-7412Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 19Wu Q. Curr. Top. Dev. Biol. 2003; 54: 167-206Crossref PubMed Google Scholar). Although the roles of the CUB domains are largely unexplored, a number of them have been shown to be involved in oligomerization and/or recognition of substrates and binding partners.The protein families from which the CUB domain derives its name are the complement serine proteases C1r, C1s, MASP-1, MASP-2, and MASP-3 and the bone morphogenetic protein-1/tolloid metalloproteases BMP-1, mTLD, mTLL-1, and mTLL-2 (or their counterparts xolloids, tolloids, and SpAN/BP10 in Xenopus, Drosophila, and sea urchin, respectively). Each consists of a catalytic domain (either N-terminal in tolloids or C-terminal in complement proteases) together with several CUB domains interspersed by calcium-binding EGF domains. In the case of the complement proteases, the CUB domains mediate dimerization and binding to the collagen-like regions of C1q (for C1r/C1s) and MBL or L- and H-ficolin (for MASP-2). The three-dimensional structures of the CUB1-EGF fragment of C1s, its homologue in MASP-2 (which also occurs as the alternatively spliced product MAp19) as well as the CUB1-EGF-CUB2 fragment of MASP-2 have been determined (20Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 21Gregory L.A. Thielens N.M. Matsushita M. Sorensen R. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2004; 279: 29391-29397Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weis W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar). These studies have revealed the presence of a calcium binding site in the CUB1 domains of both C1s and MAp19, within the loops distal to the EGF domain. In the case of MAp19, site-directed mutagenesis has shown the involvement of this region in binding to MBL and L-ficolin (21Gregory L.A. Thielens N.M. Matsushita M. Sorensen R. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2004; 279: 29391-29397Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).The BMP-1/tolloid-related metalloproteinases (BMP-1, mTLD, mTLL-1, and mTLL-2 in mammals) are members of the astacin family (M12A) of the metzincin subclan MA(M) (23Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (351) Google Scholar, 24Ge G. Greenspan D.S. Birth Defects Res. C. Embryo. Today. 2006; 78: 47-68Crossref PubMed Scopus (126) Google Scholar). Following the pro-region, which is removed by a furin-like proteinase, each variant consists of an astacin domain followed by a C-terminal region consisting of variable numbers of CUB and calcium-binding EGF domains. In the case of BMP-1, this C-terminal region has the domain organization CUB1-CUB2-EGF1-CUB3, whereas in the longer mTLD splice variant it is CUB1-CUB2-EGF1-CUB3-EGF2-CUB4-CUB5. Variants mTLL-1 and mTLL-2 share the same domain structure as mTLD but are products of separate genes. These proteinases cleave a large number of substrates, including the fibrillar procollagens I, II, III, V, and XI, the non-fibrillar procollagen VII, prolysyl oxidases, the laminin 5 γ2 chain, dentin matrix protein-1, precursor forms of the small leucine rich proteoglycans biglycan and osteoglycin, the heparan sulfate proteoglycan perlecan, growth factors myostatin and GDF11, the growth factor antagonist chordin (24Ge G. Greenspan D.S. Birth Defects Res. C. Embryo. Today. 2006; 78: 47-68Crossref PubMed Scopus (126) Google Scholar), and latent transforming growth factor-β-binding protein (LTBP), which controls transforming growth factor-β activity (25Ge G. Greenspan D.S. J. Cell Biol. 2006; 175: 111-120Crossref PubMed Scopus (203) Google Scholar).There is increasing evidence that tolloid proteinase activity is subject to regulation by a variety of extracellular proteins. During developmental patterning, for example, it has recently been shown that the frizzled-related protein sizzled is an endogenous inhibitor of BMP-1 (3Lee H.X. Ambrosio A.L. Reversade B. De Robertis E.M. Cell. 2006; 124: 147-159Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 26Muraoka O. Shimizu T. Yabe T. Nojima H. Bae Y.K. Hashimoto H. Hibi M. Nat. Cell Biol. 2006; 8: 329-338Crossref PubMed Scopus (89) Google Scholar), whereas complex formation involving the protein-twisted gastrulation stimulates the activity of BMP-1 on chordin and exposes a new cleavage site (27Scott I.C. Blitz I.L. Pappano W.N. Maas S.A. Cho K.W. Greenspan D.S. Nature. 2001; 410: 475-478Crossref PubMed Scopus (158) Google Scholar). Procollagen C-proteinase enhancers (PCPE-1 and -2) also stimulate the activities of tolloid proteinases, in a substrate-specific manner (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). PCPE-1 has no effect on cleavage of several BMP-1 substrates (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), including chordin (29Petropoulou V. Garrigue-Antar L. Kadler K.E. J. Biol. Chem. 2005; 280: 22616-22623Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), prolysyl oxidase, osteoglycin, the laminin 5 γ2 chain, procollagen VII, and the N-propeptide region of procollagen V. In contrast, both PCPE-1 and -2 have been shown to stimulate the activities of tolloid proteinases during cleavage of the C-propeptide regions of the major fibrillar procollagens (types I, II, and III) (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 30Adar R. Kessler E. Goldberg B. Collagen Rel. Res. 1986; 6: 267-277Crossref PubMed Scopus (53) Google Scholar, 31Steiglitz 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). Targeted deletion of the PCPE-1 gene has recently been shown to lead to aberrant collagen fibril formation and impaired biomechanical properties of bone tissue (32Steiglitz B.M. Kreider J.M. Frankenburg E.P. Pappano W.N. Hoffman G.G. Meganck J.A. Liang X. Hook M. Birk D.E. Goldstein S.A. Greenspan D.S. Mol. Cell Biol. 2006; 26: 238-249Crossref PubMed Scopus (41) Google Scholar).PCPEs are extracellular glycoproteins, devoid of intrinsic proteolytic activity, consisting of two CUB domains followed by a C-terminal NTR domain (31Steiglitz 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). Enzymatic removal of the NTR domain has no effect on enhancement of tolloid proteinase activity, showing that this is a property of the CUB domain region (33Kessler E. Adar R. Eur. J. Biochem. 1989; 186: 115-121Crossref PubMed Scopus (83) Google Scholar, 34Hulmes D.J.S. Mould A.P. Kessler E. Matrix Biol. 1997; 16: 41-45Crossref PubMed Scopus (49) Google Scholar). Although the mechanism of action of PCPEs is not well understood, this probably involves binding to the substrate, because maximum enhancing activity is found for PCPE: procollagen molar ratio of at least 1:1. BIAcore studies have shown that PCPE-1 binds to both the C-propeptide region as well as elsewhere in the procollagen molecule (35Ricard-Blum S. Bernocco S. Font B. Moali C. Eichenberger D. Farjanel J. Burchardt E.R. van der Rest M. Kessler E. Hulmes D.J.S. J. Biol. Chem. 2002; 277: 33864-33869Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Recent studies using a miniprocollagen III substrate have shown that enhancing activity is unaffected by removal of essentially all of the triple-helical region using highly purified bacterial collagenase (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Taken together, these data suggest that PCPE-1 binds to the non-triple-helical telopeptide region N-terminal to the BMP-1 cleavage site, in addition to the C-propeptide region, thereby inducing a conformational change that facilitates the action of BMP-1. It is also possible that PCPE-1 binds to tolloid proteinases, as recently shown for mTLL-1 (36Ge G. Zhang Y. Steiglitz B.M. Greenspan D.S. J. Biol. Chem. 2006; 281: 10786-10798Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To further explore the mechanism of action of PCPE-1, it is important to identify recognition sites within the CUB domains involved in enhancing activity. Here, we show that these involve a putative calcium binding site in the CUB1 domain, as well as a site specific to the CUB1 domain of PCPEs.EXPERIMENTAL PROCEDURESMolecular Modeling—The three-dimensional model of the CUB1 domain of human PCPE-1 (residues 12–124, numbered from the N terminus of the mature protein, i.e. after cleavage of the signal peptide) was built with the comparative molecular modeling program Geno3D (37Combet C. Jambon M. Deleage G. Geourjon C. Bioinformatics. 2002; 18: 213-214Crossref PubMed Scopus (365) Google Scholar), using the CUB domain structure with the highest sequence homology, namely the CUB2 domain of rat MASP-2 (22Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weis W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar). Ten three-dimensional structures were generated and superimposed with the ANTHEPROT three-dimensional package (38Deleage G. Combet C. Blanchet C. Geourjon C. Comput. Biol. Med. 2001; 31: 259-267Crossref PubMed Scopus (117) Google Scholar). The quality of the models was assessed using PROCHECK, and the most representative model, with the lowest energy, was selected.Recombinant Proteins—The BMP-1 FLAG construct was obtained by PCR using the BMP-1 cDNA inserted in pCEP4 as a template (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The FLAG sequence was inserted immediately contiguous to the BMP-1 sequence before the STOP codon. After sequencing and transfection in 293-EBNA cells, a clone was selected that gave the highest BMP-1 level of expression. This clone was used for further amplification and production of the protein.For site-directed mutagenesis and production of PCPE mutants, an 8-histidine tag was inserted by PCR into the human PCPE-1 cDNA (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), before the STOP codon, and the PCR product was cloned into pCR-Blunt II TOPO (Invitrogen). This construct was used as a template to generate all the PCPE-1 mutants using the QuikChange™ site-directed mutagenesis kit from Stratagene, according to the manufacturer's protocol. Overlapping oligonucleotides (Table 1) were purchased from MWG-BIOTECH (Courtaboeuf, France). The mutated inserts were then subcloned into pCEP4 (Invitrogen) using the KpnI and BamHI sites for semi-stable transfections into 293-EBNA cells (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The sequences of all mutants were confirmed by double-stranded DNA sequencing (Genome Express, France).TABLE 1Sequences of forward primers used to generate the PCPE-1 mutantsE26A5′-GTG GCA AGT GCG GGG TTC CCC-3′Y67A5′-GCC TGC CGC GCC GAT GCT CTG-3′D68A5′-GCC TGC CGC TAC GCT GCT CTG GAG G-3′T89A5′-TTT TGT GGG GCC TTC CGG CCT G-3′F90A5′-TGT GGG ACC GCC CGG CCT GCG-3′D109A5′-AGG ATG ACG ACG GCT GAG GGC ACA GGA G-3′ Open table in a new tab Protein Purification—For BMP-1-FLAG, the first purification step (Reactive Green) was as previously described for BMP-1 (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). BMP-1-FLAG-containing fractions (according to SDS-PAGE) were dialyzed against Buffer A (20 mm Hepes, pH 7.4, 2.5 mm CaCl2, 0.02% octyl-β-d-glucopyranoside) plus 0.25 m NaCl, and loaded onto an Anti-FLAG M2-agarose affinity column (Sigma) equilibrated with the same buffer. After washing with Buffer A plus 0.5 m NaCl, bound BMP-1-FLAG was eluted using 0.25 mg/ml FLAG peptide dissolved in Buffer A plus 0.5 m NaCl, then extensively dialyzed against the same buffer without peptide. We checked that the purified protein was devoid of endogenous PCPEs.For His-tagged PCPE-1 and its mutants, conditioned medium was centrifuged to remove cell debris and incubated with nickel-agarose equilibrated with 50 mm sodium phosphate, pH 8.0, 0.3 m NaCl (buffer B). The resin was then packed into a column and washed with buffer B containing 10 mm imidazole. The protein was eluted with 0.25 m imidazole in buffer B, and extensively dialyzed against phosphate-buffered saline.Enzymatic Activities—BMP-1 activity was measured as previously described (28Moali C. Font B. Ruggiero F. Eichenberger D. Rousselle P. Francois V. Oldberg A. Bruckner-Tuderman L. Hulmes D.J.S. J. Biol. Chem. 2005; 280: 24188-24194Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), using either [3H]procollagen I isolated from chick embryo fibroblasts or mini-procollagen III produced in 293 EBNA cells, as specified in the figure legends.Circular Dichroism—Far-UV (190–250 nm) CD measurements were carried out using thermostatted 0.2-mm path length quartz cells in a Jobin-Yvon CD6 instrument, calibrated with aqueous d-10-camphorsulfonic acid. Proteins (0.3–1 mg/ml) were analyzed at 25 °C in 20 mm KH2PO4/NaOH, 150 mm NaF, pH 7.2. Spectra were measured with a wavelength increment of 0.2 nm, integration time of 1 s, and bandpass of 2 nm. Protein concentrations were determined by absorbance at 280 nm (using absorbances calculated for each mutant based on the amino acid sequence) as well as by the Bradford assay, normalizing the latter to a known concentration of wild-type PCPE-1. Secondary structure analysis was carried out on the DICHROWEB server (39Whitmore L. Wallace B.A. Nucleic Acids Res. 2004; 32: W668-W673Crossref PubMed Scopus (1967) Google Scholar) using the CDSSTR program.Fluorescence—Intrinsic fluorescence was measured at 25 °C using a Photon Technology International instrument with a 10-mm path length quartz cell. Emission was recorded between 295 and 400 nm after excitation at 280 nm (excitation slits: 8 nm; emission slits: 4 nm) with a scanning rate of 1 nm/s. PCPE-1 was dialyzed against 20 mm HEPES, pH 7.4, 0.15 m NaCl and diluted to 2.5 μm in a total volume of 500 μl of the same buffer. EGTA and CaCl2 were added in 1-μl volumes. Fluorescence spectra were recorded 5 min after each addition and corrected for fluorescence of the buffer (containing or not EGTA and/or calcium). The effect of EGTA and calcium was also measured on a control solution of 5.4 μm N-acetyltryptophan amide (NATA) to check that changes in fluorescence were not due to absorption by added compounds (40Divita G. Di Pietro A. Roux B. Gautheron D.C. Biochemistry. 1992; 31: 5791-5798Crossref PubMed Scopus (17) Google Scholar).Surface Plasmon Resonance—Surface plasmon resonance analyses were carried out on a BIAcore 3000 instrument. Mini-procollagen III was covalently coupled to a CM4 sensor chip after activation with N-hydroxysulfosuccinimide and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (amine coupling) and injection in 10 mm sodium acetate, pH 5. Unreacted groups on the surface were neutralized with ethanolamine hydrochloride. The whole procedure was performed at a flow rate of 5 μl/min. A control flow cell was prepared similarly except that the mini-procollagen III solution was replaced by 10 mm sodium acetate, pH 5. The signal recorded on the control flow cell was automatically subtracted from those of the other flow cells. Prior to analysis, ligands were dialyzed against HBS-P (10 mm HEPES, pH 7.4, 0.15 m NaCl, and 0.005% P20 surfactant), and binding was monitored at 25 °C in the same buffer (containing or not 5 mm CaCl2) at a flow rate of 60 μl/min. Regeneration was carried out with 2 m guanidinium chloride when experiments were run in the absence of added calcium or with sequential injections of 0.25 m EDTA and 2 m guanidinium chloride in the presence of 5 mm calcium. Kinetic data were analyzed using BIAevaluation 4.1 software.RESULTSIdentification of Target Residues—As sequence alignment with CUB domains of known three-dimensional structure (20Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 21Gregory L.A. Thielens N.M. Matsushita M. Sorensen R. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2004; 279: 29391-29397Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weis W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar, 41Romero A. Romao M.J. Varela P.F. Kolln I. Dias J.M. Carvalho A.L. Sanz L. Topfer-Petersen E. Calvete J.J. Nat. Struct. Biol. 1997; 4: 783-788Crossref PubMed Scopus (128) Google Scholar) showed the strongest homology (33% sequence identity) with the CUB2 domain of rat MASP-2 (22Feinberg H. Uitdehaag J.C. Davies J.M. Wallis R. Drickamer K. Weis W.I. EMBO J. 2003; 22: 2348-2359Crossref PubMed Scopus (88) Google Scholar), this structure was used for molecular modeling of the human PCPE-1 CUB1 domain. As shown in Fig. 1, the predicted structure is a β-sandwich, where the root mean square deviation is 1.12 Å with respect to the template. Fig. 2 shows a sequence alignment of selected CUB domains in PCPEs and related human proteins, compared with the positions of sheets and loops in known structures, as illustrated by the MAp19 CUB domain (21Gregory L.A. Thielens N.M. Matsushita M. Sorensen R. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2004; 279: 29391-29397Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Four large loops are present on the upper face of the structure (loops L3, L5, L7, and L9, Fig. 1), as being exposed to solvent and accessible for ligand binding.FIGURE 2Alignment of CUB domains. Amino acid sequences of selected extracellular human proteins were aligned using ClustalW. Conserved cysteines are highlighted in pink, tyrosines in blue, and acidic residues involved in calcium coordination (based on known three-dimensional structures) in red. Residues in surface loops specific to PCPE CUB1 domains are highlighted in green. Secondary structure elements (β sheets and loops) are also shown based on the structure of the MAp19 CUB domain (equivalent to MASP2-CUB1). Residue numbering refers to the CUB1 domain of mature PCPE-1 (i.e. after cleavage of the signal peptide). Individual CUB domains in multi-CUB domain proteins are distinguished by numbers starting at the N terminus. TSG6, tumor necrosis factor-stimulated gene 6; CBLN, cubulin; NRPLN1, neuropilin-1. The figure was prepared using ESPript (53Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2505) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because previous studies on MAp19 indicated the importance of the loop regions in CUB domain interactions with MBL and L-ficolin (21Gregory L.A. Thielens N.M. Matsushita M. Sorensen R. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2004; 279: 29391-29397Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), we sought to identify residues involved in the interaction of the PCPE-1 CUB1 domain with procollagens by analysis of conserved and specific residues in the loops in the model shown in Fig. 1. As shown in Fig. 2, and as previously pointed out by Gregory et al. (20Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), CUB domains from several proteins, including the CUB1 domain of PCPE-1, contain three highly conserved acidic residues (Glu-45, Asp-53, and Asp-98 in C1s; Glu-52, Asp-60, and Asp-105 in MAp19; and Glu-60, Asp-68, and Asp-109 in PCPE-1) that have been shown to be involved in the coordination of a calcium atom in the three-dimensional structures of C1s (20Gregory L.A. Thielens N.M. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2003; 278: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and MAp19 (21Gregory L.A. Thielens N.M. Matsushita M. Sorensen R. Arlaud G.J. Fontecilla-Camps J.C. Gaboriaud C. J. Biol. Chem. 2004; 279: 29391-29397Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This calcium binding site is formed by the loops located on the upper face of the β-sandwich structure (Fig. 1). Furthermore, a tyrosine residue involved in stabilizing this calcium binding site through formation of an H-bonding network (Tyr-17 in C1s, Tyr-24 in MAp19) is also conserved in PCPE-1 (Tyr-32). These sequence identities therefore point to a putative calcium binding site in PCPE-1. In addition to Glu-60, Asp-68, and Asp-109, Tyr-67 in PCPE-1 CUB1 (
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