Mature bovine articular cartilage contains abundant aggrecan that is C-terminally truncated at Ala719-Ala720, a site which is readily cleaved by m-calpain

Abstract

Extracts of normal mature articular cartilage contain aggrecan molecules which bear the G1 domain (the N-terminal globular domain of aggrecan) and are C-terminally truncated by proteolysis at a number of sites. A proportion of these molecules are generated by an aggrecanase and/or matrix-metalloproteinase-mediated cleavage in the IGD (interglobular domain between the G1 and G2 domains of aggrecan). However, the proteinase(s) responsible for formation of the majority of the larger G1-G2 and glycosaminoglycan-bearing truncated species is (are) unknown. N-terminal sequencing of aggrecan core fragments generated by m-calpain digestion of bovine aggrecan has identified four novel cleavage sites: one within the CS (chondroitin sulphate)-1 domain (at one or more of the bonds Ser1229-Val1230, Ser1249-Val1250, Ser1287-Val1288, Gly1307-Val1308 and Ser1346-Val1347), two within the IGD (at bonds Ala474-Ala475 and Gly365-Gly366) and one within the KS (keratan sulphate) domain (at Ala719-Ala720). A new monoclonal antibody (SK-28) to the C-terminal neoepitope at M710VTQVGPGVA719 showed that aggrecan products generated by this cleavage are present in high abundance in mature bovine articular cartilage extracts. We conclude that m-calpain, or an unidentified proteinase with the capacity to cleave at the same site, is active during aggrecan biosynthesis/secretion by mature chondrocytes or in the matrix of mature bovine articular cartilage in vivo.

Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties

“…Chondrocyte-derived hyaluronidases may be responsible, although there is conflicting evidence for upregulated expression and activity of hyaluronidases in response to inflammatory cytokines (Chow and Knudson, 2005;Flannery, et al, 1998). Chondrocytes also express non-metalloproteinase enzymes such as cathepsin-B (Fosang, et al, 1992) and m-calpain (Oshita, et al, 2004) which have been shown to cleave the aggrecan core protein, and these enzymes may be upregulated in response to IL-1 treatment.…”

Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair

“…The calcium-dependent cysteine proteinase m-calpain cleaves aggrecan in vitro (35,36), and fragments consistent with m-calpain cleavage are present in extracts of mature bovine cartilage (36). Whether m-calpain is involved in growth plate aggrecanolysis in vivo has not been investigated.…”

Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1 Is Necessary for Normal Endochondral Ossification and Aggrecan Metabolism

“…Indeed, GAGs such as CS and keratan sulfate attached to aggrecan core protein affect the manner of cleavage by proteinases (22). Although various matrix metalloproteinases, a disintegrin and metalloproteinase with a thrombospondin motif (ADAMTS)-1, -4, -5, -8, -9, -15 and m-calpain, have been shown to cleave the aggrecan core protein at specific sites (23)(24)(25)(26), the mechanism of physiological aggrecan metabolism has not been fully elucidated. Determination of the cleavage sites may lead to identification of proteinases that participate in normal aggrecan metabolism.…”