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.     

Heparin type IV collagen interactions: equilibrium binding and inhibition of type IV collagen self-assembly

Interactions between type IV collagen and heparin were examined under equilibrium conditions with rotary shadowing, solid-phase binding assays, and affinity chromatography. With the technique of rotary shadowing and electron microscopy, heparin appeared as thin, short strands and bound to the following three sites: the NC1 domain, and in the helix, at 100 and 300 nm from the NC1 domain. By solid-phase binding assays the binding of [3H]heparin in solution to type IV collagen immobilized on a solid surface was found to be specific, since it was saturable and could be displaced by an excess of unlabeled heparin. Scatchard analysis indicated three classes of binding sites for heparin-type IV collagen interactions with dissociation constants of 3, 30, and 100 nM, respectively. Furthermore, by the solid-phase binding assays, the binding of tritiated heparin could be competed almost to the same extent by unlabeled heparin and chondroitin sulfate side chains. This finding indicates that chondroitin sulfate should also bind to type IV collagen. By affinity chromatography, [3H]heparin bound to a type IV collagen affinity column and was eluted with a linear salt gradient, with a profile exhibiting three distinct peaks at 0.18, 0.22, and 0.24 M KCl, respectively. This suggested that heparin-type IV collagen binding was of an electrostatic nature. Finally, the effect of the binding of heparin to type IV collagen on the process of self-assembly of this basement membrane glycoprotein was studied by turbidimetry and rotary shadowing. In turbidity experiments, the presence of heparin, even in small concentrations, drastically reduced maximal aggregation of type IV collagen which was prewarmed to 37 degrees C. By using the morphological approach of rotary shadowing, lateral associations and network formation by prewarmed type IV collagen were inhibited in the presence of heparin. Thus, the binding of heparin resulted in hindrance of assembly of type IV collagen, a process previously described for interactions between various glycosaminoglycans and interstitial collagens. Such regulation may influence the assembly of basement membranes and possibly modify functions. Furthermore, qualitative and quantitative changes of proteoglycans which occur in certain pathological conditions, such as diabetes mellitus, may alter molecular assembly and possibly permeability functions of several basement membranes.     

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.     

Interaction of proteoglycans with the pericellular (1 alpha, 2 alpha, 3 alpha) collagens of cartilage

Interaction between cartilage proteoglycan and the collagen(s) composed of 1 alpha, 2 alpha, and 3 alpha chains was studied in vitro. Most of the collagen was insoluble under the conditions of assay (0.15 M NaCl, 0.008 M phosphate buffer, pH 7.4; 4 degrees C) and was in the form of fibrils 20 nm in diameter or thinner. The larger fibrils had 60-70 nm periodicity, characteristic of native collagens. Proteoglycan monomers which had been labeled by incubating cartilage slices in vitro with Na2 35SO4 were used to assay the interaction. The insoluble collagen fraction bound proteoglycan from solution. At proteoglycan:collagen ratios lower than 1:2, binding was rapid and linear, and the dissociation constant was 1.7 X 10(-9) M. At higher proteoglycan:collagen ratios, more proteoglycan was bound, but at a slower rate. Binding of proteoglycan to collagen did not require fibrils, since soluble 1 alpha, 2 alpha, and 3 alpha containing collagen also bound to proteoglycan and formed an insoluble complex. Denatured collagens did not bind proteoglycan or compete for binding with normal collagen. Optimum binding occurred with intact proteoglycan, but proteoglycan which had been treated with protease was also bound at low levels. Both protease-treated proteoglycan and free chondroitin sulfate competed with intact proteoglycan in the binding assays, but neither chondroitinase ABC-treated proteoglycan nor the oligosaccharides produced by digestion of chondroitin sulfate with testicular hyaluronidase altered the binding of proteoglycan to collagen. Hyaluronic acid did not compete with radioactive proteoglycan, but heparin and dextran sulfate were extremely effective inhibitors of binding. These data suggest a relatively nonspecific interaction between sulfated polyanions and 1 alpha, 2 alpha, and 3 alpha containing collagens. However, given the location of these collagens near the chondrocyte surface, the interaction of fibrillar 1 alpha, 2 alpha, 3 alpha collagen with proteoglycan is likely to occur and to be of biological importance.     

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.     

Primary structure of the heparin-binding site of type V collagen

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.     

Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV

The organizational relationship between the recently identified alpha 3 chain of basement membrane collagen (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B.G. (1987) J. Biol. Chem. 262, 7874-7877) and collagen IV was determined. This was accomplished by the identification of subunits in hexamers of the NC1 domain of collagen IV that were immunoprecipitated with antibodies prepared against subunits M1, corresponding to alpha 1(IV)NC1 and alpha 2(IV)NC1, and M2, corresponding to alpha 3NC1, and by amino acid sequence analysis. The presence of at least two distinct types of hexamers was revealed, one enriched in M1 and the other enriched in M2, but in both types, M1 and M2 coexist. Evidence was also obtained for the existence of heterodimers comprised of M1 and M2. These results indicate that M2 is an integral component of the NC1 hexamer of collagen IV. The amino acid sequence of the NH2-terminal region of M2 was found to be highly related to the collagenous-NC1 junctional region of the alpha 1 chain of collagen IV. Therefore, M2 is designated alpha 3(IV)NC1 and its parent chain alpha 3(IV). These findings lead to a new concept about the structure of collagen IV: namely, 1) collagen IV is comprised of a third chain (alpha 3) together with the two classical ones (alpha 1 and alpha 2); the alpha 3(IV) chain exists within the same triple-helical molecule together with the alpha 1(IV) and alpha 2(IV) chains and/or within a separate triple-helical molecule, exclusive of alpha 1(IV) and alpha 2(IV) chains, but connected through the NC1 domains to the classical triple-helical molecule comprised of alpha 1(IV) and alpha 2(IV) chains. Additionally, a portion of those triple-helical molecules exclusive of alpha 1(IV) and alpha 2(IV) chains may be connected to each other through their NC1 domains; and 3) the epitope to which the major reactivity of autoantibodies are targeted in glomerular basement membrane in patients with Goodpasture syndrome is localized to the NC1 domain of the alpha 3(IV) chain.     

Quaternary organization of the goodpasture autoantigen,the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains

Goodpasture's (GP) disease is caused by autoantibodies that target the alpha3(IV) collagen chain in the glomerular basement membrane (GBM). Goodpasture autoantibodies bind two conformational epitopes (E(A) and E(B)) located within the non-collagenous (NC1) domain of this chain, which are sequestered within the NC1 hexamer of the type IV collagen network containing the alpha3(IV), alpha4(IV), and alpha5(IV) chains. In this study, the quaternary organization of these chains and the molecular basis for the sequestration of the epitopes were investigated. This was accomplished by physicochemical and immunochemical characterization of the NC1 hexamers using chain-specific antibodies. The hexamers were found to have a molecular composition of (alpha3)(2)(alpha4)(2)(alpha5)(2) and to contain cross-linked alpha3-alpha5 heterodimers and alpha4-alpha4 homodimers. Together with association studies of individual NC1 domains, these findings indicate that the alpha3, alpha4, and alpha5 chains occur together in the same triple-helical protomer. In the GBM, this protomer dimerizes through NC1-NC1 domain interactions such that the alpha3, alpha4, and alpha5 chains of one protomer connect with the alpha5, alpha4, and alpha3 chains of the opposite protomer, respectively. The immunodominant Goodpasture autoepitope, located within the E(A) region, is sequestered within the alpha3alpha4alpha5 protomer near the triple-helical junction, at the interface between the alpha3NC1 and alpha5NC1 domains, whereas the E(B) epitope is sequestered at the interface between the alpha3NC1 and alpha4NC1 domains. The results also reveal the network distribution of the six chains of collagen IV in the renal glomerulus and provide a molecular explanation for the absence of the alpha3, alpha4, alpha5, and alpha6 chains in Alport syndrome.     

Identification of noncollagenous sites encoding specific interactions and quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen: implications for Alport gene therapy

Defective assembly of alpha 3 alpha 4 alpha 5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha1/alpha 5 chimeras containing alpha 5 NC1 residues 188-227 replicated the ability of alpha 5 NC1 to bind to alpha3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5 NC1 residues 188-227 by alpha1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha3NC1 encoded binding to alpha 5 NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5 NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5 NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5 NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients.     

The alpha 4(IV) chain of basement membrane collagen. Isolation of cDNAs encoding bovine alpha 4(IV) and comparison with other type IV collagens.

Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.     

Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.     

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.     

Complete primary structure of the triple-helical region and the carboxyl-terminal domain of a new type IV collagen chain, alpha 5(IV)

We have isolated and characterized overlapping cDNA clones which code for a previously unidentified human collagen chain. Although the cDNA-derived primary structure of this new polypeptide is very similar to the basement membrane collagen alpha 1(IV) and alpha 2(IV) chains, the carboxyl-terminal collagenous/non-collagenous junction sequence does not correspond to the junction sequence in either of the newly described alpha 3(IV) or alpha 4(IV) chains (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B. G. (1987) J. Biol. Chem. 262, 7874-7877). Thus the protein presented here has been designated the alpha 5 chain of type IV collagen. Four clones encode an open reading frame of 1602 amino acids that cover about 95% of the entire chain including half of the amino-terminal 7S domain and all of the central triple-helical region and carboxyl-terminal NC1 domain. The collagenous region of the alpha 5(IV) chain contains 22 interruptions which are in most cases identical in distribution to those in both the alpha 1(IV) and alpha 2(IV) chains. Despite the relatively low degree of conservation among the amino acids in the triple-helical region of the three type IV collagen chains, analysis of the sequences clearly showed that alpha 5(IV) is more related to alpha 1(IV) than to alpha 2(IV). This similarity between the alpha 5(IV) and alpha 1(IV) chains is particularly evident in the NC1 domains where the two polypeptides are 83% identical in contrast to the alpha 5(IV) and alpha 2(IV) identity of 63%. In addition to greatly increasing the complexity of basement membranes, the alpha 5 chain of type IV collagen may be responsible for specialized functions of some of these extracellular matrices. In this regard, it is important to note that we have recently assigned the alpha 5(IV) gene to the region of the X chromosome containing the locus for a familial type of hereditary nephritis known as Alport syndrome (Myers, J.C., Jones, T.A., Pohjalainen, E.-R., Kadri, A.S., Goddard, A.D., Sheer, D., Solomon, E., and Pihlajaniemi, T. (1990) Am. J. Hum. Genet. 46, 1024-1033). Consequently, the newly discovered alpha 5(IV) collagen chain may have a critical role in inherited diseases of connective tissue.     

The Heparin-Binding Activity of Secreted Modular Calcium-Binding Protein 1 (SMOC-1) Modulates Its Cell Adhesion Properties

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.     

Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice

Type IV collagen includes six genetically distinct polypeptides named alpha1(IV) through alpha6(IV). These isoforms are speculated to organize themselves into unique networks providing mammalian basement membranes specificity and inequality. Recent studies using bovine and human glomerular and testis basement membranes have shown that unique networks of collagen comprising either alpha1 and alpha2 chains or alpha3, alpha4, and alpha5 chains can be identified. These studies have suggested that assembly of alpha5 chain into type IV collagen network is dependent on alpha3 expression where both chains are normally present in the tissue. In the present study, we show that in the lens and inner ear of normal mice, expression of alpha1, alpha2, alpha3, alpha4, and alpha5 chains of type IV collagen can be detected using alpha chain-specific antibodies. In the alpha3(IV) collagen-deficient mice, only the expression of alpha1, alpha2, and alpha5 chains of type IV collagen was detectable. The non-collagenous 1 domain of alpha5 chain was associated with alpha1 in the non-collagenous 1 domain hexamer structure, suggesting that network incorporation of alpha5 is possible in the absence of the alpha3 chain in these tissues. The present study proves that expression of alpha5 is not dependent on the expression of alpha3 chain in these tissues and that alpha5 chain can assemble into basement membranes in the absence of alpha3 chain. These findings support the notion that type IV collagen assembly may be regulated by tissue-specific factors.     

The goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.     

The complete primary structure of mouse alpha 2(IV) collagen. Alignment with mouse alpha 1(IV) collagen

We have determined the nucleotide and amino acid sequences of mouse alpha 2(IV) collagen which is 1707 amino acids long. The primary structure includes a putative 28-residue signal peptide and contains three distinct domains: 1) the 7 S domain (residues 29-171), which contains 5 cysteine and 8 lysine residues, is involved in the cross-linking and assembly of four collagen IV molecules; 2) the triple-helical domain (residues 172-1480), which has 24 sequence interruptions in the Gly-X-Y repeat up to 24 residues in length; and 3) the NC1 domain (residues 1481-1707), which is involved in the end-to-end assembly of collagen IV and is the most highly conserved domain of the protein. Alignment of the primary structure of the alpha 2(IV) chain with that of the alpha 1(IV) chain reported in the accompanying paper (Muthukumaran, G., Blumberg, B., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6310-6317) suggests that a heterotrimeric collagen IV molecule contains 26 imperfections in the triple-helical domain. The proposed alignment is consistent with the physical data on the length and flexibility of collagen IV.     

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.     

A novel synthetic peptide from the B1 chain of laminin with heparin- binding and cell adhesion-promoting activities

Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]heparin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.     

The antitumor properties of the alpha3(IV)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the alpha3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to alpha(v)beta(3) integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 alpha3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 alpha3(IV)-(185-203) peptides show a beta-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the beta-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the beta-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of alpha(v)beta(3) integrin in melanoma progression.