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PubMed1989

Characterization of mammalian type IX collagen fragments from limited pepsin digests of a transplantable swarm rat chondrosarcoma.

Collagenous fragments from type IX molecules have been solubilized by limited pepsin proteolysis of a transplantable rat chondrosarcoma and isolated by selective salt precipitation. Chromatography of the solubilized precipitate on CM-cellulose under nondenaturing conditions yielded three fractions. When examined by polarimetry, the material in all three fractions revealed native collagen helical structure with melting points which ranged from 31-37 degrees C. When the fractions were denatured and rechromatographed on a column of agarose beads, the most acidic fraction eluted as 13-kDa polypeptides with and without prior reduction and alkylation. In contrast, the second and third fractions eluted as 100-kDa and 30-kDa polypeptides prior to reduction, but on reduction and alkylation produced reducible products of 34 kDa and 10 kDa, respectively. The general compositional features of the three fractions closely resemble comparable collagenous fragments of type IX collagen from other species. The denaturation products of the 13-kDa nonreducible, the 30-kDa reducible, and the 100-kDa reducible fractions were sequentially purified by CM-cellulose and reversed-phase chromatography to resolve the chain constituents. The isolated 10-kDa, 13-kDa, and 34-kDa chains were cleaved with CNBr, and the cleavage products identified by gel-permeation chromatography. Two 13-kDa polypeptides, 13K2 and 13K3, which did not contain any methionyl residues and were not cleaved with CNBr, were digested with trypsin, and the peptide digests were resolved by reversed-phase chromatography. Comparisons of the CNBr and tryptic cleavage products demonstrate that the three major collagenous fragments are composed of three unique polypeptides. A partial amino acid sequence of an 8-kDa CNBr peptide derived from a purified 10-kDa peptide (10K1) matches identically the amino acid sequence derived from a cDNA sequence in the rat alpha 1(IX) chain (Kimura et al., 1989). These studies, then, present convenient procedures useful in the isolation of mammalian type IX collagen fragments and describe features of the rat molecule, indicating that it is similar to the avian counterpart with respect to chain composition and general molecular structure.

Amino Acid Sequence↗
PubMed1989

Problems in the immunolocalization of type IX collagen in fetal calf cartilage using a monoclonal antibody.

Monoclonal antibodies were prepared against the pepsin-resistant fragments (X1-X3) of bovine type IX collagen. One of the five hybridomas that gave a positive reaction in an enzyme-linked immunosorbent assay was selected (H1a) for structural analysis and immunolocalization of type IX collagen. The location of the epitope for H1a was deducted from immunoblots and electron microscopic observations after rotary shadowing. The H1a antibody binds to one end of the longest X2, X3, X4 molecules, and preferentially 40-55nm from one end of X1 molecules thus, on or near the noncollagenous domain, NC2. Different immunolocalizations of type IX collagen in the superficial, middle and deep zones of fetal calf epiphyseal cartilage were observed depending on the thickness of the section and on hyaluronidase digestion conditions. In the middle and deep zones, staining with H1a throughout the matrix was obtained only with thin sections (5 microns) and digestion for 1 h at 37 degrees C. With thick sections (15 microns) or with digestion for 1 h at 24 degrees C, staining was restricted to the pericellular regions. Staining throughout the matrix was obtained in the superficial zone under all experimental conditions. Without hyaluronidase treatment, no immunofluorescent staining was seen with either H1a or polyclonal antibody to type II collagen, indicating that type IX collagen is present throughout the matrix in the different zones of fetal calf cartilage. This result is in good accordance with the recent demonstration of common cross-links between type II and type IX collagen in chicken and bovine cartilage. However, the preferential unmasking of type IX collagen antigenic sites in the pericellular regions of middle and deep zones of fetal calf cartilage does not preclude the presence in that region of a special pericellular organization of the collagenous network.

Animals↗
PubMed1991

Collagen types IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins.

To examine the regulation of collagen types IX and X during the hypertrophic phase of endochondral cartilage development, we have employed in situ hybridization and immunofluorescence histochemistry on selected stages of embryonic chick tibiotarsi. The data show that mRNA for type X collagen appears at or about the time that we detect the first appearance of the protein. This result is incompatible with translational regulation, which would require accumulation of the mRNA to occur at an appreciably earlier time. Data on later-stage embryos demonstrate that once hypertrophic chondrocytes initiate synthesis of type X collagen, they sustain high levels of its mRNA during the remainder of the hypertrophic program. This suggests that these cells maintain their integrity until close to the time that they are removed at the advancing marrow cavity. Type X collagen protein in the hypertrophic matrix also extends to the marrow cavity. Type IX collagen is found throughout the hypertrophic matrix, as well as throughout the younger cartilaginous matrices. But the mRNA for this molecule is largely or completely absent from the oldest hypertrophic cells. These data are consistent with a model that we have previously proposed in which newly synthesized type X collagen within the hypertrophic zone can become associated with type II/IX collagen fibrils synthesized and deposited earlier in development (Schmid and Linsenmayer, 1990; Chen et al. 1990).

Animals↗
PubMed1985

Biosynthesis of type IX collagen during chick limb development.

We analyzed the collagens synthesized by developing chick limbs (stages 22 to 34). Type IX collagen synthesis started at stage 26, concurrently with the chondrogenic differentiation of limb mesenchyme, and gradually increased during subsequent stages. By stage 34, the central cartilaginous region of the limbs substantially synthesized type IX collagen, in addition to cartilage-specific type II collagen, while the outer non-cartilaginous region of the limbs synthesized predominantly type I collagen. The present study indicates that type IX collagen is cartilage-specific and can be used as a marker for the chondrogenic phenotype.

Animals↗
PubMed2002

Immunogenicity of recombinant type IX collagen in murine collagen-induced arthritis.

OBJECTIVE: Past attempts to isolate type IX collagen (CIX) from cartilage using limited proteolysis yielded partially degraded material. Recent application of recombinant technology, however, has allowed the preparation of intact native CIX. We used the murine collagen-induced arthritis model to characterize the immunologic properties of recombinant human CIX (rCIX) produced using a baculovirus expression system. METHODS: A panel of B10 congenic mice was immunized with rCIX emulsified with Freund's complete adjuvant (CFA). The ability of the rCIX to induce tolerance and suppress arthritis was determined by administration intravenously or orally before challenge with CII/CFA. RESULTS: None of the mice immunized with rCIX developed overt arthritis, although 2 of 5 HLA-DR1 transgenic mice developed limited digital erythema and swelling. Recombinant CIX administered by either route effectively induced suppression of arthritis, although the suppression was less pronounced than that induced with CII. Immune responses to CIX and CII were specific, suggesting that bystander suppression, rather than cross-reactivity with CII, was instrumental in suppressing arthritis. CONCLUSION: These data show that CIX down-regulates arthritis in mice while having no associated risk of inducing arthritis.

Administration, Oral↗
PubMed1992

Differential co-expression of long and short form type IX collagen transcripts during avian limb chondrogenesis in ovo.

Using RNA blot analysis of developmentally staged avian limb buds, we demonstrate that transcripts of several cartilage marker genes appear in limb tissue prior to overt chondrogenesis. Type II collagen mRNA, cartilage proteoglycan core protein mRNA, alpha 2(IX) collagen mRNA, and transcripts of the short form alpha 1(IX) collagen chain derived from the downstream promoter are co-expressed in limb tissue approximately 24-36 hours before the appearance of the respective polypeptides in differentiating cartilagenous tissue. Transcripts of the long form alpha 1(IX) collagen chain derived from the upstream promoter appear somewhat later in development; nearly coincident with the immunolocalization of type IX collagen in the cartilage elements of the limb. The spatial distribution of type II and type IX collagen transcripts was analyzed by in situ hybridization. Type II collagen and the long form alpha 1(IX) collagen transcripts co-localized in the chondrogenic elements of the developing forelimb. In contrast, short form alpha 1(IX) collagen transcripts which lack the 5' region encoding the NC4 globular amino-terminal domain were distributed throughout the non-chondrogenic, non-myogenic mesenchymal regions of the limb and were not detectable above background levels in the limb chondrogenic elements. The precocious appearance of several cartilage marker gene transcripts prior to chondrogenesis suggests that multiple levels of gene regulation including alternative promoter use, alternative RNA splicing, alternative polyadenylation, and other post-transcriptional as well as translational mechanisms are active prior to, and during avian limb chondrogenesis.

Animals↗
PubMed1993

Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen.

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer alpha 1(IX)alpha 2(IX)alpha 3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C-propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C-terminal triple helical disulfide-bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide-bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (alpha 1)3, (alpha 2)3 and alpha 1 alpha 2 alpha 3. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.

Animals↗
PubMed2006

Pathogenesis of osteoarthritis-like changes in the joints of mice deficient in type IX collagen.

OBJECTIVE: To examine the pathogenetic mechanisms of osteoarthritis (OA)-like changes in Col9a1-/- mice, which are deficient in type IX collagen. METHODS: Knee joints and temporomandibular joints (TMJs) from Col9a1-/- mice and their wild-type (Col9a1+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, degraded type II collagen, and the discoidin domain receptor 2 (DDR-2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle. RESULTS: Histologic analysis showed age-dependent OA-like changes in the knee and TMJs of Col9a1-/- mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP-13 and DDR-2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1-/- mice than in their wild-type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1-/- mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1-/- mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites. CONCLUSION: Lack of type IX collagen in Col9a1-/- mice results in age-dependent OA-like changes in the knee joints and TMJs.

Animals↗
PubMed2005

Evidence for a role of the genomic region of the gene encoding for the alpha1 chain of type IX collagen (COL9A1) in hip osteoarthritis: A population-based study.

OBJECTIVE: Type IX collagen proteoglycan is an important protein in collagen networks and has been implicated in hip osteoarthritis (OA). We studied 2 COL9A1 markers (509-8B2 and 509-12B1) in relation to radiographic OA, within the framework of the Rotterdam Study, a population-based study of 7,983 subjects ages 55 years and older. METHODS: We used 2 different designs, as follows: 1) a linkage study of 83 probands with multiple joints affected with radiographic OA and their 221 siblings, yielding 445 sibpairs who participated in the study, and 2) an association study in a series of 71 patients with radiographic hip OA and 269 controls without radiographic OA. All subjects were characterized for the 2 COL9A1 markers, 509-8B2 and 509-12B1. The mean test was used to assess the proportion of alleles shared in concordantly affected and unaffected sibpairs. The chi-square test was used to compare the allele distributions in patients and controls. RESULTS: Affected sibpairs with radiographic hip OA shared alleles identical by descent at markers 8B2 and 12B1 significantly more often than expected (mean +/- SD 0.66 +/- 0.07 and 0.65 +/- 0.08, respectively; P < 0.05). No excess sharing for radiographic OA was observed at other joint sites. When comparing the frequency of marker 8B2 and 12B1 alleles in subjects with radiographic OA and controls, the frequency of 8B2 alleles in subjects with radiographic OA differed significantly(P = 0.01) from that in controls. CONCLUSION: Our data suggest that susceptibility for hip OA is conferred within or close to the COL9A1 gene in linkage disequilibrium with the COL9A1 509-8B2 marker.

Aged↗
PubMed1986

Type IX collagen is a potent inducer of PGE2 and interleukin 1 production by human monocyte macrophages.

Type IX collagen and its collagenous fragments are potent stimulatory agents on human blood mononuclear cells for the production of prostaglandin E2 and interleukin 1/mononuclear cell factor. Type IX collagen is 2-4-fold more potent that type I and II and 1 alpha-, 2 alpha- and 3 alpha-collagens. This property may be important in the destructive process of cartilage in inflammatory diseases.

Cells, Cultured↗
PubMed1985

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.

Amino Acid Sequence↗
PubMed2003

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.

Alleles↗
PubMed1988

D-periodic distribution of collagen type IX along cartilage fibrils.

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.

Animals↗
PubMed1986

Identification of the type IX collagen polypeptide chains. The alpha 2(IX) polypeptide carries the chondroitin sulfate chain(s).

Type IX collagen has recently been shown to contain glycosaminoglycan chain(s) and furthermore to be immunologically identical with proteoglycan Lt (Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1985) J. Biol. Chem. 260, 4758-4763). Here we demonstrate that the chondroitin sulfate carrying 115-kDa polypeptide of type IX collagen corresponds to the alpha 2(IX) chain. In addition the 84- and 68-kDa polypeptides were identified as the alpha 1(IX) and the alpha 3(IX) chains, respectively. This conclusion is based on a comparison of the tryptic fingerprints of the 84-, 115-, and 68-kDa chains of type IX collagen on high performance liquid chromatography with the similarly treated C2, C3, and C5 chains of the peptic fragment HMW. In addition, we provide evidence that both the C3 and C4 components of HMW are derived from the alpha 2(IX) chain.

Amino Acids↗
PubMed1997

Immunolocalization of type IX collagen in normal and spontaneously osteoarthritic canine tibial cartilage and isolated chondrons.

OBJECTIVE: The pericellular localization of type IX collagen in avian and mammalian hyaline cartilages remains controversial, while its distribution during osteoarthritic degeneration is poorly understood. This study aimed to compare and contrast the immunohistochemical distribution of type IX collagen in normal mature and spontaneously osteoarthritic canine tibial cartilage. DESIGN: Thick vibratome sectioning techniques were evaluated and compared with isolated chondrons using a range of streptavidin-linked probes in combination with light, confocal and transmission electron microscopy. RESULTS: In normal intact samples, type IX collagen was concentrated in the pericellular microenvironment, while a weaker extracellular reaction around each chondron separated the territorial matrix from the unstained interterritorial matrix. Further differentiation was evident in isolated chondrons where the fibrous pericellular capsule stained more intensely than the tail and interconnecting segments between columnated chondrons. Two regions of type IX reactivity were identified in osteoarthritic tissue: an intensely stained superficial reactive region below the eroding margins, and normal deep layer cartilage where pericellular staining persists. The superficial reactive region was characterized by chondron swelling and chondrocyte cluster formation, a loss of pericellular type IX staining, and a significant increase in matrix staining between clusters. Disintegration and loss of fibrillar collagens was evident in both the swollen microenvironment and adjacent territorial matrices. CONCLUSIONS: The results suggest that changes in type IX distribution, expansion of the pericellular microenvironment and chondrocyte proliferation represent key elements in the chondron remodeling and chondrocyte cluster formation associated with osteoarthritic degeneration.

Animals↗
PubMed1998

Differences between the thermal stabilities of the three triple-helical domains of type IX collagen.

Fibre-forming collagens in dilute solution show highly co-operative helix-coil transitions at temperatures that are remarkably close to the body temperature of the animal from which the collagen was extracted. This close correlation holds across animal Phyla and the transition temperatures, which range from 5 degrees C to 40 degrees C, are adjusted to suit by changing the primary structure, especially the concentration of the water-bridge-enhancing hydroxyproline residue. Fibril-forming collagens are thermally stabilised by fibrillogenesis, which causes a loss of random coil configurational entropy by intermolecular and intramolecular cross-linking and by spacial confinement of the molecule within the lattice of the fibre. But this mechanism cannot apply to the full length of the type IX collagen molecule, since its COL3 arm, according to current models, projects out from the stabilising influence of the type II fibre. In this paper we examine the thermal stability of the type IX collagen molecule and its three triple-helical domains, thereby demonstrating that the COL3 arm is much more stable than the rest of the molecule. At a scanning rate of 60 deg. C/h COL3 exhibited an unfolding endotherm with a tmax at 49.0 degrees C, well above body temperature. Corresponding peak maxima for COL1 and COL2 were seen at 40.6 degrees C and 39.6 degrees C, respectively. The sizes of the thermally labile units of COL1, COL2 and COL3, calculated from the measured activation enthalpies, were 24, 28 and 28 residues, respectively, much smaller than type I (65 residues) because of the relatively short lengths of triple helix to be unfolded. However, unlike type I collagen, no regions of the required size were found completely devoid of hydroxyproline. Consequently, the intrinsic stabilities of these thermally labile units were higher than that of type I with DeltaH updownarrow DeltaS updownarrow for COL1, COL2 and COL3 being, respectively, 385 K, 371 K and 384 K, contrasting with the much lower 349 K of type I collagen. We therefore speculate that the increased thermal stability of the thermally labile units was caused by the presence of the water-bridge-enhancing residue, hydroxyproline. Finally the stabilisation of type IX collagen tissue is considered and an alternative structural organisation of the type IX molecule on the type II fibre is proposed.

Animals↗
PubMed1991

Monoclonal antibodies against bovine type IX collagen (LMW fragment): production, characterization, and use for immunohistochemical localization studies.

Four high-affinity monoclonal antibodies (MAb) which react specifically with the low molecular weight (LMW) fragment of bovine type IX collagen (BIX) have been produced in mice. On the basis of the ability of these MAb to cross-react with type IX collagen purified from human, rat, and chick cartilage and to inhibit one another in a competitive inhibition assay, we conclude that the MAb D1-9, B3-1, and B2-7 recognize unique epitopes, whereas MAb B4-5 recognizes the same epitope as B3-1. None of the MAb reacted with bovine type I, II, and XI collagen. MAb D1-9 and B3-1 were tested for their ability to bind to tissue antigen, using an immunohistochemical assay system. Positive immunoperoxidase reactions were observed in the perichondrocytic regions of human and rat costochondral cartilage. Positive responses were also detected in rat auricular cartilage, as well as in tissue obtained from the middle and inner ears of rats and mice. This report demonstrates the relative ease of producing MAb to heterologous type IX collagen and the utility of these MAb for localizing type IX collagen in cartilage and cartilage-like tissues.

Animals↗
PubMed1995

Simultaneous expression of type IX collagen and an inhibin-related antigen in proliferative myoepithelial cells with pleomorphic adenoma of canine mammary glands.

To identify an alcian blue-positive component expressed in the early stage of mammary mixed tumor and to pursue the possible involvement of a tumorigenic inducing factor, monoclonal antibodies to type IX collagen were generated and used to investigate the immunohistochemical kinetics of type IX collagen expression by the myoepithelial cell-derived chondrocyte-like cells during the development of chondrometaplasia. We also examined the expression of inhibin-related antigen using antibodies to a synthetic peptide spanning amino acids 1-30 of the inhibin alpha chain. At the earliest stage of chondrometaplasia, where myoepithelial cells began to proliferate inside the basement membrane, the cells expressed type IX collagen together with an inhibin-related antigen which was immunoreactive with the anti-inhibin peptide antibodies. The expression of the inhibin-related antigen was also demonstrated in normal embryonic chondrocytes and myoblasts, but was much less strong in mature chondrocytes and myotubes, strongly suggesting that the inhibin-related antigen is involved in the development of chondrocytes and myoblasts from undifferentiated mesenchymal cells as well as proliferating myoepithelial cells as a chondro-progenitor cell in the mammary mixed tumor. The pathophysiological significance of type IX collagen expression as a possible cell marker of the progenitor cell in myoepithelial cell-related chondrometaplasia is also discussed.

Adenoma, Pleomorphic↗