scholarly journals A sensitive assay for active link protein from cartilage

1985 ◽  
Vol 232 (2) ◽  
pp. 613-616 ◽  
Author(s):  
J D Sandy ◽  
A H Plaas
Keyword(s):  
Heat Denaturation ◽  
Sensitive Assay ◽  
Protein Activity ◽  
Link Protein ◽  
Chondrocyte Cultures ◽  
Proteoglycan Aggregates ◽  
Active Link

A new assay for the activity of cartilage link protein is described. The method is based on the finding [Plaas, Sandy & Muir (1983) Biochem. J. 214, 855-864] that addition of link protein to [35S]sulphate-labelled proteoglycan aggregates from rabbit chondrocyte cultures resulted in the formation of link-stabilized aggregates. The percentage aggregate was found to be related linearly to the amount of purified bovine link protein added in the 20-120 ng range. The assay was used to monitor loss of link-protein activity during heat denaturation and to measure binding of link protein by purified proteoglycan monomer.

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 Demonstration of link protein in proteoglycan aggregates from human intervertebral disc

1984 ◽  
Vol 222 (1) ◽  
pp. 85-92 ◽  
Author(s):  
A Tengblad ◽  
R H Pearce ◽  
B J Grimmer
Keyword(s):  
Intervertebral Disc ◽  
Density Gradient ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Intervertebral Discs ◽  
Human Articular Cartilage ◽  
Link Protein ◽  
Nasal Cartilage ◽  
Proteoglycan Aggregates ◽  
Cartilage Proteoglycans

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.

scholarly journals A proteolytic fragment from human link protein is taken up and processed by monocytes and B cells

1991 ◽  
Vol 280 (3) ◽  
pp. 679-686 ◽  
Author(s):  
H Martin ◽  
M Dean
Keyword(s):  
B Cells ◽  
Plasma Membranes ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Peritoneal Macrophages ◽  
High Uptake ◽  
Proteolytic Fragment ◽  
Link Protein ◽  
Protein Uptake ◽  
Proteoglycan Aggregates

Mild digestion of 125I-labelled human proteoglycan aggregates with trypsin or stromelysin produced specific peptides that were taken up rapidly by THP-1 monocytes. SDS/PAGE of undigested aggregate showed that the three components of molecular mass 48, 44 and 41 kDa, corresponding to isoforms of link protein originally present, had been converted into a single component of 41 kDa by trypsin treatment, and that fragments of 6-12 kDa were present in fractions containing the high-uptake peptide. Separate proteolysis of isolated proteoglycan monomer and link protein confirmed that the specific high-uptake fragment was derived from link protein. Uptake of the link fragment was rapid, reaching a maximum after 5 min, and specific, since it was blocked by metabolic or serine proteinase inhibitors and at 4 degrees C. After uptake the cleaved fragment was processed further, with 50% of the radiolabel being released as degraded peptides within 5 min. In contrast, accumulation of whole aggregate reached a maximum after 45 min and only 50% had been released after 2 h. Uptake of aggregate was less affected by inhibitors or at low temperature, suggesting that a separate mechanism existed for its turnover. The aggregate was transported to lysosomes after uptake, although the link fragment did not sediment with either lysosomes or plasma membranes, suggesting that it was present in the cytoplasm or in very labile vesicles. However, the mode of handling of the peptide by the cells remains unclear. The link fragment was taken up by several different monocytic and B cell lines, but not by mouse fibroblasts or peritoneal macrophages. These data suggest that a surface serine proteinase on monocytes and B cells enables them to process and take up a fragment of link protein derived by extracellular proteolysis.

scholarly journals Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin.

1983 ◽  
Vol 258 (20) ◽  
pp. 12280-12286 ◽  
Author(s):  
L S Lohmander ◽  
S A Fellini ◽  
J H Kimura ◽  
R L Stevens ◽  
V C Hascall
Keyword(s):  
Chondrocyte Cultures ◽  
Proteoglycan Aggregates

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 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 Metalloproteinase digestion of cartilage proteoglycan. Pattern of cleavage by stromelysin and susceptibility to collagenase

1991 ◽  
Vol 279 (3) ◽  
pp. 733-739 ◽  
Author(s):  
C Hughes ◽  
G Murphy ◽  
T E Hardingham
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Relative Viscosity ◽  
Gel Chromatography ◽  
High Concentration ◽  
Link Protein ◽  
Rapid Fall ◽  
Sds Page ◽  
Proteoglycan Aggregates ◽  
Rabbit Bone

The action of purified rabbit bone stromelysin was investigated on proteoglycan aggregates from pig laryngeal cartilage. The enzyme caused a rapid fall in viscosity of proteoglycan aggregate solution (6 mg/ml), and the products of a partial digest (60% loss of relative viscosity) and a complete digest (95% loss of relative viscosity) were characterized. Analysis by gel chromatography on Sepharose 2B under associative conditions showed that 95% of the glycosaminoglycans in the complete digest were in small-sized fragments, whereas most of the hyaluronan-binding G1 domain and link protein remained intact and bound to hyaluronan. In contrast, there was extensive digestion of the G2 domain which resulted in 76% loss in its detection by immunoassay. Analysis of the partial digest also showed considerable loss (40%) of detection of the G2 domain, but the glycosaminoglycan-rich fragments were much larger than in the complete digest. There was also much less cleavage to create small fragments containing the G1 domain. This was evident on SDS/PAGE analysis where a 58 kDa G1 domain fragment was abundant in the complete digest, but was only present in small amounts in the partial digest. There was also only very limited conversion of link protein from a 44 kDa form to a 40 kDa form. The digestion of proteoglycan aggregate (6 mg/ml) by stromelysin was unaffected by the addition of a high concentration of extra chondroitin sulphate chains (14 mg/ml), and the digestion of proteoglycan monomer showed that the G1 domain was resistant to stromelysin digestion even when not bound to hyaluronan and link protein. The results show that stromelysin degrades the proteoglycan protein core with major cleavages close to, but not within, the G1 domain, and extensive cleavage in other regions. Experiments with purified collagenase, a metalloproteinase structurally related to stromelysin, showed that it too cleaved proteoglycan at several sites within the glycosaminoglycan-rich region of the core protein. Metalloproteinase attack on proteoglycan thus not only occurs with stromelysin but also with collagenase.

Sign in / Sign up

Export Citation Format

Share Document