Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: Comparative analyses with ADAMTS-5,-9,-16 and-18

Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho) physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M, similar to 85 kDa1- ADAMTS-5(p85)) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M-r similar to 60 kDa (ADAMTS-5(p60) and M, similar to 45 kDa (ADAMTS-5(p45)). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5(p45) structural mimetic, terminating at Phe(628) and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F(628)), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu(1771)-Ala(1772)) and in the interglobular domain (at Glu(373)-Ala(374)). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9(F649)) or negligible GAG-affinity (trADAMTS-16(F647) and trADAMTS-18(F650)) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity. (C) 2006 Elsevier B.V. All rights reserved.