Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein

The actions of human recombinant stromelysins-1 and -2, collagenase, gelatinases A and B and matrilysin on neonatal human proteoglycan aggregates were examined. With the exception of gelatinase B, aggrecan was degraded extensively by most metalloproteinases studied, whereas link protein showed only limited proteolysis. Sequencing studies of modified link protein components revealed that stromelysins-1 and -2, gelatinases A and B and collagenase cleaved specifically between His16 and Ile17, and matrilysin, stromelysin-2 and gelatinase A cleaved between Leu25 and Leu26. Cleavage at the former bond generated a link protein component with the same N-terminus as that isolated from newborn human cartilage. Based on previously determined in situ cleavage sites it is evident that matrix metalloproteinases are not solely responsible for the accumulation of link protein degradation products in adult human cartilage, indicating that additional proteolytic agents are involved in the normal catabolism of human cartilage matrix.

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Link protein as a monitor in situ of endogenous proteolysis in adult human articular cartilage

Biochemical Journal ◽

10.1042/bj2780143 ◽

1991 ◽

Vol 278 (1) ◽

pp. 143-147 ◽

Cited By ~ 40

Author(s):

Q Nguyen ◽

J Liu ◽

P J Roughley ◽

J S Mort

Keyword(s):

Articular Cartilage ◽

Degradation Products ◽

Proteolytic Processing ◽

Cathepsin G ◽

Human Articular Cartilage ◽

Link Protein ◽

Adult Human ◽

Protein Components

The link protein components of proteoglycan aggregates in adult human articular cartilage show heterogeneity due to proteolysis. Cleavages near the N-terminus of the intact link proteins, before residues 17, 19 and 24, generate three proteins of slightly diminished size (LP3). Cleavages within the N-terminal disulphide-bonded loop, before residues 66 and 73 of the intact link proteins, generate proteins that yield smaller degradation products upon reduction (LP fragments). In vitro, modified link protein components of a similar size to LP3 can be generated by a variety of proteinases, but of the physiologically relevant enzymes only stromelysin, cathepsin B and cathepsin G have the ability to yield modified link proteins with N-termini identical with those observed in situ. None of the proteolytic agents tested was able to produce LP fragments with N-termini identical with those observed in situ, and the majority of proteinases were not able to cleave within the disulphide-bonded loops. Cathepsin L and hydroxyl radicals can cleave within the N-terminal disulphide-bonded loop, and have the potential of initially opening the loop to allow further proteolytic processing by other agents to generate the native cleavage sites.

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

Biochemical Journal ◽

10.1042/bj2370117 ◽

1986 ◽

Vol 237 (1) ◽

pp. 117-122 ◽

Cited By ~ 31

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.

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Variations in aggrecan structure modulate its susceptibility to aggrecanases

Biochemical Journal ◽

10.1042/bj20030609 ◽

2003 ◽

Vol 375 (1) ◽

pp. 183-189 ◽

Cited By ~ 23

Author(s):

Peter J. ROUGHLEY ◽

James BARNETT ◽

Fengrong ZUO ◽

John S. MORT

Keyword(s):

Chondroitin Sulphate ◽

Human Cartilage ◽

In Vitro Degradation ◽

Link Protein ◽

Rich Domain ◽

Sds Page ◽

Lower Ability ◽

Proteoglycan Aggregates ◽

Fetal Bovine

Proteoglycan aggregates and purified aggrecan from adult and fetal bovine cartilage and adult and neonatal human cartilage were subjected to in vitro degradation by recombinant aggrecanase-1 and aggrecanase-2. The ability of the aggrecanases to cleave within the aggrecan IGD (interglobular domain) and CS2 domain (chondroitin sulphate-rich domain 2) was monitored by SDS/PAGE and immunoblotting. Aggrecanase-2 showed a similar ability to cleave within the IGD of adult and immature aggrecan, whereas aggrecanase-1 was less efficient in cleavage in the IGD of immature aggrecan, for both the bovine and the human substrates. Both aggrecanases showed a similar ability to cleave within the CS2 domain of bovine aggrecan irrespective of age, but showed a much lower ability to cleave within the CS2 domain of human aggrecan. Equivalent results were obtained whether aggrecan was present in isolation or as part of proteoglycan aggregates. When proteoglycan aggregates were used, neither aggrecanase was able to cleave link protein. Thus, for aggrecan cleavage by aggrecanases, variations in cleavage efficiency exist with respect to the species and age of the animal from which the aggrecan is derived and the type of aggrecanase being used.

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The origin of human cartilage proteoglycan link-protein heterogeneity and fragmentation during aging

Biochemical Journal ◽

10.1042/bj2320805 ◽

1985 ◽

Vol 232 (3) ◽

pp. 805-812 ◽

Cited By ~ 31

Author(s):

J S Mort ◽

B Caterson ◽

A R Poole ◽

P J Roughley

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Articular Cartilage ◽

Human Cartilage ◽

Link Protein ◽

Cartilage Proteoglycan ◽

Disulphide Bonds ◽

Single Link ◽

Chemical Deglycosylation

Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins.

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

Biochemical Journal ◽

10.1042/bj2590061 ◽

1989 ◽

Vol 259 (1) ◽

pp. 61-67 ◽

Cited By ~ 80

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.

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

Biochemical Journal ◽

10.1042/bj2130371 ◽

1983 ◽

Vol 213 (2) ◽

pp. 371-378 ◽

Cited By ~ 35

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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