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Journal of Biochemistry Advance Access originally published online on January 29, 2007
Journal of Biochemistry 2007 141(4):469-477; doi:10.1093/jb/mvm052
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© 2007 The Japanese Biochemical Society.

G1-G2 Aggrecan Product that can be Generated by M-calpain on Truncation at Ala709–Ala710 is Present Abundantly in Human Articular Cartilage

Hideaki Maehara1, Kiichi Suzuki2, Tomohiro Sasaki1, Hidefumi Oshita3, Eriko Wada4, Toshiyuki Inoue1 and Katsuji Shimizu1,*

1Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194; 2Department of Orthopaedic Surgery, Takeda Hospital, 841-5 Higashi-Shiokoji-Machi, Shimogyo-Ku, Kyoto 600-8558; 3Department of Orthopaedic Surgery, Kizawa Memorial Hospital, 590 Shimofurui, Furui-Machi, Minokamo City, Gifu 505-8503; and 4Department of Clinical Pathology, Daiyukai General Hospital, 1-9-9 Sakura, Ichinomiya City, Aichi 491-8551, Japan

*To whom correspondence should be addressed. Tel: +81-58-230-6330, Fax: +81-58-230-6331, E-mail: shim{at}gifu-u.ac.jp

Received September 3, 2006; Accepted January 19, 2007


  Abstract

To elucidate the specific function of m-calpain in the metabolism of aggrecan in human articular cartilage, the prevalence and localization of a large glycosaminoglycan-bearing aggrecan product generated by m-calpain in human osteoarthritis (OA) cartilage were investigated. Extracts of human OA articular cartilage were analysed by immunostaining using new polyclonal anti-VPGVA antiserum that detects the COOH terminal neoepitope IVTQVVPGVA709 generated by m-calpain-related cleavage within the keratan sulphate rich region of human aggrecan. Immunoblotting analyses of aggrecan populations in guanidine hydrochloride-extracts showed that OA cartilages contained anti-VPGVA positive aggrecan products with the COOH terminal neoepitope ... VPGVA709, resulting from truncation between the Ala709–Ala710 m-calpain-related cleavage site. This aggrecan product consisted of two NH2 terminal globular domain (G1 and G2) and KS side chains. Immunohistochemical staining showed that anti-VPGVA positive staining was localized within chondrocytes and spread to the surrounding interterritorial matrix. Confocal microscopic analysis showed subcellular colocalization of anti-VPGVA and anti m-calpain. These results indicate that the aggrecan product with the COOH terminal neoepitope VPGVA709 is synthesized regularly by intracellular processing in chondrocytes, and is present abundantly as a limited form of aggrecan. M-calpain is the major candidate of the proteinase to generate this aggrecan product during the intracellular aggrecan processing.

Key Words: aggrecan, human osteoarthritis, keratan sulphate, m-calpain, proteoglycan

Abbreviations: ABTS, 2,2' (azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid); ADAMTS, a disintegrin and metalloproteinase with thrombospondin repeats; BSA, bovine serum albumin; CS, chondroitin sulphate; ELISA, enzyme-linked immunosorbent assay; GAG, glycosaminoglycan; GuHCl, guanidine hydrochloride; HRP, horseradish peroxidase; IGD, interglobular domain; KS, keratan sulphate; MMP, matrix metalloproteinase; OA, osteoarthritis


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