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.     

Domains of type X collagen: alteration of cartilage matrix by fibril association and proteoglycan accumulation

During endochondral bone formation, hypertrophic cartilage is replaced by bone or by a marrow cavity. The matrix of hypertrophic cartilage contains at least one tissue-specific component, type X collagen. Structurally type X collagen contains both a collagenous domain and a COOH-terminal non-collagenous one. However, the function(s) of this molecule have remained largely speculative. To examine the behavior and functions of type X collagen within hypertrophic cartilage, we (Chen, Q., E. Gibney, J. M. Fitch, C. Linsenmayer, T. M. Schmid, and T. F. Linsenmayer. 1990. Proc. Natl. Acad. Sci. USA. 87:8046-8050) recently devised an in vitro system in which exogenous type X collagen rapidly (15 min to several hours) moves into non-hypertrophic cartilage. There the molecule becomes associated with preexisting cartilage collagen fibrils. In the present investigation, we find that the isolated collagenous domain of type X collagen is sufficient for its association with fibrils. Furthermore, when non-hypertrophic cartilage is incubated for a longer time (overnight) with "intact" type X collagen, the molecule is found both in the matrix and inside of the chondrocytes. The properties of the matrix of such type X collagen-infiltrated cartilage become altered. Such changes include: (a) antigenic masking of type X collagen by proteoglycans; (b) loss of the permissiveness for further infiltration by type X collagen; and (c) enhanced accumulation of proteoglycans. Some of these changes are dependent on the presence of the COOH-terminal non-collagenous domain of the molecule. In fact, the isolated collagenous domain of type X collagen appears to exert an opposite effect on proteoglycan accumulation, producing a net decrease in their accumulation, particularly of the light form(s) of proteoglycans. Certain of these matrix alterations are similar to ones that have been observed to occur in vivo. This suggests that within hypertrophic cartilage type X collagen has regulatory as well as structural functions, and that these functions are achieved specifically by its two different domains.     

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 type X collagen gene. Intron sequences split the 5'-untranslated region and separate the coding regions for the non-collagenous amino-terminal and triple-helical domains

Type X collagen, expressed by hypertrophic chondrocytes, consists of homotrimeric molecules with subunits that are only about one-half the size of the polypeptides of fibrillar collagens. In this report we describe for the first time the complete primary structure of type X collagen, based on cloning and sequencing of cDNA and genomic DNA. A comparison between the nucleotide sequences of the cDNA and genomic DNA clones has also allowed determination of the complete exon structure of the type X collagen gene. Our results demonstrate that the primary translation product of the chicken type X collagen mRNA is 682 amino acid residues long with a calculated molecular mass of 67,317 Da for the nonhydroxylated form. This calculated molecular mass is in excellent agreement with the observed electrophoretic mobility of cell-free translation products with both poly(A)+ RNA isolated from chondrocytes as well as RNA transcribed in vitro from a full length cDNA construct. It is also in agreement with the observed size of type X collagen polypeptides isolated from the media of cultured hypertrophic chondrocytes. Thus, our data exclude the possibility of a high molecular weight precursor form of type X collagen. Our results also confirm that the chicken type X gene has a most unusual exon structure when compared to other vertebrate collagen genes. The gene has only three exons. One exon (97 base pairs (bp)), codes for most of the 5'-untranslated region of the mRNA, a second exon (159 bp) codes for the signal peptide and a short non-triple-helical domain, while the third exon (2136 bp) contains the coding region for the entire triple-helix and a large non-triple-helical carboxyl domain.     

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.     

Type X collagen alterations in rachitic chick epiphyseal growth cartilage

We examined collagens of both normal and vitamin D-deficient chick epiphyseal growth cartilage. Special emphasis was placed on the study of Type X collagen, a recently described product of hypertrophic chondrocytes. Scanning electron microscopy of the epiphyseal growth cartilage of vitamin D-deficient chickens showed an enlarged growth cartilage with a disorganized extracellular matrix. The cartilage collagens were solubilized by proteolytic digestion and disulfide bond reduction of both normal and rachitic growth tissues. Sequential extraction with neutral salt and acetic acid buffers followed by pepsin digestion at 4 degrees C solubilized about 12% of normal tissues and about 7% of collagen from rachitic growth cartilage. Treatment of the pepsin-resistant collagens with neutral salt-dithiothreitol buffer under nondenaturing conditions and a subsequent pepsin digestion increased the yield of solubilized collagen to greater than 95% of the total tissue collagen. Results of the biochemical studies showed a marked increase in the relative proportion of Type X collagen (from 5.6 to 27.9%), a corresponding decrease in the proportions of Types II and IX collagens, and a moderate increase in Type XI collagen in rachitic cartilage. Amino acid analysis indicated that there were no differences in the Types II and X collagens of normal and rachitic cartilage. However, an abnormality in the relative proportions of the CNBr peptides of Type X collagen was detected in the rachitic cartilage. We suggest that the increase in collagen in the rachitic state may reflect increased levels of Type X collagen synthesis by cells in the hypertrophic region. It is likely that in rickets the overproduction of Type X collagen may be a compensatory mechanism by which the hypertrophic chondrocyte attempts to provide a maximum area of calcifiable matrix for the calcium-depleted serum.     

Macromolecular organization of chicken type X collagen in vitro

The macromolecular structure of type X collagen in the matrices of primary cultures of chick hypertrophic chondrocytes was initially investigated using immunoelectron microscopy. Type X collagen was observed to assemble into a matlike structure with-in the matrix elaborated by hypertrophic chondrocytes. The process of self assembly was investigated at the molecular level using purified chick type X collagen and rotary-shadowing EM. It was shown that under neutral conditions at 34 degrees C, individual type X collagen molecules associate rapidly into multimeric clusters via their carboxy-terminal globular domains forming structures with a central nodule of carboxy-terminal domains and the triple helices radiating outwards. Prolonged incubation resulted in the formation of a regular hexagonal lattice by lateral association of the juxtaposed triple-helical domains from adjacent multimeric clusters. This extended lattice may play an important role in modifying the cartilage matrix for subsequent events occurring in endochondral bone formation.     

Differential effects of parathyroid hormone fragments on collagen gene expression in chondrocytes

The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule. To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system of fetal bovine and human chondrocytes. Expression of alpha1 (I), alpha1 (II), alpha1 (III), and alpha1 (X) mRNA was investigated by in situ hybridization and quantified by Northern blot analysis. NH2- terminal and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in alpha1 (II) mRNA in proliferating chondrocytes. In addition, the COOH-terminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on alpha1 (II) and alpha1 (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage. PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell.     

Protein consequences of the Col2a1 C-propeptide mutation in the chondrodysplastic Dmm mouse.

The Disproportionate micromelia (Dmm) mouse has a three nucleotide deletion in Col2a1 in the region encoding the C-propeptide which results in the substitution of one amino acid, Asn, for two amino acids, Lys-Thr. Western blot and immunohistochemical analyses failed to detect type II collagen in the cartilage matrix of the homozygous mice and showed reduced levels in the matrix of heterozygous mice. Type II collagen chains localized intracellularly within the chondrocytes of homozygote and heterozygote tissues. These findings provide evidence that the expression of type II procollagen chains containing the defective C-propeptide results in an intracellular retention and faulty secretion of type II procollagen molecules. A complete absence of mature type II collagen from the homozygote cartilage and an insufficiency of type II collagen in the heterozygote cartilage explains the Dmm mouse phenotype. The integrity of the C-propeptide is thus crucial for the biosynthesis of normal type II collagen by chondrocytes.     

Localization of collagen X in human fetal and juvenile articular cartilage and bone.

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilage-bone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often "spotty" distribution of collagen X with increasing cartilage "degeneration" associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and "spotty" collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Sequence analysis of a full-length cDNA for the murine pro alpha 2(I) collagen chain: comparison of the derived primary structure with human pro alpha 2(I) collagen.

Comparison of the nucleotide sequence and primary structure of murine and human pro alpha 2(I) collagen indicates a high degree of homology: 87% at the nucleotide level and 87% at the amino acid level, with the greatest degree of variability in the amino- and carboxy-pro-peptide domains. The homology is greatest in the triple helical domain, repeating [Gly-X-Y]338, exhibiting 90% homology at the amino acid level, with only X and Y position residue substitutions. The X and Y residues show 86% homology between murine and human pro alpha 2(I) collagen triple helices, with no truly nonconservative substitutions.     

Immunoperoxidase localization of type X collagen in chick tibiae

Type X collagen was prepared from medium of long-term cultures of embryonic chick tibiotarsal chondrocytes. Antibodies to type X collagen were raised and used in immunoperoxidase localization studies with embryonic and growing chick tibiotarsus. Strong anti-type X collagen reactivity was detected mainly in the region of hypertrophic chondrocytes, and to a lesser extent in the zone of calcified cartilage. No reactivity was detected in the proliferative zone nor the superficial layer of the cartilage growth plate. These results suggest that type X collagen may play a key role in matrix calcification during growth and development of the skeletal system.     

Cartilage type IX collagen-proteoglycan contains a large amino-terminal globular domain encoded by multiple exons

Type IX collagen in cartilage consists of molecules composed of three genetically distinct polypeptide subunits. One of the subunits, alpha 2(IX), contains a covalently attached glycosaminoglycan side chain whereas a second subunit, alpha 1(IX), contains a large noncollagenous, amino-terminal domain called NC4. In this report, we describe for the first time the complete primary structure of this noncollagenous domain, based on cloning and sequencing of cDNA and genomic DNA as well as amino acid sequencing of tryptic peptides. Analysis of genomic clones has also allowed determination of the exon structure of NC4. Our results demonstrate that the noncollagenous, amino-terminal domain of alpha 1(IX) chains contains 266 amino acid residues (including the signal peptide) with 5 cysteinyl residues forming two disulfide bridges. The domain is basic with an estimated pI of 9.7, thus supporting the idea that it may participate in ionic interactions with polyanionic glycosaminoglycans in cartilage. Both the sequence and exon structure of the NC4 domain is unique among collagens and there is no obvious homology with the noncollagenous domains of other types of collagen, including the propeptides of fibrillar collagens.     

Biosynthesis of a disulphide-bonded short-chain collagen by calf growth-plate cartilage.

Collagen biosynthesis by organ cultures of the hypertrophic zone of calf growth-plate cartilage was studied. It was found that this tissue devotes a large portion of its biosynthetic commitment towards production of a collagen molecule comprising short collagen chains. This collagen is similar to short-chain collagens synthesized by chick-embryo tibiotarsus, rabbit growth-plate cartilage and chick chondrocytes grown in three-dimensional gels. However, in contrast with the collagen synthesized in these three systems, the short-chain collagen synthesized by calf growth-plate hypertrophic cartilage is stabilized by disulphide bonds localized within the pepsin-resistant triple-helical collagenous domains of these molecules.     

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.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.