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J. Biol. Chem., Vol. 279, Issue 11, 10109-10119, March 12, 2004

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Altered Proteolytic Activities of ADAMTS-4 Expressed by C-terminal Processing^*

Masahide Kashiwagi, Jan J. Enghild, Christi Gendron¶, Clare Hughes||**, Bruce Caterson||, Yoshifumi Itoh, and Hideaki Nagase

From the Kennedy Institute of Rheumatology Division, Imperial College London, 1 Aspenlea Rd., Hammersmith, London W6 8LH, United Kingdom, the Department of Molecular Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark, and the ^||Connective Tissue Laboratories, Cardiff School of Biosciences, University of Cardiff, Cardiff CF1 3US, United Kingdom

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu³⁷³-Ala³⁷⁴ bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu³⁷³-Ala³⁷⁴ and Glu¹⁴⁸⁰-Gly¹⁴⁸¹ bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.

Received for publication, November 5, 2003

^* The work was supported in part by the Wellcome Trust Grants 057508 and 061707 and National Institutes of Health Grant AR 40994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of the Wellcome Trust Prize Studentship.

^** An Arthritis Research Campaign Fellow.

To whom correspondence should be addressed. Tel.: 44-20-8383-4444; Fax: 44-20-8383-4994; E-mail: h.nagase{at}imperial.ac.uk.

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