Increased degradation and alteered tissue distribution of cartilage oligomeric matrix protein in human rheumatoid and osteoarthritic cartilage

Abstract

We investigated the degradation and tissue distribution of cartilage oligomeric matrix protein in normal, osteoarthritic, and rheumatoid arthritic articular cartilage of the human knee. Cartilage was subjected to sequential extractions with buffers containing neutral salt, with EDTA, and finally with guanidine/HCl and then was analyzed by Western blotting with a polyclonal antiserum to human cartilage oligomeric matrix protein. Western blots of the nine neutral salt extracts from normal cartilage revealed mostly intact pentameric molecules of cartilage oligomeric matrix protein, in contrast to the 13 osteoarthritic and five rheumatoid arthritic cartilage samples that demonstrated marked degradation of cartilage oligomeric matrix protein as noted by a predominance of reduction-sensitive bands at approximately 150 kDa and nonreduction-sensitive bands in the 67-94 kDa range. The EDTA and guanidine/HCl extracts from all groups were similar and showed mostly intact molecules of cartilage oligomeric matrix protein, with smaller amounts of degraded cartilage oligomeric matrix protein identical to those resolved by the Western blots of the neutral salt extracts. Western blots of matched pairs of synovial fluid and cartilage extracts demonstrated cartilage oligomeric matrix protein fragments of the same molecular mass. Competitive enzyme-linked immunosorbent assay revealed significantly less cartilage oligomeric matrix protein in rheumatoid articular cartilage than in either normal or osteoarthritic cartilage. In contrast to normal cartilage, where cartilage oligomeric matrix protein was predominantly localized to the interterritorial matrix throughout all zones of the matrix, with increased staining in the deeper cartilaginous zones, the most intense staining in osteoarthritic cartilage was in the superficial zones of fibrillated cartilage, with little to no immunostaining in the midzones and relatively poor staining in the deeper cartilaginous zones. This distribution was the inverse of that for proteoglycans, as demonstrated by toluidine blue staining, where proteoglycans were depleted primarily from the superficial fibrillated cartilage. In mild to moderately affected rheumatoid cartilage, the tissue distribution of cartilage oligomeric matrix protein was similar to the distribution of proteoglycans, with relatively uniform staining of the interterritorial and territorial matrics. In more severely affected rheumatoid cartilage, the superficial zones demonstrated punctate immunostaining for cartilage oligomeric matrix protein in the interterritorial and territorial matrics, and staining was restricted to the territorial matrix in the deep cartilaginous zones. It is evident from this study that (a) noncollagenous proteins such as cartilage oligomeric matrix protein are greatly affected in arthritis, (b) degradation fragments released from the matrix into the synovial fluid reflect the processes occurring within the matrix, and (c) different zones of the articular cartilage are susceptible to degradation of cartilage oli...

ADAMTS‐7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein

“…To examine whether divalent cations were involved in this association, 5 mM Ca 2+ were added to one set of binding buffer. The bound proteins were denatured in sample buffer and separated by 12% SDS-PAGE, and COMP protein was detected by Western blotting with polyclonal rabbit anti-COMP antiserum (4,21,45).…”

“…To examine COMP degradation by full-length ADAMTS-7 and to investigate the zinc ion concentration dependence of the intact enzyme, purified COMP (200 nM) was incubated with the cell lysates prepared from Sf9 insect cells infected with either control or ADAMTS-7 bacluovirus in the presence of lower (0.1 mM) or higher (2 mM) levels of ZnCl 2 as well as in the absence of Zn 2+ by addition of 5 mM EDTA in a digestion buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl 2 , and 0.05% Brij-35, pH 7.5) at 37°C for 12 h. The digested nonreduced products were resolved by 10% SDS-PAGE, and intact COMP and COMP fragments were detected by Western blotting with polyclonal rabbit anti-COMP antiserum, as described previously (4,21,45).…”

“…This may explain the discrepancy in protein mobility and pattern in the gel (50). The polyclonal antiserum against COMP was generated using intact purified hCOMP as an antigen (4,21,45). For the CO-IP assays, the cartilage extractswere incubated with either anti-COMP antiserum (Fig.…”

“…Fragments of COMP have been detected in diseased cartilage, synovial fluid, and serum of patients with knee injuries, posttraumatic, primary OA, and rheumatoid arthritis (RA; refs [21][22][23]. The nature of COMP degradation and the enzyme(s) responsible for the production of degradative fragments in vivo, however, have yet to be identified.…”

Characterization of metalloprotease cleavage products of human articular cartilage

“…Other cartilage fragments, including those originating from cartilage oligomeric matrix protein (COMP) (23), , and fibromodulin (25), have also been described as potential biomarkers of OA. Cleavage products of aggrecan that originate from the activities of ADAMTS-4 or ADAMTS-5 have been measured in synovial fluid, and these could also serve as cartilage degradation biomarkers (26).…”