The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the alpha 1(IV) and alpha 2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.     

Identification of a multifunctional, cell-binding peptide sequence from the a1(NC1) of type IV collagen

We have previously identified three distinctive amino acid sequences from type IV collagen which specifically bound to heparin and also inhibited the binding of heparin to intact type IV collagen. One of these chemically synthesized domains, peptide Hep-I, has the sequence TAGSCLRKFSTM and originates from the a1(noncollagenous [NC1]) chain of type IV collagen (Koliakos, G. G., K. K. Koliakos, L. T. Furcht, L. A. Reger, and E. C. Tsilibary. 1989. J. Biol. Chem. 264:2313-2323). We describe in this report that this same peptide also bound to intact type IV collagen in solid-phase assays, in a dose-dependent and specific manner. Interactions between peptide Hep-I and type IV collagen in solution resulted in inhibition of the assembly process of this basement membrane glycoprotein. Therefore, peptide Hep-I should represent a major recognition site in type IV collagen when this protein polymerizes to form a network. In addition, solid phase-immobilized peptide Hep-I was able to promote the adhesion and spreading of bovine aortic endothelial cells. When present in solution, peptide Hep-I competed for the binding of these cells to type IV collagen- and NC1 domain-coated substrata in a dose-dependent manner. Furthermore, radiolabeled peptide Hep-I in solution also bound to endothelial cells in a dose-dependent and specific manner. The binding of radiolabeled Hep-I to endothelial cells could be inhibited by an excess of unlabeled peptide. Finally, in the presence of heparin or chondroitin/dermatan sulfate glycosaminoglycan side chains, the binding of endothelial cells to peptide Hep-I and NC1 domain-coated substrates was also inhibited. We conclude that peptide Hep-I should have a number of functions. The role of this type IV collagen-derived sequence in such diverse phenomena as self-association, heparin binding and cell binding and adhesion makes Hep-I a crucial domain involved in the determination of basement membrane ultrastructure and cellular interactions with type IV collagen-containing matrices.     

Use of monoclonal antibodies to locate the chondroitin sulfate chain(s) in type IX collagen

Recent results show that type IX collagen isolated from chicken cartilage is associated with one or perhaps two chondroitin sulfate chains. To locate the chondroitin sulfate chain(s) along the type IX collagen molecule, rotary shadowing was performed in the presence of monoclonal antibodies which recognize stubs of chondroitin sulfate generated after chondroitinase ABC digestion. Monoclonal antibodies 9-A-2 and 2-B-6 which recognize stubs of chondroitin 4-sulfate were found to bind specifically to the NC3 domain of type IX collagen, and this binding was dependent on prior digestion of the preparation with chondroitinase ABC. Monoclonal antibody 1-B-5, which recognizes unsulfated stubs of chondroitin sulfate, did not show any specific binding to type IX collagen either with or without chondroitinase ABC digestion. As a control, monoclonal antibody 2C2 was used, which in previous work was shown to bind specifically to an epitope located close to or at the NC2 domain. Binding of this antibody to NC2 was unaffected by chondroitinase ABC digestion, and no specific binding of the antibody to the NC3 domain was detected either before or after chondroitinase ABC digestion.     

Inhibition of laminin self-assembly and interaction with type IV collagen by antibodies to the terminal domain of the long arm

Laminin is a major glycoprotein of the basement membrane. Although its precise localization and orientation within this structure is unknown, it is presumably anchored to other macromolecules such as type IV collagen or proteoheparan sulfate. In vitro, laminin has the ability to self-assemble and to bind to type IV collagen molecules at distinct sites. To identify more precisely the domains of the complex, cross-shaped laminin molecule that are involved in these interactions, images of laminin-laminin dimers and laminin-type IV collagen complexes obtained by the rotary shadowing method were analyzed. We observed that the complex domain at the end of the long arm of laminin is predominantly involved in these interactions. By using Fab fragments of antibodies specific for a peptide fragment derived from this complex domain, it is shown that laminin self-assembly is inhibited in their presence, as measured by turbidity and by electron microscopy. In addition, these antibodies inhibit the specific interaction of laminin with type IV collagen. These data suggest that the complex domain at the end of the long arm of laminin contains binding sites of potential importance for the assembly of basement membranes.     

The role of the main noncollagenous domain (NC1) in type IV collagen self-assembly

Type IV collagen incubated at elevated temperatures in physiologic buffers self-associates (a) via its carboxy-terminal (NC1) domain, (b) via its amino-terminal (7S) domain, and (c) laterally; and it forms a network. When examined with the technique of rotary shadowing, isolated domain NC1 was found to bind along the length of type IV collagen to four distinct sites located at intervals of approximately 100 nm each. The same 100-nm distance was observed in domain NC1 of intact type IV collagen bound along the length of the collagen molecules during initial steps of network formation and in complete networks. The presence of anti-NC1 Fab fragments in type IV collagen solutions inhibited lateral association and network formation in rotary shadow images. During the process of self-association type IV collagen develops turbidity; addition of isolated domain NC1 inhibited the development of turbidity in a concentration-dependent manner. These findings indicate that domain NC1 of type IV collagen plays an important role in the process of self-association and suggest that alterations in the structure of NC1 may be partially responsible for impaired functions of basement membranes in certain pathological conditions.     

Shape and assembly of type IV procollagen obtained from cell culture.

Type IV procollagen was isolated from the culture medium of the teratocarcinoma cell line PYS-2 by affinity chromatography on heparin-Sepharose. Immunological studies showed that type IV procollagen is composed of pro-alpha 1(IV) and pro-alpha 2(IV) chains and contains two potential cross-linking sites which are located in the short triple-helical 7S domain and the globular domain NC1 . The 7S domain was also identified as the heparin binding site. Rotary shadowing visualized type IV procollagen as a single triple-helical rod (length 388 nm) with a globule at one end. Some of the procollagen in the medium, however, had formed aggregates by alignment of 2-4 molecules along their 7S domains. After deposition in the cell matrix, non-reducible cross-links between the 7S domains are formed while the globules of two procollagen molecules connect to each other. The latter may require a slight proteolytic processing of the globular domains NC1 . The shape of type IV procollagen and the initial steps in its assembly are compatible with a recently proposed network of type IV collagen molecules in basement membranes. Since both type IV collagen and laminin bind to heparin, the formation of higher ordered structures by interaction of both proteins with heparan-sulfate proteoglycan may occur in situ.     

Binding of laminin to type IV collagen: a morphological study

A mixture of laminin and type IV collagen was analyzed by rotary shadowing using carbon/platinum and electron microscopy. Laminin was found to form distinct complexes with type IV collagen: one site of interaction is located 140 nm from the COOH-terminal, noncollagenous (NC1) domain and the other is located within the NH2-terminal region. The isolated NC1 fragment of type IV collagen does not appear to interact with laminin, while pepsin-treated type IV collagen, which lacks the NC1 domain, retains its ability to form complexes with laminin. Analysis of the laminin-type IV complexes indicates that laminin binds to type IV collagen via the globular regions of either of its four arms. This finding is supported by experiments using fragment P1 of laminin which lacks the globular regions and which does not bind to type IV collagen in a specific way. In addition, after heat-denaturation of laminin no specific binding is observed.     

Isolation of a type IV collagen binding protein from human platelets.

In order to study the molecular basis of platelet interaction with collagen IV of the basement membrane separating the arterial endothelium from the underlying subendothelial connective tissue, the possibility of presence of platelet membrane protein with affinity to type IV collagen was examined by subjecting the platelet membrane extract to affinity chromatography on collagen IV-sepharose. Urea (4 M) eluate was found to contain a protein with an apparent mol. wt of 68 kDa. The radioiodinated protein was isolated and used to test its specificity. By dot blot assay on nitrocellulose disks and solid-phase assays, the 68 kDa protein was found to bind with high affinity to collagen IV. Lack of significant binding to fibronectin and laminin when compared to albumin control indicated its high specificity for collagen. The radioiodinated protein was inserted into egg yolk lecithin liposomes. While these liposomes attached to microtitre plates coated with collagen IV, there was no significant binding to fibronectin or laminin coated wells, suggesting the membrane associated character of the protein as well as its specificity for collagen. These results indicate that presence of a 68 kDa protein in platelet membrane which interacts with very high specificity to collagen IV.     

Mapping the heparin-binding sites on type I collagen monomers and fibrils

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin- binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell- directed assembly or restructuring of the collagenous extracellular matrix.     

Structure and binding properties of collagen type XIV isolated from human placenta

Collagen XIV was isolated from neutral salt extracts of human placenta and purified by several chromatographic steps including affinity binding to heparin. The same procedures also led to the purification of a tissue form of fibronectin. Collagen XIV was demonstrated by partial sequence analysis of its Col1 and Col2 domains and by electron microscopy to be a disulphide-linked molecule with a characteristic cross-shape. The individual chains had a size of approximately 210 kD, which was reduced to approximately 180 kD (domain NC3) after treatment with bacterial collagenase. Specific antibodies mainly to NC3 epitopes were obtained by affinity chromatography and used in tissue and cell analyses by immunoblotting and radioimmunoassays. Two sequences from NC3 were identified on fragments obtained after trypsin cleavage. They were identical to cDNA-derived sequences of undulin, a noncollagenous extracellular matrix protein. This suggests that collagen XIV and undulin may be different splice variants from the same gene. Heparin binding was confirmed in ligand assays with a large basement membrane heparan sulphate proteoglycan. This binding could be inhibited by heparin and heparan sulphate but not by chondroitin sulphate. In addition, collagen XIV bound to the triple helical domain of collagen VI. The interactions with heparin sulphate proteoglycan and collagen VI were not shared by the NC3 domain, or by reduced and alkylated collagen XIV. No or only low binding was observed for collagens I-V, pN- collagens I and III, and several noncollagenous matrix proteins, including laminin, recombinant nidogen, BM-40/osteonectin, plasma and tissue fibronectin, vitronectin, and von Willebrand factor. Insignificant activity was also shown in cell attachment assays with nine established cell lines.     

Characterization of type XI collagen-glycosaminoglycan interactions

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen. Pretreatment of chondrocytes with either heparin or heparinase significantly reduced attachment to type XI collagen. Incubation of denatured and cyanogen bromide-cleaved type XI collagen with radiolabeled heparin identified sites of interaction on the alpha1(XI) and alpha2(XI) chains. NH(2)-terminal sequence data confirmed that the predominant heparin-binding peptide contained the sequence GKPGPRGQRGPTGPRGSRGAR from the alpha1(XI) chain. Using rotary shadowing electron microscopy of native type XI collagen molecules and heparin-bovine serum albumin conjugate, an additional binding site was identified at one end of the triple helical region of the collagen molecule. This coincides with consensus heparin binding motifs present at the amino-terminal ends of both the alpha1(XI) and the alpha2(XI) chains. The contribution of glycosaminoglycan-type XI collagen interactions to cartilage matrix stabilization is discussed.     

A network model for the organization of type IV collagen molecules in basement membranes

Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NC1 were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously 7-S collagen domain. The pepsin-solubilized form lacked domain NC1 and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NC1 domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NC1) was estimated to be about 800 nm comprising the length of two molecules.     

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.     

Binding domain for laminin on type IV collagen

Binding of type IV collagen to laminin was studied by attaching one member of the ligand pair to a solid phase. When laminin was bound to a solid phase, type IV collagen exhibited saturable binding. Digestion of type IV collagen with high concentrations of pepsin destroyed the laminin binding activity. Type IV collagen was also found to bind to fibronectin but the binding activity was not destroyed by pepsin treatment. Rotary shadowing electron microscopy of the pepsin digested type IV collagen indicated that the carboxy terminal end region of about 100 nm is cleaved. Rotary shadowing electron microscopy studies demonstrate that the carboxy terminal end of type IV collagen has a major laminin binding site.     

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

The C-terminus type I collagen is a major binding site for heparin

The binding of collagens and fragments of type I collagen to heparin was studied by gel electrophoresis and affinity chromatography. Samples bound in 150 mM NaCl/10 mM Hepes (pH6.5) were eluted with 2 M NaCl, 6 M urea, or a linear gradient of 0.15–1.0 M NaCl. The triple-helical conformation was shown to be essential for binding. The vertebrate collagenase-generated C-terminal fragment, TC^B was shown to have greater binding affinity for heparin than the N-terminal TC^A fragment. Both type II collagen and the NC1 domain of type IV collagen bound to heparin, whereas pepsin-solubilized tetrameric type IV failed to bind.     

Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV

The laminin-nidogen complex and purified nidogen both bind collagen IV but not other collagens, as shown by solid-state ligand-binding and inhibition assays. Laminin purified from the dissociated complex and a variety of laminin proteolytic fragments failed to bind collagen IV. Complexes formed in solution between nidogen or laminin-nidogen and collagen IV were visualized by rotary shadowing which identified one major binding site about 80 nm away from the C-terminus of the collagen triple helix. A second, weaker binding site may exist closer to its N-terminus. Binding sites of nidogen were assigned to its C-terminal globular domain which also possesses laminin-binding structures. A more diverse collagen-IV-binding pattern was observed for the laminin nidogen complex, whereby interactions may involve both nidogen and short-arm structures of laminin.     

Melanoma cell CD44 interaction with the alpha 1(IV)1263-1277 region from basement membrane collagen is modulated by ligand glycosylation

Invasion of the basement membrane is believed to be a critical step in the metastatic process. Melanoma cells have been shown previously to bind distinct triple-helical regions within basement membrane (type IV) collagen. Additionally, tumor cell binding sites within type IV collagen contain glycosylated hydroxylysine residues. In the present study, we have utilized triple-helical models of the type IV collagen alpha1(IV)1263-1277 sequence to (a) determine the melanoma cell receptor for this ligand and (b) analyze the results of single-site glycosylation on melanoma cell recognition. Receptor identification was achieved by a combination of methods, including (a) cell adhesion and spreading assays using triple-helical alpha1(IV)1263-1277 and an Asp(1266)Abu variant, (b) inhibition of cell adhesion and spreading assays, and (c) triple-helical alpha1(IV)1263-1277 affinity chromatography with whole cell lysates and glycosaminoglycans. Triple-helical alpha1(IV)1263-1277 was bound by melanoma cell CD44/chondroitin sulfate proteoglycan receptors and not by the collagen-binding integrins or melanoma-associated proteoglycan. Melanoma cell adhesion to and spreading on the triple-helical alpha1(IV)1263-1277 sequence was then compared for glycosylated (replacement of Lys(1265) with Hyl(O-beta-d-galactopyranosyl)) versus non-glycosylated ligand. Glycosylation was found to strongly modulate both activities, as adhesion and spreading were dramatically decreased due to the presence of galactose. CD44/chondroitin sulfate proteoglycan did not bind to glycosylated alpha1(IV)1263-1277. Overall, this study (a) is the first demonstration of the prophylactic effects of glycosylation on tumor cell interaction with the basement membrane, (b) provides a rare example of an apparent unfavorable interaction between carbohydrates, and (c) suggests that sugars may mask "cryptic sites" accessible to tumor cells with cell surface or secreted glycosidase activities.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

High-affinity binding of the NC1 domain of collagen VII to laminin 5 and collagen IV

Anchoring functions of collagen VII depend on its ability to form homotypic fibrils and to bind to other macromolecules to form heterotypic complexes. Biosensor-based binding assays were employed to analyze the kinetics of the NC1 domain-mediated binding of collagen VII to laminin 5, collagen IV, and collagen I. We showed that collagen VII interacts with laminin 5 and collagen IV with a Kd value of 10(-9) M. In contrast, the NC1-mediated binding to collagen I was weak with a Kd value of 10(-6) M. Binding assays also showed that the NC1 domain utilizes the same region to bind to both laminin 5 and collagen IV. We postulate that the ability of the NC1 domains to bind with high affinities to laminin 5 and collagen IV facilitates stabilization of the structure of the basement membrane itself and that the NC1-collagen I interaction may be less important for stabilization of the dermal-epidermal junction.