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At least 19 recordsLinked to original sources
PubMed1984

Regulation of type I collagen fibril assembly by link protein and proteoglycans.

Link protein, a glycoprotein, that is present both in cartilaginous and non-cartilaginous tissues, has previously been shown to bind to collagen and to proteoglycan. Here, we have examined the effects of link protein and proteoglycans, both alone and in combination, on the assembly of type I collagen fibrils in vitro. Link protein alone had no effect on the kinetics of fibril formation or on the size of the fibrils. Link protein, however, modulated the effects of various proteoglycans including those from bone, cartilage, cornea and sclera. Link protein had the most significant effect on fibril assembly in the presence of the low molecular weight bone proteoglycan. Although the bone proteoglycan alone had no effect on fibril formation, the fibrils were wider in the presence of link protein and proteoglycan. Cartilage proteoglycan alone increased the extent of fibril formation and the resultant fibrils were wider in diameter with a complement of incompletely assembled fibrils. In the presence of both link protein and cartilage proteoglycan, the fibrils were fully formed with the characteristic banding pattern. Further, corneal and scleral proteoglycans alone decreased the extent of fibril formation and the width of the fibrils was either unaltered or slightly decreased in the presence of the link protein. Our results indicate that both link protein and tissue-specific proteoglycans may regulate the organization of collagen fibrils in tissues.

Animals↗
PubMed1982

Preparation and characterization of an antiserum against purified proteoglycan link proteins from avian cartilage.

Link proteins have been purified from avian xyphoid process. Cartilage was extracted in 4.0 M guanidine hydrochloride and a link fraction (A1D5) was obtained by sequential cesium chloride centrifugation. Link proteins were separated from low buoyant density proteoglycans by chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The presence of contaminating proteoglycans at various purification steps was monitored in an enzyme-linked immunosorbent assay using antiserum against avian cartilage proteoglycan monomer (anti A1D1-1400 Vo). Antiserum generated against this purified link preparation (anti link[SDS]) was characterized for its ability to bind link proteins, proteoglycan monomer, and aggregate. The serum was specific only for link proteins when tested by an enzyme-linked immunosorbent assay. Some reactivity against proteoglycan monomer was observed in a Farr-type assay.

Animals↗
PubMed1975

Immunological studies of bovine nasal cartilage proteoglycan "link proteins".

Bovine nasal cartilage proteoglycan aggregates are dissociated and separated by density gradient centrifugation in 4 M guanidine into proteoglycan subunit (PGS) and glycoprotein link (GPL) fractions, the latter containing hyaluronic acid and "link proteins" responsible for aggregate formation. It was previously concluded on the basis of immunodiffusion studies that GPL has two antigenic components, one in common with PGS and one specific for the link proteins. However, in the present study it was found that antisera to PGS, which should lack link proteins, reacted with both "subunit" and "link" components of GPL, and antisera to fragments of PGS derived from the hyaluronic acid-binding portion of the molecule reacted preferentially with the link component. Reduction and alkylation of GPL led to modification of the reactions of both anti-GPL and anti-PGS sera with its link component. These immunodiffusion results indicate that the proteoglycan subunit and the link proteins are immunologically related and suggest that the link proteins may be identical with and derived from the hyaluronic acid binding portion of the proteoglycan subunit.

Animals↗
PubMed1992

Juvenile rheumatoid arthritis patients manifest immune reactivity to the mycobacterial 65-kDa heat shock protein, to its 180-188 peptide, and to a partially homologous peptide of the proteoglycan link protein.

Immune reactivity to the 65-kDa mycobacterial heat shock protein (hsp65) has been associated with arthritis in rats and humans. In this report we evaluated patients with juvenile rheumatoid arthritis for such immunity. A high proportion of affected children showed both antibody and T lymphocyte responses to hsp65 and to two related peptides: the nonapeptide 180-188 sequence of hsp65 and a partially homologous peptide of the cartilage proteoglycan link protein. The titer of circulating antibodies was generally higher in patients with clinically active disease. In contrast to the juvenile rheumatoid arthritis patients, patients with adult rheumatoid arthritis tended to have lower responses of their peripheral blood T lymphocytes to the whole hsp65 molecule. Moreover, the adult rheumatoid arthritis patients did not respond to the peptides. Thus, there appear to be immunological differences between juvenile and adult forms of rheumatoid arthritis related to hsp65 reactivity.

Adolescent↗
PubMed1992

Exclusion of human proteoglycan link protein (CRTL1) and type II collagen (COL2A1) genes in pseudoachondroplasia.

Patients with pseudoachondroplasia have a skeletal dysplasia with marked short stature. The most common cause of this condition is an autosomal dominant mutation, although autosomal recessive inheritance has been reported. Linkage to 2 cartilage-specific candidate genes, type II collagen (COL2A1) and proteoglycan link protein genes (CRTL1), was tested in 9 autosomal dominant families with pseudoachondroplasia. Tight linkage to these candidate genes was excluded with LOD scores for COL2A1 of -2.45 at theta = 0.05 and for CRTL1 of -7.28 at theta = 0.001. Discordant inheritance of the disease phenotype with each of these genes was also observed. Thus, these 2 candidate genes can be excluded as the cause of disease in these families.

Collagen↗
PubMed1984

The effect of link protein on proteoglycan aggregate structure. An electron microscopic study of the molecular architecture and dimensions of proteoglycan aggregates reassembled from the proteoglycan monomers and link proteins of bovine fetal epiphyseal cartilage.

Proteoglycan monomer and link protein were prepared from bovine fetal epiphyseal cartilage. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronic acid in the presence or in the absence of link protein at pH 7 and at pH 5. The proteoglycan solutions were spread on nitrocellulose films and examined by electron microscopy. At pH 7, the aggregates formed in the presence of link protein showed dramatic differences in their dimensions, compared with the link protein-free aggregates. The link protein-containing aggregates were five times longer and contained three times as many monomers per aggregates. The mean distance between monomers was twice as long and the spacing between monomers was more regular in the link protein-containing aggregates. Essentially the same differences between link protein-free and link protein-containing proteoglycan aggregates were observed at pH 5. These results show that link protein increases proteoglycan aggregate size by facilitating the binding of more monomers to hyaluronic acid and influences the spacing of monomers along hyaluronic acid chains.

Animals↗
PubMed1983

Age-related changes in the structure of proteoglycan link proteins present in normal human articular cartilage.

Link proteins were identified immunologically in human articular-cartilage protein preparations from various individuals. Irrespective of age, all cartilages contained three link proteins of mol.wts. 48000, 44000 and 41000. However, with increasing age, multiple additional components of mol.wts. 26000-30000 were commonly observed under conditions where disulphide bonds were reduced.

Aging↗
PubMed1982

Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate.

The hypothesis is widely held that, in growth plate during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization, and that this is the mechanism by which a noncalcifiable matrix is transformed into a calcifiable matrix. We have evaluated this hypothesis by examining the immunofluorescent localization and concentrations of proteoglycan monomer core protein and link protein, and the concentrations of glycosaminoglycans demonstrated by safranin 0 staining, in the different zones of the bovine fetal cartilage growth plate. Monospecific antibodies were prepared to proteoglycan monomer core protein and to link protein. The immunofluorescent localization of these species was examined in decalcified and undecalcified sections containing the zones of proliferating and hypertrophic chondrocytes and in sections containing the zones of proliferating and hypertrophic chondrocytes and the metaphysis, decalcified in 0.5 M EDTA, pH 7.5, in the presence of protease inhibitors. Proteoglycan monomer core protein and link protein are demonstrable without detectable loss throughout the extracellular matrix of the longitudinal septa of the hypertrophic zone and in the calcified cartilage of the metaphysis. In fact, increased staining is observed in the calcifying cartilage. Contrary to the prevailing hypothesis, our results indicate that there is no net loss of proteoglycans during mineralization and that the proteoglycans become entombed in the calcified cartilage which provides a scaffolding on which osteoid and bone are formed. Proteoglycans appear to persist unaltered in the calcified cartilage core of the trabeculae, until at last the entire trabeculae are eroded from their surfaces and removed by osteoclasts, when the primary spongiosa is replaced by the secondary spongiosa.

Animals↗
PubMed1985

An amino acid sequence common to both cartilage proteoglycan and link protein.

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. In the course of determining the primary structure of link protein, two peptides produced by digestion of rat chondrosarcoma link protein with trypsin or chymotrypsin have been selectively purified by immunoaffinity chromatography on a column of monoclonal anti-link protein antibody (8A4) immobilized to Sepharose 4B. These peptides have been sequenced using the double-coupling dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate procedure. A consensus sequence, Cys-X-Ala-Gly-Trp-Leu-X-Asp-Gly-Ser-Val-X-Tyr-Pro-Ile-X-X-Pro, obtained by comparing the affinity-isolated tryptic peptide with the affinity-isolated chymotryptic peptide and an overlapping tryptic peptide, shows homology with a sequence obtained from the NH2-terminal of a CNBr peptide from proteo glycan core protein of bovine nasal cartilage: Ser-Ser-Ala-Gly-Trp-Leu-Ala-Asp-Arg-Ser-Val-Arg-Tyr-Pro-Ile-Ser-. We suggest that the common sequence is structurally important to the function of these proteins and may be involved in the binding of both link protein and proteoglycan to hyaluronic acid.

Amino Acid Sequence↗
PubMed1987

Cartilage proteoglycan aggregates. The link protein and proteoglycan amino-terminal globular domains have similar structures.

Cartilage proteoglycan aggregates contain two components (proteoglycan monomer and link protein) which interact with each other and with hyaluronic acid. Data from amino acid sequence analysis are presented that shows that a domain of the proteoglycan, the hyaluronic acid binding region, which interacts with link protein and hyaluronic acid is very similar to link protein in terms of its primary structure. However, the pattern of glycosylation in the hyaluronic acid binding region is different from that found in link protein. After removal of N-linked oligosaccharides, the tryptically prepared hyaluronic acid binding region from rat chondrosarcoma has a mass by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 43 +/- 2 kDa. The COOH-terminal two-thirds of rat chondrosarcoma link protein, starting at residue 105, has 41.3% identity with a similar region in the hyaluronic acid binding region. We show that, in addition to the hyaluronic acid binding region, proteoglycan contains another region with similarity to the two repeating loop structures in the COOH-terminal two-thirds of link protein. This presumably corresponds to the second globular domain reported in rotary shadowing studies of cartilage proteoglycans. We have deduced the positions of all of the disulfide bonds in the hyaluronic acid binding region and find them to be in the same positions as would be expected from comparison of these sequences with link protein.

Amino Acid Sequence↗
PubMed1987

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.

Animals↗
PubMed1979

Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan.

Link proteins from bovine nasal cartilage have been purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Baker, J.R., and Caterson, B. (1979) J. Biol. Chem. 254, 2387-2393) and used to raise antisera in rabbits. A sensitive radioimmunoassay procedure utilizing binding of 125I-labeled antigen . antibody complexes to Protein A of Staphylococcus aureus has served to demonstrate the specificity of the antisera for the link proteins. The lack of reactivity with proteoglycan fractions indicates that link proteins and proteoglycan do not share antigenic determinants. This result is in accord with published cyanogen bromide peptide cleavage data (Baker, J.R., and Caterson B. (1977) Biochem. Biophys. Res. Commun. 77, 1-10) which showed proteoglycan and link protein to be structurally dissimilar. The radioimmunoassay procedure has been used to quantitate small amounts of link protein which remain associated with proteoglycan after purification by equilibrium density gradient centrifugation in 4 M guanidine HCl and by gel chromatography in sodium dodecyl sulfate.

Animals↗
PubMed1991

Distribution of cartilage proteoglycan (aggrecan) core protein and link protein gene expression during human skeletal development.

The distribution of cartilage proteoglycan core protein (aggrecan) and cartilage proteoglycan link protein was investigated by in situ hybridization during different stages of human skeletal development. Aggrecan and link protein expression were confined to chondrocytes of the developing skeleton and other cartilaginous structures. Distribution and intensity of the signal was identical with aggrecan as compared to link protein probes. Parallel to the calcification of cartilaginous matrix, chondrocytes of this area lost the expression of aggrecan and link protein specific mRNA and stayed negative throughout the following stages of skeletal development. Highest expression was found in the lower proliferative and upper hypertrophic zone whereas the resting zone showed less expression. Aggrecan gene expression was additionally investigated in iliac crest biopsies of 3 patients with pseudoachondroplasia and compared to age-matched controls. Distribution and intensity of staining revealed no abnormalities. Thus, the phenotypic changes during chondrocyte maturation are accompanied by distinct changes in aggrecan and link protein gene expression. This pattern was maintained in the growth plate of patients with pseudoachondroplasia.

Aggrecans↗
PubMed1997

Changes in messenger RNA and protein levels of proteoglycans and link protein in human osteoarthritic cartilage samples.

OBJECTIVE: To determine the steady-state messenger RNA (mRNA) levels and corresponding protein contents of major matrix components in osteoarthritic (OA) cartilage. METHODS: Steady-state levels of gene-specific mRNA (relative to GAPDH) were measured by quantitative polymerase chain reaction (PCR), and the relative levels of the corresponding proteins were determined by Western blotting. RESULTS: All mRNA levels and corresponding protein contents of aggrecan and versican (hyaluronan-binding large proteoglycans), decorin, biglycan, fibromodulin, and lumican (small proteoglycans), and link protein were higher in OA cartilage samples than in age-matched normal samples. The ratio of increase, however, was different for each component. The mRNA and protein levels of biglycan, decorin, and fibromodulin increased synchronously, whereas message for link protein and lumican were several-fold higher than expected by their measured protein contents. Versican was also detected in OA cartilage; however, the versican protein content was associated with a relatively low mRNA level. CONCLUSION: The expression of matrix components was increased in chondrocytes of OA cartilage, especially the expression of small proteoglycans, most likely due to the repair processes. A discoordinate gene expression accompanied with imbalanced accumulation of noncollagenous matrix components may contribute to the disorganization of the cartilage and the development of OA processes.

Aged↗
PubMed1981

Cartilage proteoglycan aggregate formation. Role of link protein.

Cartilage proteoglycan aggregate formation was studied by zonal rate centrifugation in sucrose gradients. Proteoglycan aggregates, monomers and proteins could be resolved. It was shown that the optimal proportion of hyaluronic acid for proteoglycan aggregate formation was about 1% of proteoglycan dry weight. The reaggregation of dissociated proteoglycan aggregate A1 fraction was markedly concentration-dependent and even at 9 mg/ml only about 90% of the aggregates were reformed. The lowest proportion of link protein required for maximal formation of link-stabilized proteoglycan aggregates was 1.5% of proteoglycan dry weight. It was separately shown that link protein co-sedimented with the proteoglycan monomer. By competition with isolated hyaluronic acid-binding-region fragments, a proportion of the link proteins was removed from the proteoglycan monomers, indicating that the link protein binds to the hyaluronic acid-binding region of the proteoglycan monomer.

Cartilage↗
PubMed1988

Biosynthesis of cartilage proteoglycan and link protein by articular chondrocytes from immature and mature rabbits.

Chondrocytes from immature and mature rabbits have been compared in biosynthetic studies with [3H] leucine and [35S]sulfate as precursors. The time course of incorporation of [3H]leucine into general protein, proteoglycan monomer core protein, and link protein and of [35S]sulfate into proteoglycan monomer has been examined. Proteoglycan monomer was isolated from the high buoyant density (p greater than 1.60) fractions of dissociative CsCl gradients and link protein by immunoprecipitation with antibody 8A4 followed by gel electrophoresis. Results based on the period of linear isotope incorporation showed that mature cells synthesize protein at about 40% of the rate of immature cells and both proteoglycan and link protein at about 20% of the rate of immature cells. The labeling rates obtained suggest that immature cells synthesize an approximate 1:1 molar ratio of link protein to proteoglycan monomer, and for mature cells this ratio is about 0.8:1. While cell layer retention of newly synthesized proteoglycan was markedly lower in mature relative to immature cell cultures, link protein retention was high in both immature and mature cultures; this finding provides an explanation for our previous observation (Plaas, A. H. K., and Sandy, J. D. (1984) Biochem, J. 220, 337-340) that link-free monomer accumulates in the medium of mature but not immature cultures. The link protein synthesized by both ages of cells and isolated from cell layer or medium was a single major species of apparent molecular mass 48-51 kDa. The results suggest that mature chondrocytes are less efficient than immature chondrocytes in the coordinated assembly of link-stabilized proteoglycan aggregates in this culture system.

Animals↗
PubMed1982

Enzyme-linked immunosorbent assay analyses of the hyaluronate-binding region and the link protein of proteoglycan aggregate.

An enzyme-linked immunosorbent assay, in combination with an independent inhibition step, was established to quantitate two components of the proteoglycan aggregate, namely link protein and hyaluronate-binding region, at concentrations below 100 ng/ml. The presence of other components of the aggregate in the samples to be tested influenced quantitation in a specific manner. The apparent antigenicity of link protein increased 2-5 times when either purified proteoglycan monomer, purified hyaluronate-binding region, or purified hyaluronate (macromolecular or oligomers) were present in the link protein samples. These findings are interpreted as showing different states of conformation or degree of association of the link protein with other components of aggregate in solution. In separate experiments, a 2-4-fold increase in the apparent antigenicity of purified hyaluronate-binding region was observed when hyaluronate molecules with at least 20 disaccharides were present in the samples. Co-incubation of the hyaluronate-binding region or proteoglycan monomer with either purified link protein or with smaller hyaluronate oligomers did not change its antigenicity in the assay. However, when hyaluronate oligomers with 8 disaccharides were included in a mixture of macromolecular hyaluronate with hyaluronate-binding region, the increase in apparent antigenicity was blocked. The results illustrate the inherent difficulties in using the enzyme-linked immunosorbent assay for the quantitation of link protein or proteoglycan monomers in samples where these macromolecules can associate with themselves or other components of proteoglycan aggregates.

Animals↗