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 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 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 N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage

1992 ◽  
Vol 284 (2) ◽  
pp. 589-593 ◽  
Author(s):  
P Loulakis ◽  
A Shrikhande ◽  
G Davis ◽  
C A Maniglia
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Amino Acid ◽  
Interleukin 1 ◽  
Human Articular Cartilage ◽  
Terminal Amino Acid ◽  
Protein Core ◽  
Nasal Cartilage ◽  
The Media ◽  
Terminal Amino

Bovine articular cartilage was cultured both in the presence and in the absence of human recombinant interleukin-1 alpha (IL-1) (100 units/ml). Addition of this cytokine stimulated matrix degradation approx. 3-fold. This increased degradation permitted characterization of the large chondroitin sulphate proteoglycan (aggrecan) fragments accumulating in the media. When compared with controls, the proteoglycans isolated from the medium of cultures treated with IL-1 exhibited a decrease in the Kav. (control 0.25; IL-1-treated 0.37), determined by Sepharose CL-2B chromatography. This decrease in proteoglycan size was accompanied by a decreased ability of these monomers to associate with hyaluronic acid. Thus only 20% of the proteoglycans isolated from the medium of IL-1-treated cultures, compared with 39% for control cultures, had the capacity to form high-M(r) aggregates with hyaluronic acid. SDS/PAGE analysis of the proteoglycans from the media of IL-1-treated cultures demonstrated several large proteoglycan protein-core bands (M(r) 144,000-380,000). The protein-core bands with M(r) 144,000-266,000 exhibited a significantly decreased reactivity with monoclonal antibody 1-C-6 (specific for domains G1 and G2). The N-terminal amino acid sequence of four of these protein-core bands (M(r) 144,000, 173,000, 214,000 and 266,000) yielded sequences LGQRPPV-Y-PQLF(E), AGEGP(S)GILEL-GAP(S)-AP(D)M, GLG-VEL-LPGE and (A)RGSVIL-AKPDFEV-P-A. A comparison of these N-terminal amino acid sequences with the published proteoglycan sequence for bovine nasal cartilage [Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259], rat chondrosarcoma [Doege, Sasaki, Horigan, Hassell & Yamada (1987) J. Biol. Chem. 262, 17757-17769] and human articular cartilage [Doege, Sasaki, Kimura & Yamada (1991) J. Biol. Chem. 266, 894-902] permitted assignment of their relative positions on the core protein. Furthermore, on the basis of this similarity to published sequence, putative sites of enzymic cleavage were constructed. These theoretical cleavage sites revealed a glutamic acid residue in the P1 position and an uncharged polar or non-polar residue in the P1′ position.

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 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 inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage

1984 ◽  
Vol 221 (3) ◽  
pp. 637-644 ◽  
Author(s):  
P J Roughley ◽  
R J White ◽  
A R Poole ◽  
J S Mort
Keyword(s):  
Hyaluronic Acid ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Human Articular Cartilage ◽  
Human Cartilage ◽  
Adult Human ◽  
Cscl Density Gradient ◽  
Proteoglycan Aggregates

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.

scholarly journals Proteoglycan aggregate formation by articular chondrocytes. Decrease in link-protein synthesis during culture

1983 ◽  
Vol 214 (3) ◽  
pp. 855-864 ◽  
Author(s):  
A H K Plaas ◽  
J D Sandy ◽  
H Muir
Keyword(s):  
Hyaluronic Acid ◽  
Articular Chondrocytes ◽  
Core Protein ◽  
Link Protein ◽  
Monolayer Cultures ◽  
Proteoglycan Aggregates ◽  
Primary Monolayer ◽  
Primary Monolayer Cultures ◽  
Stable Aggregate ◽  
Rabbit Articular Chondrocytes

The synthesis of link-stabilized proteoglycan aggregates by rabbit articular chondrocytes was investigated by [35S]sulphate labelling of primary monolayer cultures maintained for up to 21 days. (1) At all culture times the cells secreted a high-molecular-weight cartilage-type proteoglycan monomer of which 75%-80% formed aggregates with hyaluronic acid. (2) At 2 days of culture all of the aggregates were in link-stabilized form, but by 21 days only 5% were link-stabilized, as shown by displacement of monomers from the aggregate by hyaluronic acid oligosaccharides. (3) The addition of purified link protein to 21-day culture medium increased the proportion of link-stable aggregate from 5% to 70%. (4) Analysis of [3H]serine-labelled proteoglycan aggregates in the medium showed a marked decrease with culture time in the ratio of 3H-labelled link protein to 3H-labelled core protein present. The results suggest that the secretion of proteoglycan monomers and link protein by articular chondrocytes changes independently during prolonged monolayer culture.

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