The alpha 2(VIII) collagen gene. A novel member of the short chain collagen family located on the human chromosome 1

Type VIII collagen is a major component of Descemet's membrane, the specialized basement membrane of corneal endothelial cells. Sequence analysis of a cDNA isolated from a library made with mRNA from rabbit corneal endothelial cells has indicated that type VIII molecules contain a polypeptide chain, alpha 1(VIII), consisting of a short triple-helical domain of 454 amino acid residues flanked by non-triple-helical domains of 117 and 173 amino acid residues at the amino and carboxyl ends, respectively (Yamaguchi, N., Benya, P. D., van der Rest, M., and Ninomiya, Y. (1989) J. Biol. Chem. 264, 16022-16029). The sequence of alpha 1(VIII) is strikingly similar to that of alpha 1(X) collagen, a product of hypertrophic chondrocytes. Also, characterization of the alpha 1(VIII) and alpha 1(X) collagen genes has shown that they are quite similar in their exon organization. It has been concluded, therefore, that they are homologous members of a distinct subclass of collagen genes (Yamaguchi, N., Mayne, R., and Ninomiya, Y. (1991) J. Biol. Chem. 266, 4508-4513). We have given this subclass the name short chain collagens because of the relatively small size of the triple-helical domain. In the present study, we report on the identification and characterization of a collagen gene encoding a polypeptide which is co-expressed with the alpha 1(VIII) chain in corneal endothelial cells. This collagen chain contains a triple-helical and a carboxyl non-triple-helical domain encoded by a single, large exon both in mice and humans. We conclude, therefore, that the genes encodes a novel member of the short chain collagen family, and we have given this chain the designation alpha 2(VIII) collagen. By in situ hybridization we demonstrate that the alpha 2(VIII) gene is located in the p32.3-p34.3 region of the short arm of chromosome 1.     

Isolation and sequencing of cDNAs and genomic DNAs encoding the alpha 4 chain of basement membrane collagen type IV and assignment of the gene to the distal long arm of human chromosome 2.

We cloned three overlapping cDNAs covering 2,452 base pairs encoding a new basement membrane collagen chain, alpha 4(IV), from rabbit corneal endothelial cell RNA. Nucleotide sequence analysis demonstrated that the clones encoded a triple-helical domain of 392 1/3 amino acid residues and a carboxyl non-triple-helical (NC1) domain of 231 residues. We also isolated a genomic DNA fragment for the human alpha 4(IV) chain, which contained two exons encoding from the carboxyl end of the triple-helical domain to the amino end of the NC1 domain. Identification of the clones was based on the amino acid sequence identity between the cDNA-deduced amino acid sequence and the reported amino acid sequence obtained from a fragment of the alpha 4(IV) collagen polypeptide M28+ (Butkowski, R. J., Shen, G.-Q., Wieslander, J., Michael, A. F., and Fish, A. J. (1990) J. Lab. Clin. Med. 115, 365-373). When compared with four other type IV collagen chains, the NC1 domain contained 12 cysteinyl residues in positions identical to those of the residues in those chains. The domain demonstrated 61, 70, 55, and 60% amino acid similarity with human alpha 1, human alpha 2, bovine alpha 3, and human alpha 5 chains, respectively. The human genomic DNA fragment allowed us to map the alpha 4(IV) gene (COL4A4) to the 2q35-2q37.1 region of the human genome.     

The alpha 1 (VIII) collagen gene is homologous to the alpha 1 (X) collagen gene and contains a large exon encoding the entire triple helical and carboxyl-terminal non-triple helical domains of the alpha 1 (VIII) polypeptide

We recently cloned and sequenced alpha 1 (VIII) collagen cDNAs and demonstrated that type VIII collagen is a short-chain collagen that contains both triple helical and carboxyl-terminal non-triple helical domains similar to those of type X collagen (Yamaguchi, N., Benya, P., van der Rest, M., and Ninomiya, Y. (1989) J. Biol. Chem. 264, 16022-16029). We report here on the structural organization of the gene encoding the rabbit alpha 1 (VIII) collagen chain. The alpha 1 (VIII) gene contains four exons, whose sizes are 69, 120, 331, and 2278 base pairs. The first and second exons encode only 5'-untranslated sequences, whereas the third exon codes for a very short (3 nucleotides) stretch of 5'-untranslated sequence, the signal peptide, and almost the entire amino-terminal non-triple helical (NC2) domain (109 1/3 codons). Interestingly, the last exon encodes the rest of the translated region, including 7 2/3 codons of the NC2 domains, the complete triple helical domain (COL1, 454 amino acid residues), the entire carboxyl-terminal non-triple helical domain (NC1, 173 amino acid residues), and the 3'-untranslated region. This exon-intron structure is in stark contrast to the multi-exon structure of the fibrillar collagen (types I, II, III, V, and XI) genes, but it is remarkably similar to that of the type X collagen gene (LuValle, P., Ninomiya, Y., Rosenblum, N. D., and Olsen, B. R. (1988) J. Biol. Chem. 263, 18278-18385). The data suggest that the alpha 1 (VIII) and the alpha 1 (X) genes belong to the same subclass within the collagen family and that they arose from a common evolutionary precursor.     

Type VIII collagen: heterotrimeric chain association

Two chains, alpha1(VIII) and alpha2(VIII), have been described for type VIII collagen. Early work suggested that these chains were present in a 2:1 ratio, although recent work has shown that homotrimers can form and predominate in some tissues. In order to address the question of whether the alpha1(VIII) and alpha2(VIII) chains could co-polymerise we made a shortened alpha1(VIII) chain and expressed this with full length alpha2(VIII) chain in an in vitro translation system supplemented with semi-permeabilised cells. Heterotrimers containing either two or one alpha2(VIII) were evident. Interestingly, a point mutation in the NC1 domain of the alpha1(VIII) chain abrogated trimer formation. In addition we were able to demonstrate chain association of the alpha1(X) chain of type X collagen with the shortened alpha1(VIII) chain. Variations in chain association were seen when altered ratios of message were used. These results demonstrate the importance of the NC1 domain in chain association and suggest that gene expression regulates the composition and function of type VIII collagen by varying chain composition.     

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.     

Type XII collagen. A large multidomain molecule with partial homology to type IX collagen

Three overlapping cDNAs encoding alpha 1 (XII) collagen have been isolated and sequenced. The DNAs define five sequence domains within the chain. Three domains are nontriple-helical; two are relatively short triple-helical regions. The amino acid sequences of tryptic peptides derived from 16- and 10-kDa pepsin-resistant fragments isolated from tendon extracts are in full agreement with the deduced sequences of the triple-helical regions. Two of the five sequence domains in alpha 1 (XII), one triple-helical and one nontriple-helical, show a high degree of similarity to regions in type IX collagen chains. In addition, examination of seven exons in the alpha 1 (XII) gene shows that the gene is, in part, similar to the structure of type IX collagen genes. Therefore, collagen types IX and XII are partially homologous. The alpha 1 (XII) sequence data predict an asymmetric structure for type XII collagen molecules, fully consistent with the rotary shadowing images. These images show a triple-helical 75-nm tail attached through a central globule to three finger-like structures, each 60 nm long (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156).     

Crystal structure of the collagen alpha1(VIII) NC1 trimer.

Collagen VIII is a major component of Descemet's membrane and is also found in vascular subendothelial matrices. The C-terminal non-collagenous domain (NC1) domain of collagen VIII, which is a member of the C1q-like protein family, forms a stable trimer and is thought to direct the assembly of the collagen triple helix, as well as polygonal supramolecular structures. We have solved the crystal structure of the mouse alpha1(VIII)(3) NC1 domain trimer at 1.9 A resolution. Each subunit of the intimate NC1 trimer consists of a ten-stranded beta-sandwich. The surface of the collagen VIII NC1 trimer presents three strips of partially exposed aromatic residues shown to interact with the non-ionic detergent CHAPS, which are likely to be involved in supramolecular assemblies. Equivalent strips exist in the NC1 domain of the closely related collagen X, suggesting a conserved assembly mechanism. Surprisingly, the collagen VIII NC1 trimer lacks the buried calcium cluster of the collagen X NC1 trimer. The mouse alpha1(VIII) and alpha2(VIII) NC1 domains are 71.5% identical in sequence, with the differences being concentrated on the NC1 trimer surface. A few non-conservative substitutions map to the subunit interfaces near the surface, but it is not obvious from the structure to what extent they determine the preferred assembly of collagen VIII alpha1 and alpha2 chains into homotrimers.     

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.     

Occurrence of collagen and proteoglycan forms of type IX collagen in chick embryo cartilage. Production and characterization of a collagen form-specific antibody.

Type IX collagen from chick embryonic cartilage is a proteoglycan bearing a single chondroitin sulfate chain covalently linked to the alpha 2(IX) polypeptide chain. We have isolated type IX collagen metabolically labeled with [3H]proline using an antibody to type IX collagen and have found that the molecule is synthesized in two forms, a collagen form (COLIX) and a proteoglycan form (PGIX). In cultured chondrocytes, the two forms of type IX collagen showed a different ability to be deposited in the matrix. We have suggested the possibility that both forms may arise from an alternative substitution of a chondroitin sulfate chain to the NC3 domain of the alpha 2(IX) chain. Based on the reported amino acid sequence at the NC3 domain of alpha 2(IX), we have synthesized undecapeptides containing the sequence around the glycosaminoglycan attachment site of the alpha 2(IX) chain. Antibody against the peptide, which was raised in rabbit, only recognized COLIX and made it possible to distinguish COLIX from PGIX. Evidence shows that this could be due to a difference in antigenicity of the NC3 domain of the alpha 2(IX) chain between COLIX and PGIX caused by the substitution of a chondroitin sulfate chain to the serine residue in this domain. Therefore, this antibody may be useful as a probe for studies on the functions of glycosaminoglycan substitution in type IX collagen.     

Isolation and characterization of type VIII collagen synthesized by cultured rabbit corneal endothelial cells. A conventional structure replaces the interrupted-helix model

Radioactive proline-labeled type VIII collagen was biosynthesized in the presence of beta-aminoproprionitrile by rabbit corneal endothelial cells and isolated from the culture medium. Type VIII was purified in the presence of protease inhibitors and at neutral pH by ultrafiltration, precipitation with 3.9 M NaCl, sedimentation in sucrose gradients, and DEAE-Sephacel chromatography. The major components of this collagen, VIII-1, -2, and -3, exhibited apparent molecular weights of greater than 194,000, 124,000, and 61,000, respectively, and were shown to contain identical CNBr peptides. Following separation of VIII-1, -2, and -3 from each other and any residual proteases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exposure to acetic acid led to the conversion of VIII-1 to VIII-2 and VIII-3. Thus, VIII-1 is not a continuous single peptide chain, and the preliminary interrupted-helix model of the type VIII structure (Benya, P. D. (1980) Renal Physiol. 3, 30-35) was revised. VIII-3 appears to be the parent alpha 1 (VIII)-chain, with VIII-2 and VIII-1 representing beta- and gamma-chain configurations stabilized by strong noncovalent acid-labile interactions and beta-aminoproprionitrile-insensitive covalent cross-links. Based on two-dimensional CNBr peptide mapping, the alpha-chain is composed of six peptides. Mr 5,300-19,600. The terminal peptides are pepsin sensitive and correlate with two noncollagenous domains, NC1 (Mr 14,700) and NC2 (Mr 4-5,000). NC1 contains the site of acid-labile chain association.     

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.     

Mechanism of chain selection in the assembly of collagen IV: a prominent role for the alpha2 chain

Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the alpha1(IV) and alpha2(IV) NC1 domains. We show that the differential affinity of alpha2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the alpha1(IV) NC1 domain, we conclude that the alpha2(IV) chain plays a regulatory role in directing chain composition in the assembly of (alpha1)(2)alpha2 triple-helical molecule. Detailed crystal structure analysis of the [(alpha1)(2)alpha2](2) NC1 hexamer and sequence alignments of the NC1 domains of all six alpha-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a beta-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.     

Construction and characterization of cDNA encoding the alpha 2 chain of chicken type IX collagen

We have isolated and characterized a cDNA encoding the carboxy-terminal half of one of the polypeptide subunits of a novel disulfide-bonded collagen found in hyaline cartilage. This collagen has been given the type assignment type IX, and it has several unusual characteristics. First, the polypeptide subunits are shorter than alpha-chains of the fibrillar collagens types I, II, and III. Second, type IX molecules are heterotrimers of three genetically distinct polypeptide subunits. Third, type IX molecules contain three triple-helical collagenous domains interspersed with noncollagenous domains. When chicken cartilage collagens are extracted with pepsin, type IX collagen is cleaved and gives rise to the triple-helical fragments HMW and LMW. The identification of the cDNA reported here is based on a comparison of the amino acid composition of tryptic peptides derived from LMW with the composition of tryptic peptides predicted from the nucleotide sequence of the cDNA. We also show that the amino-terminal sequence of one of the subunits of LMW is identical with the sequence predicted from the nucleotide sequence of the cDNA. Finally, we demonstrate that the amino-terminal amino acid sequence of a tryptic peptide isolated from one of the subunits of HMW is identical with a sequence predicted from the cDNA. We have given the polypeptide chain encoded by the cDNA reported here the name alpha 2(IX), and we show that it is homologous to the alpha 1(IX) chain previously characterized by us.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction.

The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.     

Alpha 1 chain of chick type VI collagen. The complete cDNA sequence reveals a hybrid molecule made of one short collagen and three von Willebrand factor type A-like domains

A cDNA library constructed from chick aorta poly(A+) RNA in the expression vector pEX1 was screened with rabbit polyclonal antisera. Additional clones were obtained by DNA-DNA hybridization with subclones from the most 5'- and 3'-ends. The overlapping clones span 4.6 kilobases and code for the entire alpha 1 (VI) chain. The nucleotide sequence reveals a 3057-base pair open reading frame that codes for 1019 amino acids. Analysis of the deduced amino acid sequence predicts that alpha 1 (VI) has one collagenous domain (COL) of 336 residues flanked by three repeated domains of about 200 residues each, one at the amino (A'3) and two at the carboxyl ends (A'2 and A'1), respectively, that are similar to the type A repeats of von Willebrand Factor. The COL domain presents two short interruptions near the carboxyl end of the triple helix and three of the six potential N-asparaginyl-linked carbohydrate attachment sites (Asn-Xaa-Ser/Thr). Furthermore, it contains 1 cysteine at position 89 that could participate in the formation of dimers and 3 Arg-Gly-Asp sequences that might be potential sites for cell adhesion. The COL domain shows an extended region, starting from position 40, within the triple helix, made of 14 Gly-Xaa-Yaa triplets that lack proline in the Y position, suggesting that it might be more flexible than the rest of the domain. At the junction of the COL with the N- and C-terminal domains, there are several cysteines that could confer the well known resistance of type VI collagen to pepsin and collagenase digestion under nonreducing conditions. The present sequence data allow a structural model for type VI collagen assembly to be proposed that is consistent with the structure implied from previous electron microscopic observation by Furthmayr et al. (Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, R., and Engel, J. (1983) Biochem. J. 221, 303-311).     

Sites of stromelysin cleavage in collagen types II, IX, X, and XI of cartilage

Human recombinant stromelysin-1 was shown to cleave four types of collagen (types II, IX, X, and XI) prepared from bovine and rat cartilages at specific sites. Stromelysin-1 cleaved salt-soluble native molecules of type IX collagen into two main triple-helical fragments, COL1 and COL2,3. Protein microsequencing identified the exact cleavage sites in the NC2 domain of all three chains, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Stromelysin-1 also acted as a "telopeptidase," in that it efficiently clipped intact molecules of types II and XI collagens at sites just inside their terminal cross-linking hydroxylysine residues. Native molecules of type X collagen were cleaved by stromelysin-1 within their triple helical domains at a COOH-terminal site that reduced the alpha 1(X) chain size by 10 kDa. These findings suggest an important role for stromelysin in the turnover and remodeling of the collagenous matrix of cartilage both normally and in degenerative joint disease.     

NC1 domain of human type VIII collagen (alpha 1) inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis.

Endostatin, a natural angiogenesis inhibitor, had been identified for years. It opened a new approach for cancer therapy. Sequence analysis revealed that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII. In this report, the cDNA of NC1 domain of type VIII collagen (alpha 1) was cloned and expressed as soluble form in Escherichia coli. The recombinant protein was purified with Ni-NTA agarose column and named as vastatin. It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner. The ED(50) of vastatin was 0.6 microg/ml, while the ED(50) of endostatin was 0.5 microg/ml. Treatment of BAE cell with vastatin caused G(0)-G(1) arrest and cell apoptosis. It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin. The structure-function relationship of these angiogenesis molecules remains to be elucidated.     

The structure of type IX collagen

We present a detailed analysis both of tryptic peptides and amino-terminal sequences of the subunits of two collagenous fragments (HMW and LMW) previously isolated from pepsin extracts of chicken cartilage (Reese, C.A., and Mayne, R. (1981) Biochemistry 20, 5443-5448). This analysis and a comparison with the nucleotide sequence of the cDNApYN1738 (Ninomiya, Y., and Olsen, B.R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3014-3018) shows that HMW and LMW are pepsin-resistant fragments of a unique collagen composed of molecules with three different polypeptide chains (alpha-chains). This collagen has been assigned the type number IX, and the alpha-chain encoded by pYN1738 has been given the designation alpha 1 (IX). Type IX collagen contains three triple-helical domains and at least two sets of interchain disulfide bridges. At the amino and carboxyl ends are noncollagenous domains which do not appear to be homologous to amino and carboxyl propeptides of interstitial collagens.