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.     

Astrocytes express type VIII collagen during the repair process of brain cold injury

We recognized for the first time upregulation of type VIII collagen gene expression during the repair process in the mouse brain cold injury model. Immunohistochemical staining showed that type VIII collagen expression was around the necrotic region, where reactive astrocytes are frequently observed. Cultured astrocytes demonstrated a high expression of type VIII collagen genes. TGF-beta1 enhanced the expression of both alpha1(VIII) and alpha2(VIII) genes by astrocytes in culture. Further, we tested selected biological activities of type VIII collagen, compared with those of type I, IV, and V collagens and fibronectin. Astrocytes adhered to type VIII collagen via receptors requiring metal ions. Astrocyte migration on type VIII collagen was more stimulated than that observed on the other ECM molecules. These data indicate that type VIII collagen plays an important role in glial scar formation during the repair process by astrocytes.     

Cloning of the human and mouse type X collagen genes and mapping of the mouse type X collagen gene to chromosome 10.

Type X collagen, a homotrimer of alpha 1 (X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously described the isolation of a small fragment of the human type X collagen gene (COL10A1) and its localization to the q21-q22 region of human chromosome 6 [Apte, S., Mattei, M.-G. & Olsen, B. R. (1991) FEBS Lett. 282, 393-396]. Using this fragment as a probe to screen genomic libraries, we report here the isolation of human and mouse genomic clones which contain the major part of the human and mouse type X collagen genes. In both species, the 14-kb genomic clones which were isolated contain a long open reading frame (greater than 2000 bp in length) which codes for the entire C-terminal non-collagenous (NC1) domain, the entire collagenous (COL) domain and part of the N-terminal non-collagenous (NC2) domain of the alpha 1(X) collagen chain. The human genomic clone contains the major part of the COL10A1 gene, in addition to the region we have previously cloned, and is highly similar to the corresponding portions of the mouse genomic clone (84.5% similarity at the nucleotide level, and 86.1% at the level of the conceptual translation product). The identification of the mouse genomic clone as the alpha 1(X) collagen gene (Col10a1) was confirmed by in situ hybridization of a fragment of the mouse genomic clone to sections from newborn mice. Hybridization was restricted to the hypertrophic chondrocytes of developing chondroepiphyses, being absent in small chondrocytes and in other tissues. Using interspecific backcross analysis, the locus for the mouse alpha 1 (X) collagen gene was assigned to chromosome 10. The cloning and chromosomal mapping of the human and mouse alpha 1 (X) collagen genes now permit the investigation of the possible role of type X collagen gene defects in the genesis of chondrodysplasias in both species and provide data essential for the generation of transgenic mice deficient in type X collagen.     

The mouse alpha 1(XII) and human alpha 1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6.

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.     

A novel and highly conserved collagen (pro(alpha)1(XXVII)) with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family

The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro(alpha)1(XXVII) chain has a domain structure similar to that of the type B clade chains (alpha1(V), alpha3(V), alpha1(XI), and alpha2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro(alpha)1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240-110 kDa in tissue extracts and collagenous bands of 150-140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro(alpha) chains.     

Assignment of the human collagen alpha 1 (XIII) chain gene (COL13A1) to the q22 region of chromosome 10

Type XIII collagen is a recently described collagen that resembles in structure the short-chain collagens of types IX, X, and XII. Unlike any other collagen, the type XIII is found in several different forms generated through alternative splicing. A 2.0-kb genomic fragment from the human alpha 1 (XIII) collagen gene was isolated and shown by DNA sequencing to contain exon 12 as counted from the 3' end. This fragment was used as a probe to localize the gene. The gene (COL13A1) was assigned to chromosome 10 by hybridization of the probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes. Furthermore, the gene was mapped to the q22 region by in situ hybridization to metaphase chromosomes.     

Cloning and chromosomal localization of human genes encoding the three chains of type VI collagen.

Type VI collagen is a heterotrimer composed of three polypeptide chains, alpha 1(VI), alpha 2(VI), and alpha 3(VI). By immunological screening of an expression cDNA library, human cDNAs specific for each chain were isolated and characterized. Major mRNA species encoding these chains have a size of 4.2 kb (alpha 1), 3.5 kb (alpha 2), and 8.5 kb (alpha 3). The cDNA clones were also used to map the genes on human chromosomes by somatic cell hybrid analysis and in situ hybridization. The alpha 1 (VI) and alpha 2(VI) collagen genes were both located on chromosome 21, in band q223. This represents a third example of a possible physical proximity of two collagen loci. The alpha 3(VI) collagen gene was localized to chromosome 2, in the region 2q37. The alpha 3(VI) collagen gene is the fifth extracellular matrix gene to be localized to 2q, as four other extracellular matrix genes--i.e., the alpha 1(III) and alpha 2(V) collagen genes, the elastin gene, and the fibronectin gene--have been previously mapped to the distal region of the long arm of chromosome 2.     

Regulation of alpha 1 (I)-collagen gene expression in response to cell adhesion in Swiss 3T3 fibroblasts

Nonadhesive conditions cause Swiss 3T3 fibroblasts to enter a quiescent state that is reversed upon reattachment to a surface. Previously, we demonstrated that adhesion in serum-free conditions is sufficient to activate suspension-arrested cells out of Go, with the induction of the growth-associated genes, c-fos, c-myc, and actin. In this study, we have employed this system to identify programs of gene expression that respond primarily to the adhesive state of the cell, rather than the growth state. We show that cells in different adhesive states can be distinguished by their patterns of protein synthesis. Analysis of one adhesion-responsive protein led to its identification as pro-alpha 1 (I)-collagen. Pro-alpha 1 (I)-collagen synthesis and mRNA levels are decreased up to 6-fold in suspension-arrested fibroblasts, but are enhanced up to 5-fold as cells approach confluence. This suggests that the reduced expression in suspension-arrested cells is not simply a result of quiescence. In addition, reattachment of suspended cells in serum-free conditions caused a 7-fold induction of collagen mRNA levels and a greater than 20-fold rise in the rate of procollagen synthesis. The expression of c-myc was induced during adhesion in serum-free medium as well as by serum addition to suspension-arrested cells. However, alpha 1 (I)-collagen gene expression was unaffected by serum in the absence of adhesion. These results indicate that collagen gene expression is directly responsive to cell adhesion, independent of the growth state.     

Alpha 1 type IV collagen gene evolved differently from fibrillar collagen genes

Type IV collagen is a major structural component in basement membranes. It is considerably different from the fibrillar collagens, types I-III. For example, unlike fibrillar collagens, the triple helical domain of type IV collagen is frequently interrupted by nonhelical regions. In this report, we demonstrate several overlapping genomic clones which cover most of the mouse alpha 1(IV) chain. Electron microscopic analysis of R-loops revealed that there were at least 28 exons within 35 kilobases of the gene segment. The sizes of six exons were determined by DNA sequence analysis to be 81, 178, 134, 73, 129, and 213 base pairs. These sizes do not appear to be related to the 54-base pair coding unit which is characteristic of fibrillar collagen exons, suggesting that the alpha 1 type IV collagen gene evolved differently from the fibrillar collagen genes.     

Chromosomal assignment of a gene encoding a new collagen type (COL15A1) to 9q21 --> q22.

The collagens constitute a large family of extracellular matrix components primarily responsible for maintaining the structure and biological integrity of connective tissue. These proteins exhibit considerable diversity size, sequence, tissue distribution, and molecular composition. Fourteen types of homo- and/or heterotrimeric molecules, thus far reported, are encoded by a minimum of 27 genes. Nineteen of these genes, including several that are closely linked, have been assigned to 10 separate autosomes, and one collagen gene has been mapped to the X chromosome. We have isolated a 2.1-kb human cDNA clone coding for a collagen molecule different in sequence and structure from types I-XIV collagens. This polypeptide has been designated the alpha 1 chain of type XV collagen. To determine the location of the corresponding gene, the cDNA clone was hybridized to rodent-human hybrid DNAs and to human metaphase chromosomes. The results obtained using the hybrid cell lines showed that this newly identified collagen gene, COL15A1, is present in the pter --> q34 region of chromosome 9. In situ hybridization allowed sublocalization to 9q21 --> q22, a region to which no other collagen genes had previously been assigned. Our data further demonstrate the complex arrangement of the many collagen genes in the human genome.     

Chondrocyte aggregation in suspension culture is GFOGER-GPP- and beta1 integrin-dependent

Isolated chondrocytes form aggregates in suspension culture that maintain chondrocyte phenotype in a physiological pericellular environment. The molecular mechanisms involved in chondrocyte aggregation have not been previously identified. Using this novel suspension culture system, we performed mRNA and protein expression analysis along with immunohistochemistry for potential cell adhesion molecules and extracellular matrix integrin ligands. Inhibition of aggregation assays were performed using specific blocking agents. We found that: (i) direct cell-cell interactions were not involved in chondrocyte aggregation, (ii) chondrocytes in aggregates were surrounded by a matrix rich in collagen II and cartilage oligomeric protein (COMP), (iii) aggregation depends on a beta1-integrin, which binds a triple helical GFOGER sequence found in collagens, (iv) integrin alpha10-subunit is the most highly expressed alpha-subunit among those tested, including alpha5, in aggregating chondrocytes. Taken together, this body of evidence suggests that the main molecular interaction involved in aggregation of phenotypically stable chondrocytes is the alpha10beta1-collagen II interaction.