Collagen type IX from human cartilage: a structural profile of intermolecular cross-linking sites.
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ABSTRACT:
SUBMITTER: Diab M
PROVIDER: S-EPMC1217044 | biostudies-other | 1996 Feb
REPOSITORIES: biostudies-other
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Project description:Type IV collagen could not be extracted from human placenta using 6M-urea containing 10mM-dithiothreitol, indicating that the type IV molecule is stabilized within the basement membrane by covalent cross-links. However, various forms of type IV collagen molecule were extractable by means of increasingly severe proteolytic conditions. Type IV collagen tetramers ('spiders') lacking only the C-terminal globular region (NC1) were further digested to the 'long-form' 7S fragment and an assortment of helical rod-like molecules derived from the 'leg' region. These molecules were separated by salt fractionation and examined by rotary-shadowing electron microscopy. Isolation of these fractions from placenta which had been reduced with NaB3H4 revealed that both the 7S (N-terminal) and C-terminal regions contained significant proportions of reducible lysine-derived cross-links. These cross-links were shown to be exclusively the stable oxo-imine hydroxylysino-5-oxonorleucine. The number of the cross-links per molecule was significantly lower than might be expected in order to fully stabilize the molecule. These results suggest that the keto-imine cross-links in type IV collagen have been stabilized in part by conversion into an unknown non-reducible form, although a sensitive immunoassay failed to show the presence of any pyridinoline. Comparison with the fibrous collagen from placenta suggested that the rate of this conversion is much greater in basement-membrane collagen.
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Project description:The location of pyridinoline in 18-month-old bovine articular cartilage was investigated by fractionation of CNBr-derived peptides by ion-exchange chromatography and gel filtration. Two peptides, PCP1 and PCP2, were isolated and were shown to contain stoichiometric amounts of pyridinoline. From its amino acid composition and sequence studies, peptide PCP1 was shown to comprise two C-terminal non-helical chains (CB14) linked through pyridinoline to the alpha 1(II)-CB12 portion of the helix. The CB14 chains appeared to be labile at their C-terminal ends, resulting in lower-than-expected amounts of homoserine, and only the N-terminal portion of the peptide was sequenced. Similar studies of peptide PCP2 showed that it contained two N-terminal non-helical chains (CB4) linked to the alpha 1(II)-CB9,7 portion of the helix. The isolated peptides therefore confirmed the function of pyridinoline in stabilizing the 4D stagger of adjacent molecules. The possibility that the cross-link could act both as an intra- and an inter-microfibrillar cross-link was considered. A mechanism of formation of pyridinoline was postulated that, together with other evidence, appears to support the view that, in cartilage, pyridinoline acts primarily as an intramicrofibrillar cross-link and does not contribute to increased stability during maturation through lateral aggregation and bonding of filaments.
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Project description:Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the ?1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K(d) values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.
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Project description:Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, ?1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving ?1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998-23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen.
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Project description:A method for the isolation and purification of pyridinoline from bone collagen was developed, with the use of sulphonated polystyrene resins. The analytical techniques were used to quantify pyridinoline, for which hydroxyallysine is a known precursor, in a wide range of tissues. The structure of pyridinoline proposed by Fujimoto, Moriguchi, Ishida & Hayashi [(1978) Biochem. Biophys. Res. Commun. 84, 52-57] was confirmed by 13C-n.m.r. spectroscopy and fast-atom-bombardment mass spectrometry. At concentrations greater than about 0.1 mM, pyridinoline exhibited altered fluorescence properties that were consistent with excimer formation. From alkali hydrolysates of several different tissues, a fluorescent compound was purified by gel filtration and ion-exchange chromatography and was shown to be galactosylpyridinoline. This derivative was very labile to acid treatment compared with the bifunctional cross-link analogues, and was completely converted into free pyridinoline by heating at 108 degrees C for 8 h in 0.1 M-HCl. Galactosylpyridinoline was also partially converted into free pyridinoline by prolonged alkali hydrolysis. This lability, which could also apply to other multifunctional cross-link derivatives, may explain the fact that no disaccharide derivatives of pyridinoline were isolated.
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Project description:Bovine epiphysial cartilage synthesizes both proteoglycan (PG) and non-PG forms of type IX collagen in a ratio of approx. 2:1. The PG form with its attached glycosaminoglycan on the alpha 2(IX) chain is the major form in the medium, whereas both forms are found in the tissue. The results are discussed with regard to cartilage matrix organization.
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Project description:The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or 'strength') was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.
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Project description:BACKGROUND: Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. RESULTS: Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. CONCLUSION: Matrilin-4, collagen XII, thrombospondin-4, fibronectin, ?ig-h3, and epiphycan are components of the in vivo collagen IX interactome. SIGNIFICANCE: We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level, whereas matrilin-4 was verified as a novel collagen IX-binding protein. Furthermore, changes in TGF?-induced protein ?ig-h3 and fibronectin abundance were found in the collagen IX knock-out but not associated with COMP ablation, indicating specific involvement in the abnormal collagen IX null cartilage. In addition, the more widespread expression of collagen XII in the collagen IX-deficient cartilage suggests an attempted compensatory response to the absence of collagen IX. Our differential proteomic analysis of cartilage is a novel approach to identify candidate matrix protein interactions in vivo, underpinning further analysis of mutant cartilage lacking other matrix components or harboring disease-causing mutations.
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Project description:An important aspect in cartilage ageing is accumulation of advanced glycation end products (AGEs) after exposure to sugars. Advanced glycation results in cross-links formation between the collagen fibrils in articular cartilage, hampering their flexibility and making cartilage more brittle. In the current study, we investigate whether collagen cross-linking after exposure to sugars depends on the stretching condition of the collagen fibrils. Healthy equine cartilage specimens were exposed to l-threose sugar and placed in hypo-, iso-, or hyper-osmolal conditions that expanded or shrank the tissue and changed the 3D conformation of collagen fibrils. We applied micro-indentation tests, contrast enhanced micro-computed tomography, biochemical measurement of pentosidine cross-links, and cartilage surface color analysis to assess the effects of advanced glycation cross-linking under these different conditions. Swelling of extracellular matrix due to hypo-osmolality made cartilage less susceptible to advanced glycation, namely, the increase in effective Young's modulus was approximately 80% lower in hypo-osmolality compared to hyper-osmolality and pentosidine content per collagen was 47% lower. These results indicate that healthy levels of glycosaminoglycans not only keep cartilage stiffness at appropriate levels by swelling and pre-stressed collagen fibrils, but also protect collagen fibrils from adverse effects of advanced glycation. These findings highlight the fact that collagen fibrils and therefore cartilage can be protected from further advanced glycation ("ageing") by maintaining the joint environment at sufficiently low osmolality. Understanding of mechanochemistry of collagen fibrils provided here might evoke potential ageing prohibiting strategies against cartilage deterioration. © 2018 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:1929-1936, 2018.
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