title: 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.
authors.affiliation: Department of Orthopaedic Surgery, Gifu University School of Medicine, 1-1 Yanagido, Gifu City 501-1194, Japan.
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Oshita
authors.forename: John D
authors.initials: JD
authors.lastname: Sandy
authors.forename: Kiichi
authors.initials: K
authors.lastname: Suzuki
authors.forename: Atsushi
authors.initials: A
authors.lastname: Akaike
authors.forename: Yun
authors.initials: Y
authors.lastname: Bai
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sasaki
authors.forename: Katsuji
authors.initials: K
authors.lastname: Shimizu
mesh_terms: D053674:Aggrecans; D000409:Alanine / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites; D015153:Blotting, Western / Q000379:methods; D002154:Calpain / Q000378:metabolism*; D002358:Cartilage, Articular / Q000737:chemistry*; D002417:Cattle; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000276:immunology; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D020539:Sequence Analysis, Protein / Q000379:methods; D013552:Swine
chemical_list: D053674:Aggrecans; D000911:Antibodies, Monoclonal; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D010446:Peptide Fragments; D010455:Peptides; D011509:Proteoglycans; D002154:Calpain; C449000:m-calpain; D000409:Alanine