Short arm region of laminin-5 gamma2 chain: structure, mechanism of processing and binding to heparin and proteins.

Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4-6, that flank a globular L4m module containing the cleavage site. Recombinant analysis of L4m, which includes an additional imperfect LE module essential for proper folding, demonstrated an unusual pattern of disulfide bonding. These connectivities prevented the release of gamma2LE1-3L4 m after BMP-1 cleavage which required in addition disulfide reshuffling by isomerases. The liberated segment bound through its L4 m module to heparin, nidogen-1, fibulin-1 and fibulin-2. A further heparin/sulfatide-binding site could be attributed to some arginine residues in module LE1. The gamma2LE4-6 segment remaining in processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo.

Recombinant domains of mouse nidogen-1 and their binding to basement membrane proteins and monoclonal antibodies.

The basement membrane protein, nidogen-1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin-1.

Mapping of binding sites for nidogens, fibulin-2, fibronectin and heparin to different IG modules of perlecan.

Perlecan, a major basement membrane proteoglycan, has a complex modular structure designed for the binding of many cellular and extracellular ligands. Its domain IV, which consists of a tandem of immunoglobulin-like modules (IG2-IG15), is rich in such binding sites, which have been mapped to different modules obtained by recombinant production. Heparin/sulfatide binding was restricted to IG5 and shown to depend on four arginine residues that are close in space in beta strands B and E of the C-type IG fold. The nidogen-1 and nidogen-2 isoforms bind to IG3 with high affinity (K(d) approximately 10 nM). This interaction depends on the globular nidogen domain G2 and is crucial for the formation of ternary complexes with laminins. Two loops of IG3 located between beta strands B/C and F/G, which are spatially close, make a major contribution to binding. Fibronectin binding was localized to IG4-5 and fibulin-2 binds to IG2 and IG13-15 with different affinities. This implicates a complex cluster of heterotypic interaction sites apparently important for the supramolecular organization of perlecan in tissues.

Perlustrin, a Haliotis laevigata (abalone) nacre protein, is homologous to the insulin-like growth factor binding protein N-terminal module of vertebrates.

The 84-amino-acid-long sequence of perlustrin showed homology of the abalone nacre protein to the N-terminal domain of mammalian insulin-like growth factor binding proteins (IGFBPs). Despite the evolutionary distance between mollusks and mammals, the sequence identity was 40% including 12 conserved cysteines. However, the residues which were suggested recently to bind IGF-II in a complex with IGFBP-5 were conserved only partially. Nevertheless, perlustrin bound human IGFs with K(D) approximately 10(-7) M. This was the same affinity range as measured before for the interaction of isolated IGFBP-5 N-terminal domains with IGFs. Moreover, perlustrin bound bovine insulin with only approximately two- to sevenfold lower affinity than IGFs. Sequence similarity and growth factor binding identified perlustrin unequivocally as a member of the IGFBP family, the first found in an invertebrate biomineral. Nacre is known to contain proteinaceous factors which promote bone formation in vitro and in vivo. Bone contains IGFBPs which influence bone metabolism in many ways by modulating either IGF effects or IGF independently. Thus, perlustrin may provide a first clue at the molecular level to what these two phylogenetically rather distant biomineralization systems have in common.

Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1.

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.

Structural and functional analysis of the recombinant G domain of the laminin alpha4 chain and its proteolytic processing in tissues.

The C-terminal G domains of laminin alpha chains have been implicated in various cellular and other interactions. The G domain of the alpha4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules, alpha4LG1-3 and alpha4LG4-5. The recombinant fragments were shown to fold into globular structures and could be distinguished by specific antibodies. Both fragments were able to bind to heparin, sulfatides, and the microfibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for cell adhesion and had only a low affinity for the alpha-dystroglycan receptor when compared with the G domains of the laminin alpha1 and alpha2 chains. Yet antibodies to alpha4LG1-3 but not to alpha4LG4-5 clearly inhibited alpha(6)beta(1) integrin-mediated cell adhesion to laminin-8, indicating the participation of alpha4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic processing within a link region between the alpha4LG3 and alpha4LG4 modules was shown to occur during recombinant production and in endothelial and Schwann cell culture. Cleavage could be attributed to three different peptide bonds and is accompanied by the release of the alpha4LG4-5 segment. Immunohistology demonstrated abundant staining of alpha4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for alpha4LG4-5, indicating their loss from tissues. Immunogold staining demonstrated an association of the alpha4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin alpha2 chains appear primarily associated with various basement membranes.

Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V.

The C-terminal perlecan domain V of about 90 kDa consists of laminin-type G domain modules (LG) (25 kDa) and epidermal growth factor-like modules (EG) (4 kDa) in the tandem arrangement LG1-EG1-EG2-LG2-EG3-EG4-LG3. Several shorter fragments have been prepared by recombinant production in mammalian cells and used to map the single glycosaminoglycan (GAG) substitution site and the binding of several carbohydrate and protein ligands. This identified a Ser3511 residue located in a short link region between EG4 and LG3 as being involved in GAG attachment. Electron microscopy provided evidence that the same substitution exists in tissue forms of perlecan. Heparan sulphate attached to this site was shown to bind to the alpha1LG4 module of laminin-1, indicating a role in basement membrane assembly and cell-matrix interactions. This site is also close to an Asn-Asp bond which is readily cleaved by an endogenous protease that depends on the presence of Asp and the LG2 module. A weak heparin binding site was shown to include the EG2 module, which contains five basic residues. Binding to sulphatides and the alpha-dystroglycan receptor was much stronger and required at least two LG modules. However, single LG modules appear to be sufficient for the interaction with the laminin-nidogen complex, while EG3-4 and some flanking regions are apparently involved in fibulin-2 binding. These observations indicate that a complex modular structure is required for domain V in order to provide a rich repertoire of potential biological functions.

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.

Recombinant domain IV of perlecan binds to nidogens, laminin-nidogen complex, fibronectin, fibulin-2 and heparin.

Domain IV of mouse perlecan, which consists of 14 immunoglobulin superfamily (IG) modules, was prepared from recombinant human cell culture medium in the form of two fragments, IV-1 (IG2-9, 100 kDa) and IV-2 (IG10-15, 66 kDa). Both fragments bound to a heparin column, being eluted at ionic strengths either below (IV-2) or above (IV-1) physiological level, and could thus be readily purified. Electron microscopy demonstrated an elongated shape (20-25 nm), and folding into a native structure was indicated by immunological assay and CD spectroscopy. Solid-phase and surface plasmon resonance assays demonstrated strong binding of fragment IV-1 to fibronectin, nidogen-1, nidogen-2 and the laminin-1-nidogen-1 complex, with Kd values in the range 4-17 nM. The latter binding apparently occurs through nidogen-1, as shown by the formation of ternary complexes. Only moderate binding was observed for fibulin-2 and collagen IV and none for fibulin-1 and BM-40. Fragment IV-2 showed a more restricted pattern of binding, with only weaker binding to fibronectin and fibulin-2. None of these activities could be demonstrated for recombinant fragments corresponding to the N-terminal perlecan domains I to III. This indicates a special role for domain IV in the integration of perlecan into basement membranes and other extracellular structures via protein-protein interactions.

Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.

The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly.

Nidogen-2: a new basement membrane protein with diverse binding properties.

Human nidogen-2 was cloned and sequenced (1375 residues) and found to share 46% sequence identity and a similar domain arrangement with the previously characterized basement membrane protein nidogen-1. Recombinant nidogen-2 was purified as a 200 kDa protein from transfected mammalian cell medium, showed a high level of N and O-glycosylation, and could be clearly distinguished from nidogen-1 (150 kDa) by specific antibodies. Electron microscopy demonstrated that the two isoforms have a similar shape, consisting of three globular domains connected by two threads, but differ somewhat in length. Northern blots and immunological assays demonstrated co-expression of the nidogens in various tissues and cultured cells. Immunofluoresence revealed colocalization in vessel walls and other basement membrane zones but some differences in heart and skeletal muscle. Nidogen-2 interacted with collagens I and IV, and perlecan at a comparable level to nidogen-1 but failed to bind to fibulins. Nidogen-2 bound to laminin-1, but only moderately to the epitope on the laminin gamma1 chain, which promotes high-affinity binding of nidogen-1. Both nidogens were cell-adhesive for a restricted number of cell lines, with nidogen-2 having a higher activity. Together, these data suggest that nidogen-2 can compensate for some but not all functional activities ascribed to nidogen-1.

The N-terminal globular domain of the laminin alpha1 chain binds to alpha1beta1 and alpha2beta1 integrins and to the heparan sulfate-containing domains of perlecan.

The N-terminal domains VI plus V (62 kDa) and V alone (43 kDa) of the laminin alpha1 chain were obtained as recombinant products and shown to be folded into a native form by electron microscopy and immunological assays. Domain VI alone, which corresponds to an LN module, did not represent an autonomously folding unit in mammalian cells, however. Fragment alpha1VI/V, but not fragment alpha1V, bound to purified alpha1beta1 and alpha2beta1 integrins, to heparin, and to heparan sulfate-substituted domains I and V of perlecan. This localized the binding activities to the LN module, which contains two basic sequences suitable for heparin interactions.

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.

Mapping of the binding of platelet-derived growth factor to distinct domains of the basement membrane proteins BM-40 and perlecan and distinction from the BM-40 collagen-binding epitope.

A surface plasmon resonance assay was used to analyze the binding of platelet-derived growth factor (PDGF)-AA and PDGF-BB to various proteins of the extracellular matrix. This identified several collagen types; laminin-1, nidogen, perlecan and BM-40 as potential ligands for PDGF with Kd values in the range 2-3200 nM. Perlecan and BM-40 were used to examine the domain specificity and other parameters of the interactions. Recombinant human and mouse BM-40 were shown to bind both PDGFs in a similar manner with a Kd of about 5-10 nM. Studies with deletion mutants of human BM-40 demonstrated binding to its C-terminal extracellular calcium-binding (EC) module, yet the interaction did not require calcium. This distinguishes this from the binding of the EC module to various collagen types, which is strictly calcium dependent. Furthermore, deletion of helix alphaC or two point mutations in helix alphaA of the EC module either enhanced or abolished binding to collagen IV. Since these mutations had no effects on binding to PDGF, it demonstrated the presence of two different binding epitopes. Binding of PDGF-BB to the perlecan core protein could be mapped to its domain III-2 (Kd = 8 nM) with lower affinities shown for domains I, IV-1 and V (Kd = 34-64 nM). Other perlecan domains (II, III-1, III-3, IV-2) were inactive. PDGF-AA was also shown to bind domain III-2 but not III-1. Neither nidogen, BM-40 or perlecan domain III-2 interfered with the binding of PDGF to its alpha and beta receptors, however, suggesting that these interactions may be mainly used for storage of PDGF in the extracellular matrix.

Crystal structure and mapping by site-directed mutagenesis of the collagen-binding epitope of an activated form of BM-40/SPARC/osteonectin.

The extracellular calcium-binding domain (positions 138-286) of the matrix protein BM-40 possesses a binding epitope of moderate affinity for several collagen types. This epitope was predicted to reside in helix alphaA and to be partially masked by helix alphaC. Here we show that deletion of helix alphaC produces a 10-fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix. The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 A resolution. This constitutively activated mutant was used to map the collagen-binding site following alanine mutagenesis at 13 positions. Five residues were crucial for binding, R149 and N156 in helix alphaA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM-40. These residues are spatially close and form a flat ring of 15 A diameter which matches the diameter of a triple-helical collagen domain. The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens. Selected mutations in the non-activated mutant DeltaI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM-40.

Mapping of a defined neurocan binding site to distinct domains of tenascin-C.

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a KD value of 17 nM for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.

Binding of fibulin-1 to nidogen depends on its C-terminal globular domain and a specific array of calcium-binding epidermal growth factor-like (EG) modules.

The calcium-binding basement membrane protein fibulin-1C was shown to bind nidogen in a calcium-dependent fashion. Fibulin-1C consists of small N (domain 1) and C-terminal (domain III) globular structures connected by a central rod (domain II) composed of nine epidermal growth factor (EG) modules, eight of which possess a consensus sequence for calcium binding. Several point and deletion mutants and chimeric protein constructs were used to define the nidogen binding epitope of fibulin-1C by surface plasmon resonance and solid phase assays. All recombinant products were obtained from transfected kidney cells in a folded form as shown by CD spectroscopy, electron microscopy and proteolysis. They were used to demonstrate that calcium-binding is essentially due to the EG modules possessing the consensus binding sequence. Deletion of domain III caused a 30-fold reduction in nidogen binding, whereas deletion of domain I had no effect, yet domain III alone was also inactive. Successive deletions of two to seven EG modules of domain II also caused partial of complete inactivation of binding depending on how many were deleted or their position relative to domain III. Site-directed mutagenesis within the calcium binding consensus sequences demonstrated a similar dependence. Replacement of seven of the calcium-binding modules by a similar tandem array from a related protein showed a distinct (fibulin-2) to almost complete loss of binding (fibrillin-1). This indicates a complex epitope structure involving domains II and III, which each may provide binding epitopes or stabilize each other.

Structural and functional characterization of the extracellular calcium-binding protein BM-40/secreted protein, acidic, rich in cysteine/osteonectin from the nematode Caenorhabditis elegans.

Caenorhabditis elegans BM-40 (positions 19-264) and its extracellular calcium-binding domain (positions 139-264) were obtained in recombinant form from human kidney cells using an episomal expression vector. The purified proteins showed single bands of 33 kDa [BM-40-(19-264)-peptide] or 14 kDa [BM-40-(139-264)-peptide] on electrophoresis, contained internal disulfide bonds and a helices and were relatively resistant to matrix metalloproteinases. Hexosamine analysis indicated substitution by one N-linked and two O-linked oligosaccharides and recombinant BM-40 was indistinguishable in its immunological epitopes from nematode tissue-derived BM-40, suggesting that it was obtained in native form. Both recombinant C. elegans proteins showed a distinct binding activity for human collagens I and IV in solid-phase and surface-plasmon-resonance assays with an affinity (Kd = 1-2 microM), comparable to that of mammalian BM-40. However, calcium-binding studies revealed only a low-affinity site (Kd = 6.2 mM) and failed to show the characteristic conformational change upon addition of EDTA. These and a few other differences are apparently due to two extra disulfide bonds and two deletions/insertions in C. elegans BM-40 and can be partly interpreted from the X-ray structure of a large part of human BM-40. The immunological assays available and the predictions of the location of the collagen-binding epitope should facilitate a molecular and genetic approach to understand the function of BM-40 in the development of C. elegans.

Dimer model for the microfibrillar protein fibulin-2 and identification of the connecting disulfide bridge.

Fibulin-2 is a novel extracellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrillin. The entire protein and its predicted domains were obtained as recombinant products and examined by ultracentrifugation and electron microscopy. This demonstrated a disulfide-linked homodimer of 175 kDa subunits. Partial reduction to monomers identified specifically an odd Cys574 residue responsible for dimer formation in one of three anaphylatoxin-like modules that constitute the central globular domain I (13 kDa) of fibulin-2. Furthermore, a Cys574-Ser mutation abolished disulfide connection but not non-covalent dimerization of fibulin-2. The C-terminal region (85 kDa) was shown to represent a 35-nm-long rod consisting of 11 calcium-binding EGF-like modules (domain II) and a small terminal globe (domain III). The unique N-terminal domain N (55 kDa) was also rod-shaped (approximately 38 nm) and rich in galactosamine indicating extensive O-glycosylation. A dimer model is proposed indicating mainly a rod-like shape of 80 nm length based on an anti-parallel association of two subunits through their domains I. This model also implies alignment of domains II and N between different subunits. This was demonstrated by surface plasmon resonance assay which showed a distinct interaction between domains N and II with a Kd of approximately 0.7 microM.

Recombinant and tissue-derived mouse BM-40 bind to several collagen types and have increased affinities after proteolytic activation.

The calcium-binding extracellular matrix protein BM-40 was obtained as a mouse cDNA product from a stably transfected kidney cell clone. Electrophoresis and N-terminal sequence analysis demonstrated absence of the proteolytic processing previously observed for a mouse tumour-derived BM-40. Yet the two forms of BM-40 were very similar in their CD spectra, their calcium-dependent change in alpha helix content and their immunological epitopes. In surface plasmon resonance assays, recombinant mouse BM-40 showed distinct binding to the triple-helical domains of collagens I, II, III, IV and V with Kd = 1-4 microM but no binding to collagen VI. These interactions were abolished in the presence of EDTA. Tissue-derived mouse BM-40, however, bound collagens I and IV with Kd = 0.1-0.2 microM. Activation of collagen binding to give a similar Kd could be achieved for recombinant mouse BM-40 by treatment with the matrix metalloproteinase collagenase-3. The major cleavage site was located in helix C of the extracellular calcium-binding module of BM-40 and other less prominent cleavages occurred close to the N-terminus. The sensitive helix C site was just one residue away from that sensitive to endogenous tissue proteolysis, suggesting that cleavage could be a physiological mechanism to modulate collagen binding.