The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

Evolution of human von Willebrand factor: cDNA sequence polymorphisms, repeated domains, and relationship to von Willebrand antigen II

Four cDNAs extending into the 5'-noncoding region of the human von Willebrand factor cDNA have been characterized. Thirty-four residues of amino-terminal protein sequence for von Willebrand antigen II matched that predicted from the cDNA sequence, confirming that the propeptide of von Willebrand factor is von Willebrand antigen II. Among the known cDNA sequences there are four confirmed single nucleotide differences, of which two may be in linkage disequilibrium, and two would alter the protein sequence. Based on comparisons among the four repeated D domains, an evolutional model has been proposed to account for the distribution of these sequence elements in prepro-von Willebrand factor.     

The complete primary structure of type XII collagen shows a chimeric molecule with reiterated fibronectin type III motifs, von Willebrand factor A motifs, a domain homologous to a noncollagenous region of type IX collagen, and short collagenous domains with an Arg-Gly-Asp site

Extracellular matrix molecules are generally categorized as collagens, elastin, proteoglycans, or other noncollagenous structural/cell interaction proteins. Many of these extracellular proteins contain distinctive repetitive modules, which can sometimes be found in other proteins. We describe the complete primary structure of an alpha 1 chain of type XII collagen from chick embryonic fibroblasts. This large, structurally chimeric molecule identified by cDNA analysis combines previously unrelated molecular domains into a single large protein 3,124 residues long (approximately 340 kD). The deduced chicken type XII collagen sequence starts at the amino terminus with one unit of the type III motif of fibronectin, which is followed by one unit homologous to the von Willebrand factor A domain, then one more fibronectin type III module, a second A domain from von Willebrand factor, 6 units of type III motif and a third A domain, 10 consecutive units of type III motif and a fourth A domain, a domain homologous to the NC4 domain peptide of type IX collagen, and finally two short collagenous regions previously described as part of the partially sequenced collagen type XII molecule; an Arg-Gly-Asp potential cell adhesive recognition sequence is present in a hydrophilic region at the terminus of one collagenous domain. Antibodies raised to type XII collagen synthesized in a bacterial expression system recognized not only previously reported bands (220 kD et cetera) in tendons, but also bands with apparently different molecular sizes in fibroblasts and 4-d embryos. The antibodies stained a wide variety of extracellular matrices in embryos in patterns distinct from those of fibronectin or interstitial collagens. They prominently stained extracellular matrix associated with certain neuronal tissues, such as axons from dorsal root ganglia and neural tube. These studies identify a novel chimeric type of molecule that contains both adhesion molecule and collagen motifs in one protein. Its structure blurs current classification schemes for extracellular proteins and underscores the potentially large diversity possible in these molecules.     

Mosaic structure of globular domains in the human type VI collagen alpha 3 chain: similarity to von Willebrand factor, fibronectin, actin, salivary proteins and aprotinin type protease inhibitors.

Human collagen alpha 3(VI) chain mRNA (approximately 10 kb) was cloned and shown by sequence analysis to encode a 25 residue signal peptide, a large N-terminal globule (1804 residues), a central triple helical segment (336 residues) and a C-terminal globule (803 residues). Some of the sequence was confirmed by Edman degradation of peptides. The N-terminal globular segment consists of nine consecutive 200 residue repeats (N1 to N9) showing internal homology and also significant identity (17-25%) to the A domains of von Willebrand Factor and similar domains present in some other proteins. Deletions were found in the N3 and N9 domains of several cDNA clones suggesting variation of these structures by alternative splicing. The C-terminal globule starts immediately after the triple helical segment with two domains C1 (184 residues) and C2 (248 residues) being similar to the N domains. They are followed by a proline rich, repetitive segment C3 of 122 residues, with similarity to some salivary proteins, and domain C4 (89 residues), which is similar to the type III repeats present in fibronectin and tenascin. The most C-terminal domain C5 (70 residues) shows 40-50% identity to a variety of serine protease inhibitors of the Kunitz type. The whole sequence contains 29 cysteines which are mainly clustered in short segments connecting domains N1, C1, C2 and the triple helix, and in the inhibitor domain. Five putative Arg-Gly-Asp cell-binding sequences are exclusively localized in the triple helical segment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The complete cDNA sequence of bovine coagulation factor V.

Lack of availability of a primary structure for bovine factor V has hindered detailed analysis of a vast majority of structure-function correlations on this molecule. To determine the primary structure of bovine factor V, we used liver mRNA as a template for the synthesis of three cDNA libraries. The sequences of seven overlapping cDNA clones infer two bovine factor V variants. Variant 1 results in a 6910-basepair (bp) cDNA including 103 bp of 5'-untranslated sequence, 6633 bp of coding sequence and 171 bp of 3'-untranslated sequence with a putative polyadenylation site. Variant 2 differs only in the size of the coding sequence (6618 bp). The open reading frame translates to factor V consisting of 2211 (or 2206) amino acids including a 28-amino acid signal peptide. Comparison of the amino acid sequences with human factor Va reveals 84% identity for the heavy and 86% for the light chains. In contrast, the B domain (connecting region) exhibits only 59% identity relative to the human molecule. The bovine B domain contains two repeats of a 14-amino acid structure that is contained only once in the human sequence. Bovine factor V lacks one of the nine amino acid repeats and one of the 17 amino acid repeats present in the human B domain. Factor V has little homology to the factor VIII molecule in the B domain. The 17-amino acid repeat missing in bovine factor V allows identification of an 18-amino acid sequence that is homologous to the B domain of human factor VIII. These 18 amino acids may either constitute the unique vestige of a divergent evolution between the B domains of factors V and VIII or reveal the convergent evolution toward a critical epitope involved in the activation of both procofactors.     

cDNA sequences for human von Willebrand factor reveal five types of repeated domains and five possible protein sequence polymorphisms

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened with two previously described cDNA inserts for human von Willebrand factor. Among 16 positive isolates, two that hybridized with a probe corresponding to the amino terminus of von Willebrand factor were sequenced. Together, these four cDNA inserts span 6.5 kilobases of the von Willebrand factor mRNA sequence, completely specifying the 2050 amino acids of the subunit of mature, secreted von Willebrand factor and 24 residues of a precursor peptide. Approximately 77% of the sequence is contained in five types of repeated domains. Domain A consists of 193-220 amino acids and is present in three tandem copies between residues 497 and 1111. Domain B contains 25-35 amino acids and is present in three copies between residues 1533 and 1636. Domain C consists of 116-119 amino acids and is duplicated between residues 1637 and 1899. In contrast to the essentially contiguous repetition of domains A-C, the two copies of domains D and E are each separated by 804 and 1383 amino acids, respectively. Domain D1 contains 289 amino acids between residues 79 and 367, while domain D2 consists of 270 amino acids between residues 1171 and 1440. Domain E1 consists of 46 amino acids between residues 25 and 70, and domain E2 consists of 46 amino acids between residues 1453 and 1498. The triplicated A domains are notably poor in Cys content, while the remaining domains are Cys-rich. The A domains appear to be homologous to a 225-residue segment of complement factor B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drosophila substrate adhesion molecule: sequence of laminin B1 chain reveals domains of homology with mouse

Laminin, a substrate adhesion molecule in vertebrates, is a large glycoprotein complex in basement membranes that promotes cell adhesion, cell migration, and neurite outgrowth. Here we report on the cloning of the genes encoding the three subunits of Drosophila laminin. Sequence analysis of cDNA clones encoding the Drosophila B1 chain reveals a multidomain structure similar to that of its mouse homolog. The Drosophila sequence has only 25% amino acid identity with the mouse sequence in domains I, II, and IV. However, in one of the putative collagen-binding regions (domain VI) and the two cysteine-rich domains of EGF-like repeats (domains III and V), the amino acid identity between these two evolutionarily distant species jumps to 55%. Moreover, the number, length, and unique amino acid sequences of each of the 13 EGF-like repeats are highly conserved between Drosophila and mouse, suggesting that each may serve a unique function in protein-protein interactions.     

Human laminin B2 chain. Comparison of the complete amino acid sequence with the B1 chain reveals variability in sequence homology between different structural domains

The complete amino acid sequence of the human laminin B2 chain has been determined by sequencing of cDNA clones. The six overlapping clones studied cover approximately 7.5 kilobases of which 5312 nucleotides were sequenced from the 5' end. The open reading frame codes for a 33-residue signal peptide and a 1576-residue B2 chain proper, which is 189 residues less than in the highly homologous B1 chain (Pikkarainen, T., Eddy, R., Fukushima, Y., Byers, M., Shows, T., Pihlajaniemi, T., Saraste, M., and Tryggvason, K. (1987) J. Biol. Chem. 262, 10454-10462). Computer analysis revealed that the B2 chain consists of distinct domains that contain helical structures, cysteine-rich repeats, and globular regions, as does the B1 chain. However, domain alpha and domain beta of the B1 chain have no counterpart in B2, and the number of cysteine-rich repeats is 12, or 1 less than in the B1 chain. The degree of homology between the two chains is highest in the cysteine repeat-containing domains III and V where 40% of the residues match. However, results demonstrate that the B1 and B2 chains of laminin are highly homologous proteins that are probably the products of related genes.     

Amino acid sequence of the murine Mac-1 alpha chain reveals homology with the integrin family and an additional domain related to von Willebrand factor.

Clones encoding the Mac-1 alpha chain were selected from a mouse macrophage cDNA library by screening with oligonucleotide probes based on the sequence of a genomic clone encoding the N-terminus of the mature protein. The sequence of overlapping clones (4282 nt) was determined and translated into a protein of 1137 amino acids and a signal peptide of 15 amino acids. The Mac-1 sequence was found to be related to the alpha chain sequences of three other members of the integrin family of cell adhesion receptors, i.e. the fibroblast receptors for fibronectin and vitronectin and the platelet glycoprotein IIb/IIIa. All four sequences share a number of structural features, like the position of 13 cysteine residues, a transmembrane domain near the C-terminus and the location of three putative binding sites for divalent cations. Furthermore, a conserved sequence motif is repeated seven times in the N-terminal half of the molecule and three of these repeats include putative Ca/Mg-binding sites of the general structure DXDXDGXXD. On the other hand, Mac-1 contains a unique domain of 220 amino acids inserted into the N-terminal part of the integrin structure. This additional domain is homologous to a repeated domain found in von Willebrand factor, cartilage matrix protein and in the complement factors B and C2. In two of these proteins, the homologous domain is likely to be involved in binding to collagen fibrils. Therefore, Mac-1 may also bind to collagen, which could play a role in the interaction of leukocytes with the subendothelial matrix.     

Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926

A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250,000 relative mass (Mr) which was secreted into the medium as a 220,000 Mr protein. A cDNA library was constructed in lambda gt11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.     

The amino acid sequence of chick skin collagen alpha1-CB7.

The amino acid sequence of chick skin collagen alpha1-CB7, the 268 CNBr peptide from the helical portion of the alpha1 chain, has been determined by automatic and manual degradation of tryptic and chymotryptic peptides, and of the COOH-terminal fragment produced by cleavage with animal collagenase. The resulting sequence shows 94% identity with that of the corresponding peptide from calf skin collage (Fietzek, P. P., Rexrodt, F. W., Hopper, K. E., and Kühn, K. (1973), Eur. J. Biochem. 38, 396). The bond cleaved by animal collagenase has been identified as Gly-Ile at residues 221-222 of alpha1-CB7.     

The cloning and sequencing of alpha 1(VIII) collagen cDNAs demonstrate that type VIII collagen is a short chain collagen and contains triple-helical and carboxyl-terminal non-triple-helical domains similar to those of type X collagen

We have isolated two overlapping cDNA clones covering 2425 base pairs encoding a short type VIII collagen chain synthesized by rabbit corneal endothelial cells. The cDNAs encode an open reading frame of 744 amino acid residues containing a triple-helical domain of 454 residues flanked by 117- and 173-residue amino and carboxyl non-triple-helical domains (called NC2 and NC1, respectively). Based on the identity between the DNA-derived amino acid sequence and the amino acid sequence of a type VIII collagen CNBr peptide obtained from rabbit corneal Descemet's membrane, we conclude that the cDNAs code for a type VIII collagen chain. We give this chain the designation alpha 1(VIII). The alpha 1(VIII) triple-helical domain contains eight imperfections in the Gly-X-Y repeated structure with Gly-X instead of a full triplet. The length of the triple-helical domain and number and relative locations of these imperfections are remarkably similar to those of chicken alpha 1(X) collagen. The amino acid sequence of the carboxyl three-quarters of the NC1 domain has high sequence similarity to that of alpha 1(X) collagen. These data suggest that the triple-helix coding portions and carboxyl three-quarters of the NC1 domains of the alpha 1(VIII) and alpha 1(X) genes have a common evolutionary origin.     

A Bethlem myopathy Gly to Glu mutation in the von Willebrand factor A domain N2 of the collagen alpha3(VI) chain interferes with protein folding.

A single G1679E mutation in the amino-terminal globular domain N2 of the alpha3 chain of type VI collagen was found in a large family affected with Bethlem myopathy. Recombinant production of N2 ( approximately 200 residues) in transfected mammalian cells has now been used to examine the possibility that the mutation interfered with protein folding. The wild-type form and a G1679A mutant were produced at high levels and shown to fold into a stable globular structure. Only a small amount of secretion was observed for mutants G1679E and G1679Q, which apparently were efficiently degraded within the cells. Homology modeling onto the related von Willebrand factor A1 structure indicated that substitution of G1679 by the bulky E or Q cannot be accommodated without considerable changes in the folding pattern. This suggests protein misfolding as a molecular basis for this particular mutation in Bethlem myopathy, in agreement with radioimmunoassay data showing reduced levels of domain N2 in cultured fibroblasts from two patients.     

The binding of von Willebrand factor type C domains of Chordin family proteins to BMP-2 and Tsg is mediated by their SD1 subdomain

The VWC domain of Chordin family proteins consists of subdomains SD1 and SD2. In previous experiments with VWC1 from CV-2 SD-1 was shown to be crucial for BMP interaction. Now the SD1 from VWC1 and VWC3 of Chordin and CHL2 were established to confer BMP affinity and specificity to these proteins also. In addition, these SD1 subdomains are mediating binding to Tsg. Mutational analysis revealed similar binding epitopes of the various SD1 proteins for BMP-2 and Tsg. Inhibitory activity of CHL2 in C2C12 cells is reduced by mutations in SD1 of VWC1 and even more of VWC3. These results together provide strong evidence that the SD1 subdomain module of about 40 residues represents the crucial binding partner for BMPs and Tsg in these Chordin family proteins and likely in other BMP-binding VWC domains also.     

The complete primary structure for the alpha 1-chain of mouse collagen IV. Differential evolution of collagen IV domains

We report here the complete nucleotide and amino acid sequences for the alpha 1-chain of mouse collagen IV which is 1669 amino acids in length, including a putative 27-residue signal peptide. In comparison with the amino acid sequence for the alpha 2-chain (Saus, J., Quinones, S., MacKrell, A. J., Blumberg, B., Muthkumaran, G., Pihlajaniemi, J., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6318-6324), the two chains of collagen IV are 43% identical. Most of the interruptions of the Gly-X-Y repeat are homologously placed but strikingly show no sequence similarity between the two chains. Availability of the amino acid sequences for human collagen IV allows a detailed comparison of the primary structure of collagen IV and reveals evolutionarily conserved domains of the protein. Between the two species, the alpha 1 (IV) chains are 90.6% and the alpha 2 (IV) chains are 83.5% identical in sequence. We discuss these data with respect to differential evolution between and within the collagen IV chain types.