Molecular cloning of rat and human type IX collagen cDNA and localization of the alpha 1(IX) gene on the human chromosome 6

Type IX collagen is found in hyaline cartilage, where it is associated with type II collagen in quarter-staggered collagen fibrils. Chicken type IX collagen has been extensively characterized and shown to contain molecules with three triple-helical domains, interspersed with non-triple-helical sequences. The molecule contains three, genetically distinct, subunits and one of these subunits carries a covalently bound glycosaminoglycan side chain. In the present report, we describe for the first time the primary structure of mammalian type IX collagen chains, based on cloning and sequencing of cDNA from rat and human cDNA libraries. The results suggest that mammalian alpha 1(IX) chains have the same multi-domain structure as the avian protein. We also demonstrate, by in situ hybridization of chromosome spreads, that the human alpha 1(IX) collagen gene is located on the long arm of chromosome 6. The cloning of human type IX collagen cDNA provides a probe for molecular studies of human chondrodysplasias that may involve abnormalities in this extracellular collagen-proteoglycan.     

Identification of cross-linking sites in bovine cartilage type IX collagen reveals an antiparallel type II-type IX molecular relationship and type IX to type IX bonding.

Type IX collagen functions in covalent cross-linkage to type II collagen in cartilage (Eyre, D. R., Apone, S., Wu, J. J., Ericsson, L. H., and Walsh, K. A. (1987) FEBS Lett. 220, 337-341). To understand this molecular relationship better, an analysis of all cross-linking sites labeled by [3H]borohydride was undertaken using the protein prepared from fetal bovine cartilage. Sequence analysis of tryptic peptides containing the 3H-labeled cross-links showed that each of the chains of type IX collagen, alpha 1(IX), alpha 2(IX), and alpha 3(IX), contained a site of cross-linking at the amino terminus of the COL2 triple-helix to which the alpha 1(II)N-telopeptide could bond. The alpha 3(IX)COL2 domain alone also had an attachment site for the alpha 1(II)C-telopeptide. The distance between the alpha 1(II)N-telopeptide and alpha 1(II)C-telopeptide interaction sites, 137 residues, is equal to the length of the hole zone (0.6D) in a type II collagen fibril. This implies an antiparallel type II to type IX cross-linking relationship. Peptide analysis also revealed an unknown amino acid sequence linked to the COL2 cross-linking domains in both the alpha 1(IX) and alpha 3(IX) chains. Using antibodies to this novel peptide, its origin in the collagen alpha 3(IX)NC1 domain was established. In summary, the results confirm extensive covalent cross-linking between type IX and type II collagen molecules and reveal the existence of type IX-type IX bonding. These data provide a molecular basis for the proposed function of type IX collagen as a critical contributor to the mechanical stability and resistance to swelling of the collagen type II fibril framework of cartilage.     

Matrix deposition of tryptophan-containing allelic variants of type IX collagen in developing human cartilage.

Genetic polymorphisms that encode a tryptophan (Trp) residue in the triple-helical domain of the alpha2 (Trp2) or alpha3 chain (Trp3) of human type IX collagen have been linked to risk of degenerative intervertebral disc disease. To determine whether these two allelic variants express protein that may affect the extracellular matrix of cartilage in vivo, we examined the properties of resident type IX collagen in an anonymous collection of embryonic and fetal human cartilage samples screened for Trp genotypes. No difference was found in the yield and electrophoretic properties of pepsin-solubilized type IX collagen between Trp2, Trp3 and non-Trp cartilage samples. On Western blot analysis, a polyclonal antiserum raised against a synthetic peptide matching the immediate Trp-containing sequence of the Trp3 allele reacted specifically with the alpha3(IX) chain prepared from Trp3 cartilage samples. Two-dimensional peptide mapping of type IX collagen in CNBr-digests of whole tissue gave indistinguishable fingerprints for Trp2, Trp3 and control tissues, including the yield of cross-linked peptides. Analysis of one cartilage sample that was homozygous for the Trp2 allele also gave a normal yield of collagen IX, including its alpha2 chain and a normal profile of cross-linked peptides. Together, the findings indicate that both Trp2 and Trp3 allelic products are incorporated into the cross-linked fibrillar network of developing human cartilage apparently normally. Any pathological consequences are likely, therefore, to be long-term and indirect rather than from overt misassembly of matrix.     

Type X collagen, a product of hypertrophic chondrocytes.

The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment.     

Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse

Molecular mechanisms controlling the assembly of cartilage-specific types II, IX and XI collagens into a heteropolymeric network of uniformly thin, unbanded fibrils are not well understood, but collagen XI has been implicated. The present study on cartilage from the homozygous chondrodysplasia (cho/cho) mouse adds support to this concept. In the absence of alpha1(XI) collagen chains, thick, banded collagen fibrils are formed in the extracellular matrix of cho/cho cartilage. A functional knock-out of the type XI collagen molecule has been assumed. We have re-examined this at the protein level to see if, rather than a complete knock-out, alternative type XI chain assemblies were formed. Mass spectrometry of purified triple-helical collagen from the rib cartilage of cho/cho mice identified alpha1(V) and alpha2(XI) chains. These chains were recovered in roughly equal amounts based on Coomassie Blue staining of SDS-PAGE gels, in addition to alpha1(II)/alpha3(XI) collagen chains. Using telopeptide-specific antibodies and Western blot analysis, it was further shown that type V/XI trimers were present in the matrix cross-linked to each other and to type II collagen molecules to form heteropolymers. Cartilage from heterozygous (cho/+) mice contained a mix of alpha1(V) and alpha1(XI) chains and a mix of thin and thick fibrils on transmission electron microscopy. In summary, the results imply that native type XI collagen molecules containing an alpha1(XI) chain are required to form uniformly thin fibrils and support a role for type XI collagen as the template for the characteristic type II collagen fibril network of developing cartilage.     

Characterization of recombinant human type IX collagen. Association of alpha chains into homotrimeric and heterotrimeric molecules.

As type IX collagen is a minor cartilage component, it is difficult to purify sufficient amounts of it from tissues or cultured cells to study its structure and function. Also, the conventional pepsin digestion used for fibrillar collagens cannot be utilized for purifying type IX collagen, because it contains several interruptions in its collagenous triple helix. A baculovirus expression system was used here to produce recombinant human type IX collagen by coinfecting insect cells with three viruses containing full-length cDNAs for the alpha1(IX), alpha2(IX), and alpha3(IX) collagen chains together with a double promoter virus for the alpha and beta subunits of human prolyl 4-hydroxylase. Correctly folded recombinant type IX collagen was secreted, consisting of the three alpha chains in a 1:1:1 ratio and showing the expected biphasic thermal melting profile. When the individual alpha chains were expressed, disulfide-bonded homotrimers and homodimers of the alpha chains were observed. When the cells were coinfected with the viruses for all three alpha chains, heterotrimers of alpha1(IX), alpha2(IX), and alpha3(IX) were detected in cell culture medium, and the other possible combinations were less prominent. When any two of the alpha chains were co-expressed, in addition to the homodimers and homotrimers, only alpha1(IX) and alpha3(IX) chains were disulfide-bonded. The results thus suggest that the most favored molecular species is an alpha1(IX)alpha2(IX)alpha3(IX) heterotrimer, but the chains are also able to form disulfide-bonded heterotrimers of alpha1(IX) and alpha3(IX) chains and (alpha1(IX))(3), (alpha2(IX))(3), and (alpha3(IX))(3) homotrimers.     

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.     

Occurrence in chick embryo vitreous humor of a type IX collagen proteoglycan with an extraordinarily large chondroitin sulfate chain and short alpha 1 polypeptide

We have prepared a high buoyant density proteoglycan fraction from the vitreous humor of 13-day-old chick embryos. Using immunoblot analysis coupled with chondroitinase digestion, we demonstrate that the purified preparation is composed predominantly of type IX collagen-like chondroitin sulfate proteoglycan with an alpha 1(IX) chain Mr approximately 23,000 shorter than the known alpha 1 in cartilage type IX. Also different from cartilage type IX is the size of the chondroitin sulfate chain attached to the alpha 2(IX) polypeptide; its Mr is approximately 350,000 indicating that it is approximately 10 times larger in vitreous humor than in cartilage. Examination of vitreous bodies at different developmental stages indicates that a transition occurs in the size of alpha 1(IX) in a well defined temporal pattern; at about stage 31, a cartilage-type alpha 1(IX) of Mr 84,000 is the predominant species, whereas at stage 36 and thereafter, a Mr 61,000 species appears with a concomitant disappearance of the Mr 84,000 species. Immunostaining for type IX collagen followed by electron microscopic observation of 13-day-old chick embryo vitreous humor reveals a regular D-periodic arrangement of vitreous type IX collagen proteoglycan along thin fibrils. It seems possible that the chondroitin sulfate chains of extraordinarily high viscosity and high molecular weight may extend away from the fibrils, thus contributing to structural as well as functional properties of this unique matrix.