Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1.

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triple-helical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis.

Mapping of potent and specific binding motifs, GLOGEN and GVOGEA, for integrin α1ß1 using collagen toolkits II and III.

Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2ß1, but with lower affinity for α1ß1. Here, to identify specific ligands for α1ß1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the α1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin α1ß1. We also identified a new α1ß1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated α1 I domain, but not to the α2 I domain or to C2C12 cells expressing α2ß1 or α11ß1. Thus, GVOGEA is specific for α1ß1. Although recognized by both α2ß1 and α11ß1, GLOGEN is a better ligand for α1ß1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the α1 I domain from collagen IV (IC(50) ∼3 µm), GFOGER is much less potent (IC(50) ∼90 µm), as shown previously. These data confirm the selectivity of GFOGER for α2ß1 and establish GLOGEN as a high affinity site for α1ß1.

Procollagen C-proteinase enhancer stimulates procollagen processing by binding to the C-propeptide region only.

Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner. Here, we performed a detailed quantitative study of the binding of PCPE-1 and of its minimal active fragment (CUB1-CUB2) to three regions of the procollagen III molecule: the triple helix, the C-telopeptide, and the C-propeptide. Contrary to results described elsewhere, we found the PCPE-1-binding sites to be located exclusively in the C-propeptide region. In addition, binding and enhancing activities were found to be independent of the glycosylation state of the C-propeptide. These data exclude previously proposed mechanisms for the action of PCPEs and also suggest new mechanisms to explain how these proteins can stimulate BMP-1/tolloid-like proteinases by up to 20-fold.

Synergism between platelet collagen receptors defined using receptor-specific collagen-mimetic peptide substrata in flowing blood.

Exposed subendothelial collagen acts as a substrate for platelet adhesion and thrombus formation after vascular injury. Synthetic collagen-derived triple-helical peptides, designated collagen-related peptide (CRP), GFOGER, and VWF-III, can specifically engage the platelet collagen receptors, glycoprotein VI and integrin alpha(2)beta(1), and plasma von Willebrand factor (VWF), respectively. Hitherto, the role of these 3 collagen-binding axes has been studied indirectly. Use of these uniform peptide substrates, rather than collagen fibers, provides independent control of each axis. Here, we use confocal imaging and novel image analysis techniques to investigate the effects of receptor-ligand engagement on platelet binding and activation during thrombus formation under flow conditions. At low shear (100s(-1) and 300s(-1)), both GFOGER and CRP are required for thrombus formation. At 1000s(-1), a combination of either CRP or GFOGER with VWF-III induces comparable thrombus formation, and VWF-III increases thrombus deposition at all shear rates, being indispensable at 3000s(-1). A combination of CRP and VWF-III is sufficient to support extensive platelet deposition at 3000s(-1), with slight additional effect of GFOGER. Measurement of thrombus height after specific receptor blockade or use of altered proportions of peptides indicates a signaling rather than adhesive role for glycoprotein VI, and primarily adhesive roles for both alpha(2)beta(1) and the VWF axis.

First analysis of a bacterial collagen-binding protein with collagen Toolkits: promiscuous binding of YadA to collagens may explain how YadA interferes with host processes.

The Yersinia adhesin YadA mediates the adhesion of the human enteropathogen Yersinia enterocolitica to collagens and other components of the extracellular matrix. Though YadA has been proposed to bind to a specific site in collagens, the exact binding determinants for YadA in native collagen have not previously been elucidated. We investigated the binding of YadA to collagen Toolkits, which are libraries of triple-helical peptides spanning the sequences of type II and III human collagens. YadA bound to many of them, in particular to peptides rich in hydroxyproline but with few charged residues. We were able to block the binding of YadA to collagen type IV with the triple-helical peptide (Pro-Hyp-Gly)(10), suggesting that the same site in YadA binds to triple-helical regions in network-forming collagens as well. We showed that a single Gly-Pro-Hyp triplet in a triple-helical peptide was sufficient to support YadA binding, but more than six triplets were required to form a tight YadA binding site. This is significantly longer than the case for eukaryotic collagen-binding proteins. YadA-expressing bacteria bound promiscuously to Toolkit peptides. Promiscuous binding could be advantageous for pathogenicity in Y. enterocolitica and, indeed, for other pathogenic bacteria. Many of the tightly binding peptides are also targets for eukaryotic collagen-binding proteins, and YadA was able to inhibit the interaction between selected Toolkit peptides and platelets. This leads to the intriguing possibility that YadA may interfere in vivo with host processes mediated by endogenous collagen-binding proteins.

Identification of a major GpVI-binding locus in human type III collagen.

We have analyzed the adhesion of human and murine platelets, and of recombinant human and murine GpVI ectodomains, to synthetic triple-helical collagen-like peptides. These included 57 peptides derived from the sequence of human type III collagen and 9 peptides derived from the cyanogen bromide fragment of bovine type III collagen, alpha1(III)CB4. We have identified several peptides that interact with GpVI, in particular a peptide designated III-30 with the sequence GAOGLRGGAGPOGPEGGKGAAGPOGPO. Both human and murine platelets bound to peptide III-30 in a GpVI-dependent manner. III-30 also supported binding of recombinant GpVI ectodomains. Cross-linked III-30 induced aggregation of human and murine platelets, although with a lower potency than collagen-related peptide. Modifications of the peptide sequence indicated that the hydroxyproline residues play a significant role in supporting its GpVI reactivity. However, many peptides containing OGP/GPO motifs did not support adhesion to GpVI. These data indicate that the ability of a triple-helical peptide to bind GpVI is not solely determined by the presence or spatial arrangement of these OGP/GPO motifs within the peptides.

Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.

The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.

OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice.

Osteoclasts are terminally differentiated leukocytes that erode the mineralized bone matrix. Osteoclastogenesis requires costimulatory receptor signaling through adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), such as Fc receptor common γ (FcRγ) and DNAX-activating protein of 12 kDa. Identification of these ITAM-containing receptors and their ligands remains a high research priority, since the stimuli for osteoclastogenesis are only partly defined. Osteoclast-associated receptor (OSCAR) was proposed to be a potent FcRγ-associated costimulatory receptor expressed by preosteoclasts in vitro, but OSCAR lacks a cognate ligand and its role in vivo has been unclear. Using samples from mice and patients deficient in various ITAM signaling pathways, we show here that OSCAR costimulates one of the major FcRγ-associated pathways required for osteoclastogenesis in vivo. Furthermore, we found that OSCAR binds to specific motifs within fibrillar collagens in the ECM that become revealed on nonquiescent bone surfaces in which osteoclasts undergo maturation and terminal differentiation in vivo. OSCAR promoted osteoclastogenesis in vivo, and OSCAR binding to its collagen motif led to signaling that increased numbers of osteoclasts in culture. Thus, our results suggest that ITAM-containing receptors can respond to exposed ligands in collagen, leading to the functional differentiation of leukocytes, which provides what we believe to be a new concept for ITAM regulation of cytokine receptors in different tissue microenvironments.

Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III.

Immune responses are tightly controlled by the opposing actions of activating and inhibitory immune receptors. Previously we identified collagens as ligands for the inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1), revealing a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens. This interaction can be blocked by LAIR-2, a secreted member of the LAIR-1 family. LAIR-1 specifically interacts with synthetic trimeric peptides containing 10 repeats of glycine-proline-hydroxyproline (GPO) residues which can directly inhibit immune cell activation in vitro. Here we studied the interaction of human LAIR-1 and LAIR-2 with collagen in more detail by using novel overlapping synthetic trimeric peptides (Toolkits) encompassing the entire triple-helical domain of human collagens II and III. LAIR-1 and LAIR-2 bind several of these collagen-like peptides, with LAIR-2 being able to bind more than LAIR-1. LAIR binding to trimeric collagen peptides was influenced by GPO content of the peptide, although additional non-GPO triplets contributed to the interaction. Furthermore, we identified several trimeric peptides that were potent LAIR-1 ligands and could efficiently induce inhibition of T cell activation and FceRI-induced degranulation of RBL-2H3 cells through binding to LAIR-1. A detailed understanding of the LAIR recognition motifs within collagen may lead to the development of potent reagents that can be used in in vitro, ex vivo, and in vivo functional studies to dissect the biology and function of the collagen/LAIR-1 interaction.

Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.

Chondrocyte aggregation in suspension culture is GFOGER-GPP- and beta1 integrin-dependent.

Isolated chondrocytes form aggregates in suspension culture that maintain chondrocyte phenotype in a physiological pericellular environment. The molecular mechanisms involved in chondrocyte aggregation have not been previously identified. Using this novel suspension culture system, we performed mRNA and protein expression analysis along with immunohistochemistry for potential cell adhesion molecules and extracellular matrix integrin ligands. Inhibition of aggregation assays were performed using specific blocking agents. We found that: (i) direct cell-cell interactions were not involved in chondrocyte aggregation, (ii) chondrocytes in aggregates were surrounded by a matrix rich in collagen II and cartilage oligomeric protein (COMP), (iii) aggregation depends on a beta1-integrin, which binds a triple helical GFOGER sequence found in collagens, (iv) integrin alpha10-subunit is the most highly expressed alpha-subunit among those tested, including alpha5, in aggregating chondrocytes. Taken together, this body of evidence suggests that the main molecular interaction involved in aggregation of phenotypically stable chondrocytes is the alpha10beta1-collagen II interaction.

The soluble leukocyte-associated Ig-like receptor (LAIR)-2 antagonizes the collagen/LAIR-1 inhibitory immune interaction.

Leukocyte-associated Ig-like receptor (LAIR)-1 is a collagen-receptor that inhibits immune cell function upon collagen binding. Next to LAIR-1, the human genome encodes LAIR-2, a putative soluble homolog. In this study we show, for the first time, that the LAIR-2 gene is broadly transcribed in human PBMC, mirroring the expression profile of LAIR-1. LAIR-2 protein is expressed as a soluble receptor exhibiting high affinity for various collagen molecules to which it binds in a hydroxyproline-dependent manner. In vitro stimulation of PBMC induces secretion of LAIR-2. We detect high amounts of LAIR-2 in urine of pregnant women, indicating that the soluble receptor is indeed produced in vivo and can be cleared from the body via urine. Furthermore, LAIR-2 levels are increased in synovial fluid of patients with rheumatoid arthritis as compared with osteoarthritis patients. We hypothesize that soluble LAIR-2 may function as a natural competitor for LAIR-1, thereby regulating its inhibitory potential. Indeed, LAIR-2 prevents binding of human LAIR-1 to collagens and LAIR-1 cross-linking in vitro, suggesting that the protein has an immunoregulatory function in vivo. Hence, we reveal a novel mechanism of immune regulation by a soluble LAIR receptor regulating the inhibitory potential of the membrane-bound LAIR-1 via competition for ligands.

Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens.

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix alphaA of the extracellular calcium-binding domain of SPARC and is partially masked by helix alphaC. Previously, we found that the removal of helix alphaC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site approximately 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven alpha2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among alpha chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.

Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions.

Fibrillar collagens provide the most fundamental platform in the vertebrate organism for the attachment of cells and matrix molecules. We have identified specific sites in collagens to which cells can attach, either directly or through protein intermediaries. Using Toolkits of triple-helical peptides, each peptide comprising 27 residues of collagen primary sequence and overlapping with its neighbours by nine amino acids, we have mapped the binding of receptors and other proteins on to collagens II or III. Integrin alpha2beta1 binds to several GXX'GER motifs within the collagens, the affinities of which differ sufficiently to control cell adhesion and migration independently of the cellular regulation of the integrin. The platelet receptor, Gp (glycoprotein) VI binds well to GPO (where O is hydroxyproline)-containing model peptides, but to very few Toolkit peptides, suggesting that sequence in addition to GPO triplets is important in defining GpVI binding. The Toolkits have been applied to the plasma protein vWF (von Willebrand factor), which binds to only a single sequence, identified by truncation and amino acid substitution within Toolkit peptides, as GXRGQOGVMGFO in collagens II and III. Intriguingly, the receptor tyrosine kinase, DDR2 (discoidin domain receptor 2) recognizes three sites in collagen II, including its vWF-binding site, although the amino acids that support the interaction differ slightly within this motif. Furthermore, the secreted protein BM-40 (basement membrane protein 40) also binds well to this same region. Thus the availability of extracellular collagen-binding proteins may be important in regulating and facilitating direct collagen-receptor interaction.

Structural requirements for heparin/heparan sulfate binding to type V collagen.

Collagen-proteoglycan interactions participate in the regulation of matrix assembly and in cell-matrix interactions. We reported previously that a fragment (Ile824-Pro950) of the collagen alpha1(V) chain, HepV, binds to heparin via a cluster of three major basic residues, Arg912, Arg918, and Arg921, and two additional residues, Lys905 and Arg909 (Delacoux, F., Fichard, A., Cogne, S., Garrone, R., and Ruggiero, F. (2000) J. Biol. Chem. 275, 29377-29382). Here, we further characterized the binding of HepV and collagen V to heparin and heparan sulfate by surface plasmon resonance assays. HepV bound to heparin and heparan sulfate with a similar affinity (KD approximately 18 and 36 nM, respectively) in a cation-dependent manner, and 2-O-sulfation of heparin was shown to be crucial for the binding. An octasaccharide of heparin and a decasaccharide of heparan sulfate were required for HepV binding. Studies with HepV mutants showed that the same basic residues were involved in the binding to heparin, to heparan sulfate, and to the cell surface. The contribution of Lys905 and Arg909 was found to be significant. The triple-helical peptide GPC(GPP)5G904-R918(GPP)5GPC-NH2 and native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.

Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III.

A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN). Two mutant alpha(2) I-domains were tested: the inactive T221A mutant, which recognized no peptides, and the constitutively active E318W mutant, which bound a larger subset of peptides. Adhesion of activated human platelets to GER-containing peptides was greater than that of resting platelets, and HT1080 cells bound well to more of the peptides compared with platelets. Binding of cells and recombinant proteins was abolished by anti-alpha(2) monoclonal antibody 6F1 and by chelation of Mg(2+). We describe two novel high affinity integrin-binding motifs in human collagen III (GROGER and GLOGEN) and a third motif (GLKGEN) that displays intermediate activity. Each motif was verified using shorter synthetic peptides.

A single high-affinity binding site for von Willebrand factor in collagen III, identified using synthetic triple-helical peptides.

The essential event in platelet adhesion to the injured blood vessel wall is the binding to subendothelial collagen of plasma von Willebrand factor (VWF), a protein that interacts transiently with platelet glycoprotein Ibalpha (GPIbalpha), slowing circulating platelets to facilitate firm adhesion through collagen receptors, including integrin alpha2beta1 and GpVI. To locate the site in collagen that binds VWF, we synthesized 57 overlapping triple-helical peptides comprising the whole triple-helical domain of collagen III. Peptide no. 23 alone bound VWF, with similar affinity to that of native collagen III. Immobilized peptide no. 23 supported platelet adhesion under static and flow conditions, processes blocked by an antibody that prevents collagen from binding the VWF A3 domain. Truncated and alanine-substituted peptides derived from no. 23 either strongly interacted with both VWF and platelets or lacked both VWF and platelet binding. Thus, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF-binding sequence in collagen III.