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 The action of human articular-cartilage metalloproteinase on proteoglycan and link protein. Similarities between products of degradation in situ and in vitro

1986 ◽  
Vol 237 (1) ◽  
pp. 117-122 ◽  
Author(s):  
I K Campbell ◽  
P J Roughley ◽  
J S Mort
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Interleukin 1 ◽  
Human Articular Cartilage ◽  
Link Protein ◽  
Protein Components ◽  
Stimulation Of ◽  
Hyaluronic Acid Binding

Interleukin 1 stimulation of human articular cartilage in organ culture produced the concomitant release of proteoglycan fragments and latent metalloproteinase. The released fragments ranged in size from that of almost intact proteoglycan subunits to the product of limiting digestion generated by the activated metalloproteinase. None of the fragments possessed the ability to interact with hyaluronic acid. Analysis of proteoglycan aggregate digested with the activated metalloproteinase showed that isolated hyaluronic acid-binding regions were produced from the proteoglycan subunits, and that the two higher-Mr link-protein components (Mr 48,000 and 44,000) were converted into the lowest-Mr component (Mr 41,000). Link protein extracted from cartilage under stimulation with interleukin 1 showed a similar conversion. These results suggest that interleukin 1 stimulates the release of latent metalloproteinase from chondrocytes and that a proportion of the enzyme is activated in situ in the cartilage matrix. The mode of action of the activated enzyme is compatible with a role in the changes in proteoglycan structure seen in aging.

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 The properties of proteoglycan prepared from human articular cartilage by using associative caesium chloride gradients of high and low starting densities

1985 ◽  
Vol 232 (1) ◽  
pp. 111-117 ◽  
Author(s):  
M T Bayliss ◽  
P J Roughley
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Human Articular Cartilage ◽  
Adult Human ◽  
Cscl Density Gradient ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

Proteoglycan was extracted from adult human articular cartilage from both the knee and the hip, and A1 preparations were prepared by CsCl-density-gradient centrifugation at starting densities of 1.69 and 1.5 g/ml. Irrespective of whether the cartilage was diced to 1 mm cubes or sectioned to 20 micron slices there was always a lower proportion of both protein and proteoglycan aggregate in the A1 preparation prepared at 1.69 g/ml. Furthermore, the addition of exogenous hyaluronic acid to the extracts before centrifugation did not improve the yield of aggregate at 1.69 g/ml. These results were not affected by the presence of proteinase inhibitors in the extraction medium. It appears that adult human articular cartilage contains a high proportion of low-density proteoglycan subunits and hyaluronic acid-binding proteins that make most of the re-formed proteoglycan aggregates of a lower density than is usually encountered with younger human and mammalian hyaline cartilages.

scholarly journals Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Protein fragmentation, amino acid modification and hyaluronic acid cleavage

1989 ◽  
Vol 259 (3) ◽  
pp. 805-811 ◽  
Author(s):  
C R Roberts ◽  
P J Roughley ◽  
J S Mort
Keyword(s):  
Hyaluronic Acid ◽  
Amino Acid ◽  
Metal Ion ◽  
Major Product ◽  
Species Variation ◽  
Human Articular Cartilage ◽  
Acid Modification ◽  
H2o2 Treatment ◽  
Link Protein ◽  
Proteoglycan Aggregates

We have previously shown that treatment of neonatal human articular-cartilage proteoglycan aggregates with H2O2 results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid and in fragmentation of the link proteins [Roberts, Mort & Roughley (1987) Biochem. J. 247, 349-357]. We now show the following. (1) Hyaluronic acid in proteoglycan aggregates is also fragmented by treatment with H2O2. (2) Although H2O2 treatment results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid, the loss of this function is not attributable to substantial cleavage of the hyaluronic acid-binding region of the proteoglycan subunits. (3) In contrast, link proteins retain the ability to bind to hyaluronic acid following treatment with H2O2. (4) The interaction between the proteoglycan subunit and link protein is, however, abolished. (5) N-Terminal sequence analysis of the first eight residues of the major product of link protein resulting from H2O2 treatment revealed that cleavage occurred between residues 13 and 14, so that the new N-terminal amino acid is alanine. (6) In addition, a histidine (residue 16) is converted into alanine and an asparagine (residue 21) is converted into aspartate by the action of H2O2. (7) Rat link protein showed no cleavage or modifications in similar positions under identical conditions. (8) This species variation may be related to the different availability of histidine residues required for the co-ordination of the transition metal ion involved in hydroxyl-radical generation from H2O2. (9) Changes in function of these structural macromolecules as a result of the action of H2O2 may be consequences of both fragmentation and chemical modification.

scholarly journals Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage

1981 ◽  
Vol 199 (1) ◽  
pp. 17-29 ◽  
Author(s):  
S Björnsson ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Cell Cultures ◽  
Low Ph ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycan ◽  
Proteoglycan Aggregates ◽  
Cartilage Proteoglycans ◽  
Free Monomer

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.

scholarly journals Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage

1985 ◽  
Vol 231 (1) ◽  
pp. 129-138 ◽  
Author(s):  
P J Roughley ◽  
R J White ◽  
A R Poole
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Binding Protein ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Acid Binding Protein ◽  
Adult Human ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.

scholarly journals Cartilage proteoglycan binding region and link protein. Radioimmunoassays and the detection of masked determinants in aggregates

1983 ◽  
Vol 213 (2) ◽  
pp. 371-378 ◽  
Author(s):  
A Ratcliffe ◽  
T Hardingham
Keyword(s):  
Heat Treatment ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Binding Region ◽  
Link Protein ◽  
Cartilage Proteoglycan ◽  
Antigenic Sites ◽  
Proteoglycan Aggregates ◽  
Protein Components

Antibodies have been raised in rabbits to the hyaluronate-binding region and link-protein components of aggregated proteoglycans from pig laryngeal cartilage. The anti-(binding region) antibodies did not bind 125I-labelled link protein, nor was 125I-labelled binding region bound by the anti-(link protein) antibodies. The antisera were applied in sensitive inhibition radioimmunoassays to determine binding region and link protein in purified proteoglycan preparations. With intact proteoglycan aggregates, the antigenic sites of link protein, and to a lesser extent binding region, were masked. Heat treatment in the presence of sodium dodecyl sulphate (0.025%, w/v) was found to overcome this masking, thereby allowing the determination of link protein and binding region in aggregated proteoglycan preparations in pure and impure samples.

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