Molecular Features of the Collagen V Heparin Binding Site
1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês
10.1074/jbc.273.24.15069
ISSN1083-351X
AutoresFrédéric Delacoux, Agnès Fichard, C. Geourjon, R. Garrone, Florence Ruggiero,
Tópico(s)Collagen: Extraction and Characterization
ResumoA heparin binding region is known to be present within the triple helical part of the α1(V) chain. Here we show that a recombinant α1(V) fragment (Ile824 to Pro950), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual α1(V) chains and HepV are eluted at identical NaCl concentrations (0.35m) from a heparin-Sepharose column, and this binding can be inhibited specifically by the addition of free heparin or heparan sulfate. In contrast, a shorter 23-residue synthetic peptide, containing the putative heparin binding site in HepV, fails to bind heparin. Interestingly, HepV promotes cell attachment, and HepV-mediated adhesion is inhibited specifically by heparin or heparan sulfate, indicating that this region might behave as an adhesive binding site. The same site is equally functional on triple helical molecules as shown by heparin-gold labeling. However, the affinities for heparin of each of the collagen V molecular forms tested are different and increase with the number of α1(V) chains incorporated in the molecules. Molecular modeling of a sequence encompassing the putative HepV binding sequence region shows that all of the basic residues cluster on one side of the helical face. A highly positively charged ring around the molecule is thus particularly evident for the α1(V) homotrimer. This could strengthen its interaction with the anionic heparin molecules. We propose that a single heparin binding site is involved in heparin-related glycosaminoglycans-collagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues. A heparin binding region is known to be present within the triple helical part of the α1(V) chain. Here we show that a recombinant α1(V) fragment (Ile824 to Pro950), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual α1(V) chains and HepV are eluted at identical NaCl concentrations (0.35m) from a heparin-Sepharose column, and this binding can be inhibited specifically by the addition of free heparin or heparan sulfate. In contrast, a shorter 23-residue synthetic peptide, containing the putative heparin binding site in HepV, fails to bind heparin. Interestingly, HepV promotes cell attachment, and HepV-mediated adhesion is inhibited specifically by heparin or heparan sulfate, indicating that this region might behave as an adhesive binding site. The same site is equally functional on triple helical molecules as shown by heparin-gold labeling. However, the affinities for heparin of each of the collagen V molecular forms tested are different and increase with the number of α1(V) chains incorporated in the molecules. Molecular modeling of a sequence encompassing the putative HepV binding sequence region shows that all of the basic residues cluster on one side of the helical face. A highly positively charged ring around the molecule is thus particularly evident for the α1(V) homotrimer. This could strengthen its interaction with the anionic heparin molecules. We propose that a single heparin binding site is involved in heparin-related glycosaminoglycans-collagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues. Collagen V is a fibrillar collagen that plays an important role in fibrillogenesis, and it also acts as an adhesive substrate for a large variety of cells and binds to a number of extracellular components through its major triple helical domain (1Fichard A. Kleman J.-P. Ruggiero F. Matrix Biol. 1994; 14: 515-531Crossref Scopus (167) Google Scholar). Collagen V interacts with matrix proteoglycans such as the two small proteoglycans decorin and biglycan (2Whinna H.C. Choi H.U. Rosenberg L.C. Church F.C. J. Biol. Chem. 1993; 268: 3920-3924Abstract Full Text PDF PubMed Google Scholar), the proteoglycan form of macrophage colony-stimulating factor (3Suzu S. Ohtsuki T. Makishima M. Yanai N. Kawashima T. Nagata N. Motoyoshi K. J. Biol. Chem. 1992; 267: 16812-16815Abstract Full Text PDF PubMed Google Scholar), the cell surface proteoglycan syndecan-1 (4Koda J.E. Rapraeger A. Bernfield M. J. Biol. Chem. 1985; 260: 8157-8162Abstract Full Text PDF PubMed Google Scholar, 5San Antonio J.D. Karnovsky M.J. Gay S. Sanderson R.D. Lander A.D. Glycobiology. 1994; 3: 327-332Crossref Scopus (54) Google Scholar), and as shown recently, the membrane spanning proteoglycan NG2 (6Tillet E. Ruggiero F. Nishiyama A. Stallcup W.B. J. Biol. Chem. 1997; 272: 10769-10776Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Some of these interactions are mediated by the core proteins, but others depend on the glycosaminoglycan chains such as the heparan sulfate chains. Apart from in vitro binding of collagen V to membrane-spanning proteoglycans, the suggestion that heparan sulfate interacts with collagen V was supported by inhibition experiments showing a reduction of cell attachment to collagen V in the presence of heparin (7LeBaron R.G. Höök A. Esko J.D. Gay S. Höök M. J. Biol. Chem. 1989; 264: 7950-7956Abstract Full Text PDF PubMed Google Scholar). It has been shown already that cell focal adhesion on fibronectin requires the cooperation of both cell transmembrane proteoglycans and integrin receptors (8Woods A. Couchman J.R. Mol. Biol. Cell. 1994; 5: 183-192Crossref PubMed Scopus (279) Google Scholar). Because we have demonstrated already that cell-collagen V interactions involved integrins (9Ruggiero F. Champliaud M.-F. Garrone R. Aumailley M. Exp. Cell Res. 1994; 210: 215-223Crossref PubMed Scopus (57) Google Scholar, 10Ruggiero F. Comte J. Cabañas C. Garrone R. J. Cell Sci. 1996; 109: 1865-1874Crossref PubMed Google Scholar), the binding of membrane-spanning proteoglycans could reinforce cell attachment to collagen V and, in that sense, would be of physiological importance. Therefore, to understand the role of collagen V-heparan sulfate proteoglycan interactions, it appears essential to characterize the specific domain(s) of the molecule responsible for binding to heparin, a glycosaminoglycan related to heparan sulfate. Collagen V is a typical fibrillar collagen containing a 300-nm-long triple helical domain that presents different molecular forms in tissue. The predominant molecular form found in most tissues is the heterotrimer [α1(V)]2α2(V), whereas the α1(V)α2(V)α3(V) molecule is only extracted from human placenta (1Fichard A. Kleman J.-P. Ruggiero F. Matrix Biol. 1994; 14: 515-531Crossref Scopus (167) Google Scholar). The homotrimer [α1(V)]3 occurs in cultures of hamster lung cells and was suggested to be present in embryonic tissue (11Haralson M.A. Mitchell W.M. Rhodes R.K. Kresina T.F. Gay R. Miller E.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5206-5210Crossref PubMed Scopus (65) Google Scholar, 12Haralson M.A. Mitchell W.M. Rhodes R.K. Miller E.J. Arch. Biochem. Biophys. 1984; 229: 509-518Crossref PubMed Scopus (15) Google Scholar, 13Moradi-Améli M. Rousseau J.-C. Kleman J.-P. Champliaud M.-F. Boutillon M.-M. Bernillon J. Wallach J. van der Rest M. Eur. J. Biochem. 1994; 221: 987-995Crossref PubMed Scopus (57) Google Scholar). It was produced recently as a recombinant molecule (14Fichard A. Tillet E. Delacoux F. Garrone R. Ruggiero F. J. Biol. Chem. 1997; 272: 30083-30087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The heterotrimeric [α1(V)]2α2(V) molecular form was shown previously to bind to heparin at physiological salt concentrations. This activity was attributed to a proteolytic NH2-terminal 30-kDa fragment of the α1(V) chain (15Yaoi Y. Hashimoto K. Koitabashi H. Takahara K. Ito M. Kato I. Biochim. Biophys. Acta. 1990; 1035: 139-145Crossref PubMed Scopus (43) Google Scholar). Aside from a requirement for the α1(V) chain, the features of the different stoichiometries of the collagen V triple helix necessary for heparin binding remain unknown. Such studies have been hampered by the fact that determination of minimal sites on the collagenous triple helix is difficult. Thus in this manuscript, to locate more precisely the heparin binding site, we have combined several approaches: (i) recombinant technology, in which collagenous domains and the molecule isotypes can be engineered and generated in quantities sufficient for biochemical and functional analysis; (ii) a synthetic peptide to narrow the sequence involved in heparin-collagen V recognition; (iii) electron microscopy to visualize the site on triple helical molecules. Using these approaches it has been possible to establish that a recombinant fragment of the α1(V) chain (Ile824I to Pro950) but not a synthetic peptide encompassing the putative heparin binding site binds to heparin and heparan sulfate. Moreover, we provide evidence for a cell adhesive function of this fragment through a cell surface heparan sulfate proteoglycan. This region corresponds to a heparin binding site common to the three known collagen V molecular forms, although increasing heparin affinities were correlated positively with the number of α1(V) chains incorporated in the molecule. Based on molecular modeling of the portion of the α chain which includes the putative binding sequence, we propose a model to explain the different affinities observed for the distinct collagen V molecular forms. The HepV module cDNA (nucleotides 2595–2976) was generated by polymerase chain reaction using as template the clone 302 kindly provided by Dr. Takahara (16Takahara K. Sato Y. Okasawa K. Okamoto N. Noda A. Yaoi Y. Kato I. J. Biol. Chem. 1991; 266: 13124-13129Abstract Full Text PDF PubMed Google Scholar). Two oligonucleotides flanking the desired sequence were designed. One corresponding to the 5′-end of the module carried an Eco RI site (5′-TATGAATTCCCATCAAGGGTGATCGGGGGGAGA-3′), and the second, corresponding to the 3′-end of the module, introduced a Pst I site and a stop codon (5′-TATCTGCAGATTAGGGTCCCCGTTCACCAGGAGGGCCAGCTGG-3′). The resulting polymerase chain reaction product of 399 base pairs was subcloned in the Eco RI and Pst I sites of a pT7-7 expression vector (17Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1074-1078Crossref PubMed Scopus (2456) Google Scholar). The plasmid obtained, named pHepV, thus encoded the heparin binding site of α1(V) under the control of theEscherichia coli phage T7 promoter. The sequence of the recombinant DNA was checked thoroughly. To obtain the recombinant protein, pHepV was transformed in theE. coli host strain BL21(DE3). This strain carries the T7 RNA polymerase gene under the control of the lac promoter/operator region which allows induction by isopropyl-β-d-thiogalactopyranoside and subsequently the transcription of the recombinant plasmid. E. coli cells harboring the plasmid pHepV were grown at 37 °C to anA 600 of 0.7 in Luria-Bertani medium containing 50 μg/ml ampicillin. The culture was then supplemented with 0.4 mm isopropyl-β-d-thiogalactopyranoside to induce protein expression. The incubation was then maintained at the same conditions for an additional period of 6 h. Cells were harvested by centrifugation at 8,920 × g for 20 min, resuspended in 10 mm Tris/HCl, 1 mm EDTA, pH 8.0, and sonicated by two 30-s pulses at intensity level 3, using a Branson Sonifier-250 (Branson Ultrasonics). After centrifugation, supernatants and pellets were analyzed by 15% SDS-PAGE 1The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; CAPS, 3-(cyclohexylamino)propanesulfonic acid; CHO, Chinese hamster ovary. according to Laemmli (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar) followed by Coomassie Blue staining. For purification of the recombinant fragment HepV, the bacterial supernatant was dialyzed against 35 mm Tris/HCl, pH 7.4, filtered, and applied to a HPLC cation exchange chromatography on a 1-ml Resource-S column (Amersham Pharmacia Biotech) (Waters 625 LC system) and eluted by a linear NaCl gradient (0–300 mm) in the same buffer. The fraction eluted at 200 mm NaCl and containing HepV was purified further by anion exchange chromatography through a HiTrapQ (Amersham Pharmacia Biotech) column. The column was equilibrated in 200 mm NaCl, 35 mm Tris/HCl, pH 7.4. The unbound fractions contained purified HepV as analyzed by 15% SDS-PAGE. Collagen V [α1(V)]2α2(V) heterotrimer was extracted with pepsin and purified as described previously (9Ruggiero F. Champliaud M.-F. Garrone R. Aumailley M. Exp. Cell Res. 1994; 210: 215-223Crossref PubMed Scopus (57) Google Scholar). Human placental pepsinized collagen V containing [α1(V)]2α2(V) and α1(V)α2(V)α3(V) heterotrimers in equal ratio were purchased from Sigma. The production in 293-EBNA cells of recombinant homotrimer [α1(V)]3 and subsequent purification are described elsewhere (14Fichard A. Tillet E. Delacoux F. Garrone R. Ruggiero F. J. Biol. Chem. 1997; 272: 30083-30087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The peptide GTPGKPGPRGQRGPTGPRGERGP referred to as HepP was synthesized according to standard protocols using Fmoc (N -(9-fluorenyl)methoxycarbonyl) chemistry in a Milligen 9050 synthesizer. The peptide was purified by reverse phase HPLC and was shown by amino acid analysis to have the correct primary structure. Glycosaminoglycans were obtained from commercial sources: heparin and chondroitin sulfate C from Sigma and heparan sulfate from Jacques Boy, Reims, France. Heparin-Sepharose affinity columns (HiTrap Heparin, Amersham Pharmacia Biotech) were equilibrated in phosphate-buffered saline (PBS) (buffer A) or in 35 mmTris/HCl, pH 7.4 containing 200 mm NaCl (buffer B). Protein samples were loaded onto a column, and a programmed linear gradient of 150–500 mm NaCl in buffer A and 200–500 mmNaCl in buffer B was applied at a flow rate of 0.5 ml/min. To determine heparin affinities to individual chains, collagen V samples were heat-denatured (60 °C for 20 min) before loading. Fractions (1 ml) were collected, and the elution profile of protein samples was determined by monitoring the absorbance at 206 nm. The different fractions were then analyzed by SDS-PAGE as described above except fractions obtained with the peptide, which were analyzed by amino acid analysis. Analysis of the capacity of glycosaminoglycans to inhibit HepV binding to heparin and the synthetic peptide to release HepV bound to heparin were both carried out in batch with heparin-Sepharose CL-6B resin (Amersham Pharmacia Biotech). The resin was equilibrated with 50 mm Tris/HCl, pH 7.5 containing 150 mm NaCl for glycosaminoglycans inhibition experiments. For glycosaminoglycan inhibition experiments, 5 μg of HepV was incubated in the absence or presence of free heparin, heparan sulfate, or chondroitin sulfate for 2 h at room temperature before being applied to the resin for an additional 30 min. Unbound material was recovered by gentle centrifugation after the addition of 1 ml of starting buffer to the samples. Elution of bound material was achieved by adding 0.5 m NaCl to the starting buffer. All fractions were analyzed by 15% SDS-PAGE. For peptide inhibition, 50 μl of the resin was incubated with 5 μg of HepV for 30 min at room temperature and washed with 1 ml of starting buffer. Bound material was then incubated with various concentrations of the synthetic peptide HepP, and the released material was recovered by gentle centrifugation. The resin was then washed with starting buffer, and the elution of the remaining bound material was achieved by adding 1 m NaCl to the starting buffer. Fractions were analyzed by 15% SDS-PAGE. Amino acid compositions were determined after hydrolysis under vacuum (6 N HCl, 115 °C, 24 h) in presence of 2-mercaptoethanol in a Pico Tag system (Waters) with a Beckman amino acid analyzer. For NH2-terminal amino acid sequencing, proteins were electrotransferred onto polyvinylidene difluoride membrane (Problott, Applied Biosystems) for 4 h at 60 V in 10 mm CAPS, 5% methanol, pH 11, and the band of interest was excised from the membrane after brief staining with 0.2% Ponceau S in 1% acetic acid (Sigma). Amino acid sequence analysis was performed by automated Edman degradation using an Applied Biosystems 473A protein sequencer. Collagen solutions (collagen V samples and collagen I as control) were dialyzed overnight against PBS at 4 °C and then incubated with heparin-BSA-gold 10-mm particles (Sigma) for 3 h at room temperature. Samples were dialyzed against 1m ammonium acetate and finally diluted to 10 μg/ml with the same buffer. After the addition of an equal volume of glycerol, the solutions were sprayed onto freshly cleaved mica sheet and were placed immediately on the holder of a MED 010 evaporator (Balzers). Rotary shadowing was carried out as described previously (19Ruggiero F. Petit B. Ronzière M.-C. Farjanel J. Hartmann D.J. Herbage D. J. Histochem. Cytochem. 1993; 41: 867-875Crossref PubMed Scopus (41) Google Scholar). Observations of replicas were performed with a Philips CM120 microscope at the CMEABG (Centre de Microscopie Electronique Appliquée à la Biologie et à la Géologie, Université Claude Bernard, Lyon I). Chinese hamster ovary (CHO) cells were maintained in monolayer cultures in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, glutamine, nonessential amino acids, and a mixture of antibiotics. Before the adhesion assay, cells were harvested with 1% EDTA in PBS or with 0.05% trypsin, 0.02% EDTA in PBS, pH 7.4, when indicated. Multiwell plates (96-well tissue culture plates, Costar, France) were coated by overnight adsorption at 4 °C with substrates at concentrations ranging from 0 to 40 μg/ml (100 μl/well). After saturation of the wells with 1% bovine serum albumin, freshly suspended cells in serum-free Dulbecco's modified Eagle's medium (4 × 105 cells/ml) were plated onto coated wells (100 μl/well) and allowed to attach for 30–40 min at 37 °C. The attached cells were washed with PBS, fixed in 1% glutaraldehyde, and stained for 25 min with 0.1% crystal violet in water. After extensive washing, the dye absorbed to the cells was solubilized with 0.2% Triton X-100, and optical density was read with an enzyme immunosorbent assay reader (Dynex MRX) at 570 nm. Each assay point was carried out in triplicate. For inhibition assays, the coated wells were preincubated with 10 μg/ml glycosaminoglycans (heparin, heparan sulfate, or chondroitin sulfate) for 1 h at 37 °C. Freshly suspended cells were then added, and the wells were incubated for 30–40 min. For synthetic peptide inhibitions, cells were first mixed with 500 μmRGDS peptide before being seeded onto the coated wells. The assays were then continued as described above. The two-dimensional projection used to represent the three-dimensional structure of α-helices is called a helical wheel. It allows, in the case of heparin binding consensus sequences, visualization of clusters of basic residues (20Cardin A.D. Weintraub H.J.R. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar). The helical wheel representation for collagen helix differs from a true α helix and thus was represented as described previously (21Deprez P.N. Inestrosa N.C. J. Biol. Chem. 1995; 270: 11043-11046Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). For the representation of collagen V triple helices ([α1(V)]2α2(V) and [α1(V)]3), the helical wheels were positioned with the glycyl residues at the center of the molecule. For the molecular modeling of the peptide (Gly904 to Arg924), the coordinates of the three NH2-terminal GPP* (P* for hydroxyproline) triplets of the collagen-like peptide (PP*G)4PP*A(PP*G)5 were used to calculate interproton distance (22Bella J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (887) Google Scholar). The Protein Data Bank code number is 1CAG. Each value was allowed to vary from −0.3 to +0.1 Å around the actual interproton distance. In this way a total of 683 constraints was introduced for the triple helical region, 105 of which were interchain constraints. In addition, average (Φ, Ψ) dihedral angles deduced from the crystal structure were also introduced with the following values: for Gly Φ = −72 ± 15° and Ψ = 174 ± 25°; for X position Φ = −72 ± 15° and Ψ = 164 ± 20°; for Y position Φ = −60 ± 15° and Ψ = 150 ± 18° (see Table I in Ref. 22Bella J. Eaton M. Brodsky B. Berman H.M. Science. 1994; 266: 75-81Crossref PubMed Scopus (887) Google Scholar; note that the reported standard deviations were doubled for each angle). Three-dimensional structures were generated from set of constraints with the X-PLOR 3.8.5 program (23Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar) using the default parameter sets, except for some minor modifications to increase the duration of the molecular dynamic simulations and the number of energy minimization steps. Structure superimposition, three-dimensional graphic displays, and manipulations were accomplished using ANTHEPROT 2.0 software (24Geourjon C. Deléage G. J. Mol. Graphics. 1995; 13: 209-212Crossref PubMed Scopus (50) Google Scholar). The Protein Data Bank numbers are 1a89 for the homotrimer and 1a9a for the heterotrimer. We designed a fragment, referred to as HepV, which encompasses the complete NH2-terminal part of the 30-kDa CNBr peptide defined by Yaoi et al. (15Yaoi Y. Hashimoto K. Koitabashi H. Takahara K. Ito M. Kato I. Biochim. Biophys. Acta. 1990; 1035: 139-145Crossref PubMed Scopus (43) Google Scholar) and the sequence around the endoproteinase Glu-C cleavage site which was found to be determinant for heparin binding (Fig. 1). The resulting heparin binding site was thus narrowed down from the COOH-terminal end of the 30-kDa fragment to a 12-kDa polypeptide, HepV. An expression vector pT7-7, which encodes amino acids Ile824 to Pro950 of human α1(V) chain was constructed as described under "Experimental Procedures." After induction with isopropyl-β-d-thiogalactopyranoside, SDS-PAGE analysis of the soluble and insoluble cellular extracts showed the presence of an additional protein band of 17 kDa in soluble extracts which was not found in transformed E. coli cells before induction. The difference between the apparent molecular mass and the predicted polypeptide mass of 12 kDa was attributed to the peculiar structure of collagen chains migrating slower than the globular standards, as well as the additional NH2-terminal amino acid residues originating from the construct. Passage of the supernatant over a Mono S column separated the HepV domain from most of the contaminating bacterial proteins. Final purification was achieved by rechromatography on a Mono Q column (Fig. 2 A ). Analysis of the NH2-terminal amino acid sequence of the purified band indicated the sequence XRIPIKGD, which agreed with that of the HepV construct. The four first amino acid residues correspond to NH2-terminal extension added for cloning purposes.Figure 2Panel A , 15% SDS-PAGE analysis of purified recombinant HepV fragment. Lane 1 , protein pattern of the loaded material. Lanes 2 and 3 , HepV purification by two-step ion exchange chromatography: bound fraction to a cation exchange column eluted at 0.2 m NaCl contains HepV (lane 2 ); unbound fraction to the anion exchange column contains purified recombinant fragment (lane 3 ). Panel B , a HPLC heparin affinity column was used to determine the affinity of heparin for HepV. Purified HepV was bound to the column and eluted with a linear NaCl gradient (dashed line ). The fraction eluted at 0.35 m NaCl contained HepV as shown by 15% SDS-PAGE analysis of the major peak.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The binding interaction of heparin with collagen V fragments was studied using their affinities on a heparin-Sepharose column. We showed that HepV bound to the heparin-Sepharose column at physiological pH and ionic strength. Moreover, HepV was eluted from the column at 0.35m NaCl (Fig. 2 B ), which is exactly the concentration required to elute isolated native α1(V) chains. In addition, binding to heparin is inhibited by incubation of HepV with free heparin or heparan sulfate but not with chondroitin sulfate before loading on the heparin-Sepharose column (Fig.3 A ). Furthermore, to assess the importance of the length of the sequence flanking the putative heparin binding site, we designed a peptide, HcpP, containing the basic amino acid clusters KPGPRGQR but also including the basic residues close to the endoproteinase Glu-C cleavage site (at the carboxyl group of glutamate residue) (Fig. 1). Only 5% of the corresponding synthetic peptide was able to bind to a heparin-Sepharose column as indicated by the amino acid composition of the bound and unbound fractions (data not shown). Also, the peptide (added at concentrations up to 1 mg/ml; corresponding to a 200-fold molar excess) was unable to detach HepV bound to heparin-Sepharose (Fig. 3 B ) or to inhibit heparin binding to collagen V in a solid phase assay (not shown). We showed that isolated α1(V), but not α2(V) and α3(V) chains, was able to bind heparin at physiological pH and ionic strength and was eluted from the column at exactly 0.35m NaCl (Fig. 4 A ). To evaluate whether the binding site present in α1(V) is available when incorporated into the triple helix, different collagen V molecular forms were passed through a heparin column: the native heterotrimeric molecular form [α1(V)]2α2(V) from bovine bone and from human placenta, also containing in a 1:1 ratio the molecular form α1(V)α2(V)α3(V). To circumvent the problem of the α1(V) homotrimer, which is difficult to obtain from tissue, we used the recombinant homotrimer expressed and purified as described elsewhere (14Fichard A. Tillet E. Delacoux F. Garrone R. Ruggiero F. J. Biol. Chem. 1997; 272: 30083-30087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The results demonstrate that the affinities of each molecular form for heparin in terms of NaCl concentrations necessary for elution followed this order: α1(V)α2(V)α3(V) (0.28m) < [α1(V)]2α2(V) (0.35 m) < [α1(V)]3 (0.45 m) (Fig. 4 B ). These results indicate that the affinity of the different molecular forms could be modulated in relation to the number of α1(V) chains present in each subtype, two α1(V) chains in the triple helix being necessary to obtain the same affinity determined with isolated α1(V) chain. Because an active site might be available in small fragments or individual chains but masked in the context of the whole molecule, we then examined, by electron microscopy, the location of the heparin binding site on triple helices using heparin as a probe (according to Ref. 5San Antonio J.D. Karnovsky M.J. Gay S. Sanderson R.D. Lander A.D. Glycobiology. 1994; 3: 327-332Crossref Scopus (54) Google Scholar). For this purpose, collagen V, and collagen I as control, were complexed with heparin-BSA-gold and prepared for rotary shadowing as mentioned under "Experimental Procedures." Pepsinized [α1(V)]2α2(V) collagen V-heparin gold complexes were often observed as large intermolecular aggregates for which it was difficult to identify the precise location of heparin binding. However, selected areas contained a sufficient proportion of individual molecules to determine the position of the gold particles on collagen V molecules. The binding site was located at about 100 nm from one of the extremities of the molecules (Fig.5 A ). This corresponds exactly to the α1(V) binding site position if we consider that the distance is measured from the NH2-terminal extremity of the collagen molecule. This binding site is specific to collagen V molecules since it was not observed on collagen I molecules labeled with heparin-gold (data not shown). Attempts to determine the polarity of collagen V ends using the recombinant [α1(V)]3 homotrimer encompassing the entire N -propeptide were not successful. Indeed, the presence of the N -propeptide seemed to enhance the formation of intermolecular aggregates associated with heparin-gold, and individual labeled molecules were rarely observed. Therefore mapping experiments were undertaken with a 200-nm fragment product of the recombinant [α1(V)]3 homotrimer (Fig. 5 B ). We have shown previously that this fragment resulted from a proteolytic cleavage occurring close to a flexible region present in the triple helical domain of the homotrimer (14Fichard A. Tillet E. Delacoux F. Garrone R. Ruggiero F. J. Biol. Chem. 1997; 272: 30083-30087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Interestingly, the NH2-terminal sequence of this fragment was found to start at residue Asp759, which is 65 amino acid residues upstream from the heparin binding site we have determined (Ile824 to Pro950) (14Fichard A. Tillet E. Delacoux F. Garrone R. Ruggiero F. J. Biol. Chem. 1997; 272: 30083-30087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). This would correspond to about 20 nm from the NH2-terminal end of the fragment and allow the orientation of the molecules. As expected, when this fragment was applied to heparin-Sepharose it was retained on the column and was eluted in the same conditions as the entire recombinant homotrimer, thus confirming the presence of the heparin binding site (data not shown). Mapping experiments on the fragments clearly showed a single location of heparin gold particles at a site very close to one end of the fragment, thus identified as the NH2-terminal extremity (Fig. 5). It i
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