scholarly journals Cartilage proteoglycan aggregate formation Role of link protein

1981 ◽  
Vol 197 (3) ◽  
pp. 669-674 ◽  
Author(s):  
A Franzén ◽  
S Björnsson ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Dry Weight ◽  
Aggregate Formation ◽  
Binding Region ◽  
Link Protein ◽  
Cartilage Proteoglycan ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

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.

scholarly journals Degradation of proteoglycan aggregate by a cartilage metalloproteinase. Evidence for the involvement of stromelysin in the generation of link protein heterogeneity in situ

1989 ◽  
Vol 259 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Q Nguyen ◽  
G Murphy ◽  
P J Roughley ◽  
J S Mort
Keyword(s):  
Hyaluronic Acid ◽  
Protein Component ◽  
Human Articular Cartilage ◽  
Terminal Sequence ◽  
Link Protein ◽  
Cartilage Proteoglycan ◽  
Binding Regions ◽  
Proteoglycan Aggregates ◽  
Protein Components ◽  
Hyaluronic Acid Binding

Cartilage proteoglycan aggregates were subjected to degradation by a metalloproteinase, capable of degrading proteoglycan, released from cartilage in culture. This proteinase was demonstrated to be immunologically identical with fibroblast stromelysin. An early release of hyaluronic acid-binding region and large glycosaminoglycan-attachment regions was observed. With increasing time the glycosaminoglycan-attachment regions were digested into smaller fragments and the hyaluronic acid-binding regions accumulated. The degradation of link proteins also occurred concomitantly with these events. Link proteins were converted into a component of similar size to that of the smallest native link protein component. N-Terminal sequence analysis of the three human link protein components indicated that they are all derived from the same protein core, which is closely homologous to that of the rat chondrosarcoma link protein. The two larger link proteins (Mr 48,000 and 44,000) contain the same N-terminal sequence, but they differ by the apparent presence of an N-linked oligosaccharide at residue 6 of the largest link protein component. The smallest link protein (Mr 41,000), however, has an N-terminal sequence equivalent to that commencing at residue 17 in the larger link proteins. It was found that the cartilage metalloproteinase cleaves link proteins in human neonatal cartilage proteoglycan aggregates at the His-16-Ile-17 bond, the same position at which the smallest link protein component appears to be derived naturally from the two larger link protein components. These results suggest that stromelysin secreted by chondrocytes can account for the increased accumulation of hyaluronic acid-binding regions and much of the degradation of link protein observed during aging within human articular cartilage.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Radioimmunoassay of the molecules and the substructures

1980 ◽  
Vol 187 (3) ◽  
pp. 687-694 ◽  
Author(s):  
J Wieslander ◽  
D Heinegárd
Keyword(s):  
Hyaluronic Acid ◽  
Uronic Acid ◽  
Chondroitin Sulphate ◽  
Relative Content ◽  
Binding Region ◽  
Rich Region ◽  
Link Protein ◽  
Specific Radioimmunoassay ◽  
Cartilage Proteoglycans ◽  
Hyaluronic Acid Binding

Antibodies specifically reacting with the link proteins, the hyaluronic acid-binding region and chondroitin sulphate-peptides were used to design specific radioimmunoassay procedures. The sensitivity of the method used for the link protein was about 20 ng/ml, and the other two components could be determined at concentrations of about 2 ng/ml. The radioimmunoassay procedures were tested by using proteoglycan subfractions or fragments thereof. The procedures used to quantify link protein and hyaluronic acid-binding region showed no cross-interference. Fragments of trypsin-digested proteoglycan monomers still reacted in the radioimmunoassay for hyaluronic acid-binding region. Subfractions of proteoglycan monomers separated according to size had a gradually higher relative content of the hyaluronic acid-binding region compared with both chondroitin sulphate-peptides and uronic acid, when the molecules were smaller. The proteoglycans therefore may contain a variably large chondroitin sulphate-rich region, which has a constant substitution with polysaccharide side chains.

scholarly journals The role of link-protein in the structure of cartilage proteoglycan aggregates

1979 ◽  
Vol 177 (1) ◽  
pp. 237-247 ◽  
Author(s):  
T E Hardingham
Keyword(s):  
Analytical Ultracentrifugation ◽  
Free Form ◽  
Link Protein ◽  
Physiological Conditions ◽  
Neutral Ph ◽  
Laryngeal Cartilage ◽  
Cartilage Proteoglycan ◽  
Proteoglycan Aggregates

Proteoglycan fractions were prepared from pig laryngeal cartilage. The effect of link-protein on the properties of proteoglycan-hyaluronate aggregates was examined by viscometry and analytical ultracentrifugation. Aggregates containing link-protein were more stable than link-free aggregates at neutral pH, at temperatures up to 50 degrees C and in urea (up to 4.0M). Oligosaccharides of hyaluronate were able to displace proteoglycans from link-free aggregates, but not from the link-stabilized aggregates. Both types of aggregate were observed in the ultracentrifuge, but at the concentration investigated (less than 2 mg/ml) the link-free form was partially dissociated and the proportion aggregated varied with the pH and temperature and required more hyaluronate for saturation than did link-stabilized aggregate. The results showed that link-protein greatly strengthened the binding of proteoglycans to hyaluronate and suggest that under physiological conditions it ‘locks’ proteoglycans on to the hyaluronate chain.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Antigenic determinants of substructures

1979 ◽  
Vol 179 (1) ◽  
pp. 35-45 ◽  
Author(s):  
J Wieslander ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Antigenic Site ◽  
Trypsin Digestion ◽  
Antigenic Determinants ◽  
Binding Region ◽  
Keratan Sulphate ◽  
Link Protein ◽  
Before And After ◽  
Hyaluronic Acid Binding

Antibodies were raised in rabbits by injection of cartilage proteoglycan monomers, isolated hyaluronic acid-binding region, polysaccharide-peptides prepared by trypsin digestion of proteoglycans and link-protein. The rabbits injected with the proteoglycan monomers made antibodies reacting with the intact proteoglycan. The antiserum contained antibodies specific for, and also reacting with, the isolated hyaluronic acid-binding region and the keratan sulphate-rich region. In addition there were probably antibodies reacting with other structures of the proteoglycan monomer. When isolated hyaluronic acid-binding region was used for immunization the antibodies obtained reacted specifically with the hyaluronic acid-binding region. The antibodies obtained from rabbits immunized with the polysaccharide-peptides reacted with the proteoglycan monomers and showed a reaction identical with that of the chondroitin sulphate-peptides isolated after trypsin digestion of proteoglycans. The antibodies prepared with the link-protein as the antigen reacted only with the link-protein and not with any preparation from the proteoglycan monomer. Neither did any of the antisera raised against the proteoglycan monomer or its substructures react with the link-protein. Separately it was shown that the peptide ‘maps’ prepared from trypsin digests of the link-protein and the hyaluronic acid-binding region were different. Therefore it appears that the link-protein is not structurally related to the proteoglycan or the hyaluronic acid-binding region. Digestion of proteoglycan monomers or isolated hyaluronic acid-binding region with trypsin did not destroy the antigenic sites of the hyaluronic acid-binding region. In contrast trypsin digests of previously reduced and alkylated preparations did not react with the anti-(hyaluronic acid-binding region). The trypsin digests, however, reacted with both the antibodies directed against the chondroitin sulphate-peptides and those against the keratan sulphate-peptides. Trypsin digestion of the link-proteins destroyed the antigenic site and the reactivity with the antibodies. By combining immunoassay of proteoglycan preparations before and after trypsin digestion it is feasible to quantitatively determine its substructures by using the antisera described above.

scholarly journals Light and electron microscopic studies on the localization of hyaluronic acid in developing rat cerebellum.

1988 ◽  
Vol 106 (3) ◽  
pp. 845-855 ◽  
Author(s):  
J A Ripellino ◽  
M Bailo ◽  
R U Margolis ◽  
R K Margolis
Keyword(s):  
Hyaluronic Acid ◽  
Chondroitin Sulfate ◽  
Adult Brain ◽  
Developmental Changes ◽  
Chondroitin Sulfate Proteoglycan ◽  
Sulfate Proteoglycan ◽  
Binding Region ◽  
Electron Microscopic ◽  
Link Protein ◽  
Hyaluronic Acid Binding

The hyaluronic acid-binding region was prepared by trypsin digestion of chondroitin sulfate proteoglycan aggregate from the Swarm rat chondrosarcoma, and biotinylated in the presence of hyaluronic acid and link protein. After isolation by gel filtration and HPLC in 4 M guanidine HCl, the biotinylated hyaluronic acid-binding region was used, in conjunction with avidin-peroxidase, as a specific probe for the light and electron microscopic localization of hyaluronic acid in developing and mature rat cerebellum. At 1 w postnatal, there is strong staining of extracellular hyaluronic acid in the presumptive white matter, in the internal granule cell layer, and as a dense band at the base of the molecular layer, surrounding the parallel fibers. This staining moves progressively towards the pial surface during the second postnatal week, and extracellular staining remains predominant through postnatal week three. In adult brain, there is no significant extracellular staining of hyaluronic acid, which is most apparent in the granule cell cytoplasm, and intra-axonally in parallel fibers and some myelinated axons. The white matter is also unstained in adult brain, and no staining was seen in Purkinje cell bodies or dendrites at any age. The localization of hyaluronic acid and its developmental changes are very similar to that previously found in immunocytochemical studies of the chondroitin sulfate proteoglycan in nervous tissue (Aquino, D. A., R. U. Margolis, and R. K. Margolis. 1984. J. Cell Biol. 99:1117-1129; Aquino, D. A., R. U. Margolis, and R. K. Margolis. J. Cell Biol. 99:1130-1139), and to recent results from studies using monoclonal antibodies to the hyaluronic acid-binding region and link protein. The presence of brain hyaluronic acid in the form of aggregates with chondroitin sulfate proteoglycans would be consistent with their similar localizations and coordinate developmental changes.

Effect of link protein and free hyaluronic acid binding region on spacing of proteoglycans in aggregates

1994 ◽  
Vol 12 (5) ◽  
pp. 612-620 ◽  
Author(s):  
Anthony Kahn ◽  
Ari D. Taitz ◽  
Lawrence A. Pottenger ◽  
Gregory M. Alberton
Keyword(s):  
Hyaluronic Acid ◽  
Binding Region ◽  
Link Protein ◽  
Hyaluronic Acid Binding

scholarly journals The hyaluronic acid binding region as a specific probe for the localization of hyaluronic acid in tissue sections. Application to chick embryo and rat brain.

1985 ◽  
Vol 33 (10) ◽  
pp. 1060-1066 ◽  
Author(s):  
J A Ripellino ◽  
M M Klinger ◽  
R U Margolis ◽  
R K Margolis
Keyword(s):  
Hyaluronic Acid ◽  
Gel Filtration ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Staining Reaction ◽  
Binding Region ◽  
Electron Microscopic ◽  
Link Protein ◽  
Tissue Sections ◽  
Hyaluronic Acid Binding

The hyaluronic acid binding region was prepared by clostripain digestion of chondroitin sulfate proteoglycan isolated from the Swarm rat chondrosarcoma, and biotinylated in the presence of associated hyaluronic acid and link protein. After removal of hyaluronic acid by gel filtration in 4 M guanidine HCl, the biotinylated binding region-link protein complex was used as a specific histochemical probe in conjunction with avidin-peroxidase. Its utility was initially evaluated by comparison with Alcian blue staining of the axial region of 2 to 5 day chick embryos, where staining was seen in the dorsolateral area between the neural tube and the ectoderm, in the perichordal mesenchyme, and in developing limb buds. Light and electron microscopic studies of early postnatal rat cerebellum indicate that hyaluronic acid is primarily localized in the extracellular space of immature brain. Staining specificity was demonstrated by the ability of hyaluronic acid oligosaccharides of appropriate size to block the staining reaction, and by the absence of staining after treatment of tissue sections with protease-free Streptomyces hyaluronidase, which degrades only this glycosaminoglycan.

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