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PubMed1998

Physical and linkage mapping of the gene for the alpha3 chain of type IX collagen, COL9A3, to human chromosome 20q13.3.

Type IX collagen is a minor cartilage component which associates with mixed fibrils of types II/XI collagen. We have determined the precise physical and genetic locations for the gene encoding the alpha3 chain of type IX collagen, COL9A3. Utilizing fluorescence in situ hybridization, radiation hybrid mapping, and multipoint linkage analysis, we have mapped COL9A3 to human chromosome 20q13.3, 13 cM telomeric to D20S173.

Cartilage↗

PubMed2004

Gene expression of collagen types IX and X in the lumbar disc.

OBJECTIVE: To study gene expression of collagen types IX and X in human lumbar intervertebral discs during aging and degeneration and to explore the role of collagen types IX and X in disc degeneration. METHODS: Fetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene. RESULTS: In fetal intervertebral discs, positive mRNA hybridization signals of type IX collagen were concentrated in the nucleus pulposus and the inner layer of anulus fibrosus. Interstitial matrix of the nucleus pulposus also showed positive type X collagen staining. Positive mRNA hybridization signals of types IX and X were not detected in the middle and outer layers of anulus fibrosus. In adult specimens, expression of type IX collagen mRNA was markedly decreased. No hybridization signals of type X collagen was observed. As for pathological specimens, there was no gene expression of type IX collagen. In severe degenerated discs from adults, there were focal positive expressions of type X collagen. CONCLUSIONS: Obvious changes of collagen gene expression occur with aging. Expression of type IX collagen decreases in adult and pathological discs. Results of type X collagen expression suggest that type X collagen is expressed only in older adult and senile discs (i.e., when disc degeneration has already reached a terminal stage), indicating the terminal stage of degeneration.

Adolescent↗

PubMed1999

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.

Amino Acids↗

PubMed1984

Cartilage type IX collagen is cross-linked by hydroxypyridinium residues.

Type IX collagen, a recently discovered, unusual protein of cartilage, has a segmented triple-helical structure containing interchain disulfides. Its polymeric form and function are unknown. When prepared by pepsin from bovine articular cartilage, type IX collagen was found to contain a high concentration of hydroxypyridinium cross-links, similar to that in type II collagen. Fluorescence spectroscopy located the hydroxylysyl pyridinoline and lysyl pyridinoline cross-linking residues exclusively in the high-molecular-weight collagen fraction, from which they were recovered predominantly in a single CNBr-derived peptide. The results point to a structural role for type IX collagen in cartilage matrix, possibly as an adhesion material to type II collagen fibrils.

Animals↗

PubMed2006

Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16-->p15.

The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs) equally distributed in the gene were detected in a mutation scan of eight unrelated Hanoverian warmblood stallions, including one SNP that affects the amino acid sequence of COL9A2. Comparative analyses between horse, human, mouse and rat indicate that the chromosomal location of equine COL9A2 is in agreement with known chromosomal synteny relationships. The comparison of the gene structure and transcript revealed a high degree of conservation towards the other mammalian COL9A2 genes. We chose three informative SNPs for association and linkage disequilibrium tests in three to five paternal half-sib families of Hanoverian warmblood horses consisting of 44 to 75 genotyped animals. The test statistics did not reach the significance threshold of 5% and so we could not show an association of COL9A2 with equine osteochondrosis.

Amino Acid Sequence↗

PubMed1995

Molecular cloning of the alpha 3 chain of human type IX collagen: linkage of the gene COL9A3 to chromosome 20q13.3.

Type IX collagen is composed of three polypeptides derived from the human genes COL9A1, COL9A2, and COL9A3 that assemble to form a mature collagen molecule with the structure alpha 1(IX)alpha 2(IX)alpha 3(IX). We have identified overlapping cDNA and genomic clones that encode for the entire alpha 3 chain of human type IX collagen. Tryptic peptides from the human alpha 3(IX) collagen chain were subjected to N-terminal amino acid sequencing, and a stretch of 124 contiguous amino acids that included the NC1, COL1, and NC2 domains was obtained. Degenerate oligonucleotide primers were designed based on the amino acid sequences of the human tryptic peptides as well as bovine peptides and sequences from chicken cDNA clones. These primers were used to amplify three overlapping PCR products that covered the majority of the human alpha 3(IX) collagen. PCR products were then used to identify overlapping cDNA clones from a human chondrocyte library. A lambda genomic clone was identified that contained the 5'-most exon that encodes the signal peptide to complete the entire structure of the human alpha 3(IX) collagen chain. Genomic amplification identified a single-strand conformational polymorphism in COL1 that was used to map COL9A3 to chromosome 20q13.3 by linkage analysis. The present study completes the structure of human type IX collagen, and linkage for COL9A3 completes the genomic mapping of cartilage collagen genes. These data will greatly assist the genetic screening of families with degenerative cartilage and eye diseases by allowing investigators to screen for a complete set of candidate collagen gene markers.

Amino Acid Sequence↗

PubMed1992

Cloning and developmental expression of the alpha 3 chain of chicken type IX collagen.

Fibrous and nonfibrous collagens comprise two major groups within the collagen family and both groups are found in a diverse variety of tissue fabrics. Type IX collagen is in the nonfibrous group; three different subunits of type IX collagen have been identified and the alpha 1 and alpha 2 subunits have been cloned. Using molecular cloning methods we have isolated, from an embryonic chicken cartilage library, cDNA clones which code for the entire alpha 3 chain of chicken type IX collagen. The cDNA clones encompass 2416 base pairs which have a conceptual open reading frame for a protein containing 675 amino acids including 193 Gly-X-Y repeats. These collagen repeats are in three separate domains which are interspersed with four major noncollagen domains. The collagen repeats also have four minor interruptions. This chain organization directly aligns with both the alpha 1 and alpha 2 chains of chicken type IX collagen. Comparison of the deduced amino acid sequence with peptide sequences of type IX collagens shows identity with 95 of the 96 known residues of the chicken alpha 3 chain and 81 of the 98 known residues of the bovine alpha 3 chain. The identical residues match those in five peptide fragments, two from the bovine protein and three from the chicken protein. The chicken and bovine alpha 3 chains have conserved cross-linking sites, separated by 137 residues which span 40 nm, the length of the hole zone in a collagen fibril. The NC3 domain of the chicken alpha 3 chain contains a repeat Cys-Pro motif which is present in both vertebrate and invertebrate nonfibrillar collagens. Northern blot hybridization exhibits a major mRNA of about 3.3 kilobases; this transcript is found in cartilaginous tissues in the embryo, including the developing limb and is not detected in other tissues or in the precondensation stage of limb development. The composite data delineate the primary structure of the alpha 3 chain of chicken type IX collagen, show its close relationship to the alpha 1 and alpha 2 chains, demonstrate its mRNA transcript, and show the appearance of that transcript in tissues of the developing chick embryo.

Amino Acid Sequence↗

PubMed1993

The role of type IX collagen in osteoarthritis and rheumatoid arthritis.

Articular cartilage consists of a cellular and an extracellular compartment. The extracellular compartment is composed of collagen, proteoglycans, and noncollagenous matrix proteins. Collagen resists tensile forces and serves as an organizing skeleton that helps maintain the structural integrity of cartilage. Fourteen types of collagen have been identified. The cartilage-specific collagens are type II (the principal component), type IX, type X, and type XI. Type IX collagen is hypothesized to be the "glue" that holds together the type II collagen latticework of articular cartilage. Degradation of type IX collagen by proteolytic enzymes has been observed in the primary stages of osteoarthritis and rheumatoid arthritis. This degradation is thought to represent an "ungluing" of the collagen scaffold and has been proposed as the mechanism for the degenerative changes seen in osteoarthritic and rheumatoid cartilage.

Arthritis, Rheumatoid↗

PubMed1985

Type IX collagen from sternal cartilage of chicken embryo contains covalently bound glycosaminoglycans.

Type IX collagen was isolated as a native protein from chicken embryo sternal cartilages and purified to homogeneity. Chondroitin and/or dermatan sulfate were bound covalently to one of the three polypeptide chains present in this protein containing collagenous and noncollagenous domains. Type IX collagen could be metabolically labeled with both radioactive sulfate and glycine. The protein containing either of these labels was sensitive to digestion by bacterial collagenase as well as chondroitinase ABC. Besides the glycosaminoglycans, type IX collagen contains asparagine-linked carbohydrate chains because the protein could be labeled with radioactive mannose and no glycosaminoglycans other than those mentioned above were present. The melting curve indicated that, in contrast to interstitial collagens, this molecule contains at least two disulfide-bonded collagenous domains with distinct thermal stabilities.

Animals↗

PubMed1987

A comparison of the effects of different substrata on chondrocyte morphology and the synthesis of collagen types IX and X.

Embryonic chick sternal chondrocytes were cultured either within three dimensional gels of type I collagen, type II collagen or agar, or as monolayers on plastic dishes coated with air-dried films of these matrix macromolecules. It was observed that cell shape and cell growth varied markedly between the different culture conditions. Flattened monolayers of cells on plastic or films of type I or type II collagen, proliferated more rapidly and reached a higher final cell density per culture than the more rounded cells found in the cultures on agar films or within three-dimensional gels. Biosynthetic studies demonstrated that in addition to the synthesis of type II collagen, all the cultures were producing collagen types IX and X. Chondrocytes cultured on plastic or films of the different matrix macromolecules all showed a similar expression of types IX and X collagen, independent of whether they displayed a flattened or round cell morphology. In contrast, marked variations in the proportions of the minor collagens, particularly type X collagen, were observed when the cells were cultured within three-dimensional gels. The data suggest that direct interaction of the cell surface with matrix constituents displaying a particular spatial array could be an important aspect in the control of type IX and X collagen expression by chondrocytes.

Animals↗

PubMed1988

The structure of human collagen type IX and its organization in fetal and infant cartilage fibrils.

Human collagen type IX was isolated from the media of organ cultures of fetal or infant hyaline cartilage. It consisted of three distinct, disulfide-bonded polypeptides of 115, 84, and 72 kDa, respectively. Digestion with chondroitinase ABC reduced the apparent molecular mass of the 115-kDa chain to about 65 kDa demonstrating that also human collagen type IX is a proteoglycan. In the electron microscope, the molecule had a rigid rod-like structure with characteristic kinks and with a globular domain at one end. Digestion of human collagen type IX with pepsin leads to somewhat heterogeneous fragments. Affinity-purified antibodies to the mixture of fragments specifically reacted with the fragment HMW without cross-reaction with chicken HMW. LMW of both species were recognized to the same low extent. Mechanically generated fibril fragments from human fetal cartilage were heterogeneous in diameter. Significantly, they could be immunostained for collagen type IX in a D-periodic pattern and regardless of the fibril diameter. Some fibrils were poorly labeled, again independently of the diameter. Therefore, the role of collagen type IX in cartilage probably is not to control directly the lateral growth during fibrillogenesis but rather to stabilize the fibril network.

Animals↗

PubMed1998

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development.

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.

Animals↗

PubMed1989

Degradation of type IX collagen by matrix metalloproteinase 3 (stromelysin) from human rheumatoid synovial cells.

The degradation of type IX collagen, a minor collagen in cartilage, was examined by treatment with three different types of matrix metalloproteinases (MMPs) purified from the culture medium of rheumatoid synovial cells. Neither MMP-1 (collagenase) nor MMP-2 (so-called 'gelatinase') could digest type IX collagen, but MMP-3 (stromelysin) readily degraded it into smaller fragments. This suggests that MMP-3 may be responsible for the pathological degradation and/or normal turnover of type IX collagen.

Animals↗

PubMed1999

Type IX collagen immunoreactive peptides in synovial fluids from arthritis patients.

OBJECTIVES: To determine whether type IX collagen-related peptides can be detected in the synovial fluids of arthritis patients and to assess their potential as molecular markers of arthritis. PATIENTS/METHODS: Synovial fluids from a set of carefully diagnosed arthritis patients and from healthy volunteers were used. Hydroxyproline assays were carried out to determine the content and concentration of collagen. Collagen cross-link determinations were conducted by reversed-phase HPLC. SDS PAGE and immunoblotting were used to identify the collagenous components, and N-terminal sequencing was performed to confirm these identities. RESULTS: All the synovial fluids were found to contain measurable amounts of collagen at similar concentrations. This appeared to be mainly high-molecular-weight material consisting of type I and type IX collagens, but not type II collagen. However, other smaller molecular weight type IX immunoreactive peptides were detected which were more apparent in the synovial fluids from arthritis patients. These peptides were also found to contain non-collagenous material. Collagen cross-links were also present in the arthritis synovial fluids. CONCLUSION: Collagenous material can be detected in all synovial fluids and the presence of pyridinoline cross-links indicates that at least some of this is derived from a mature collagen matrix. Type IX immunoreactive peptides were identified, but were found to contain significant amounts of non-collagenous material, and their presence, even at lower levels, in synovial fluids from normal subjects limits their potential for use as molecular markers of disease. Nevertheless, this is the first report of type IX collagen-related fragments in synovial fluids.

Adult↗

PubMed2000

Cartilage oligomeric matrix protein interacts with type IX collagen, and disruptions to these interactions identify a pathogenetic mechanism in a bone dysplasia family.

Cartilage oligomeric matrix protein (COMP) and type IX collagen are key structural components of the cartilage extracellular matrix and have important roles in tissue development and homeostasis. Mutations in the genes encoding these glycoproteins result in two related human bone dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, which together comprise a "bone dysplasia family." It has been proposed that these diseases have a similar pathophysiology, which is highlighted by the fact that mutations in either the COMP or the type IX collagen genes produce multiple epiphyseal dysplasia, suggesting that their gene products interact. To investigate the interactions between COMP and type IX collagen, we have used rotary shadowing electron microscopy and real time biomolecular (BIAcore) analysis. Analysis of COMP-type IX collagen complexes demonstrated that COMP interacts with type IX collagen through the noncollagenous domains of type IX collagen and the C-terminal domain of COMP. Furthermore, peptide mapping identified a putative collagen-binding site that is associated with known human mutations. These data provide evidence that disruptions to COMP-type IX collagen interactions define a pathogenetic mechanism in a bone dysplasia family.

Amino Acid Sequence↗

PubMed1991

Type IX collagen gene expression during limb cartilage differentiation.

Changes in the steady-state levels of mRNAs for the alpha 1(IX) and alpha 2(IX) polypeptide chains of cartilage-characteristic type IX collagen were examined during the course of chick limb chondrogenesis in vitro and in vivo. Cytoplasmic type IX collagen mRNAs begin to accumulate at the onset of overt chondrogenesis in high density micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of type IX collagen mRNA accumulation at condensation coincides with the initiation of accumulation of cartilage proteoglycan core protein mRNA and with a striking increase in type II collagen mRNA accumulation. Following condensation in vitro, there is a concomitant progressive increase in cytoplasmic type IX collagen, core protein, and type II collagen mRNA levels which parallels the progressive accumulation of cartilage matrix. Type IX collagen mRNAs also begin to accumulate at the initiation of overt chondrogenesis in vivo in the chondrogenic central core of the developing limb bud. In contrast, little, or no type IX collagen mRNAs are detectable in the nonchondrogenic peripheral regions of the developing limb bud.

Animals↗

PubMed1992

Extraction and characterisation of the intact form of bovine vitreous type IX collagen.

We provide the first biochemical characterisation of intact type IX collagen extracted from bovine vitreous. It possesses a shortened alpha 1(IX) chain (M(r) 64K) compared to its cartilage counterpart (M(r) 84K). All the vitreous type IX collagen is in a proteoglycan form, its glycosaminoglycan constituent being a chondroitin/dermatan sulphate component of M(r) 15-60K attached to the alpha 2(IX) chain. This contrasts with previous findings in chick vitreous where a very long glycosaminoglycan chain of M(r) approximately 350K was demonstrated.

Animals↗

PubMed1998

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.

Animals↗