ADAMTS‐4 activity on a proteoglycan vs. a polypeptide is controlled by the ancillary domains

Introduction  In combination with the catalytic domain, the ancillary domains of the ADAMTS' are proposed to regulate activity via interactions with sulfated GAGs, the extracellular matrix (ECM) and cell surface. Interactions with both GAGs and the ECM have been attributed to the thrombospondin (TSP) type 1 motifs and spacer region (Kuno and Matsushima 1998; Flannery et al. 2002). ADAMTS-1, -4 and -5, all undergo cleavage within their respective spacer regions (Flannery et al. 2002; Rodriguez-Manzaneque et al. 2000; Georgiadis et al. 2002), an event which has been reported to increase activity towards the interglobular domain of aggrecan and decrease the heparin affinity of ADAMTS-4 (Flannery et al. 2002; Gao et al. 2002). Materials and methods  V5- and His-tagged recombinant human ADAMTS-4 constructs terminating after the catalytic (▵DTS), disintegrin-like (▵TS), TSP (▵S) or spacer region (Full) were expressed in High-Five cells. Proteoglycanase activities of the resultant proteins were assayed with solution digests of aggrecan and a polyacrylamide-entrapped aggrecan particle assay. Proteolytic activity was measured using a novel, nonglycosylated, reporter substrate assay. Results  All forms of ADAMTS-4 were active to varying degrees in the reporter substrate assay. Digestion of aggrecan in solution digests was apparent in all proteins with the exception of the catalytic domain in isolation (▵DTS). Activity towards aggrecan decreased with increasing truncation of the protein. Discussion  Removal of the cysteine-rich-spacer domain and further C-terminal truncations decrease the activity of ADAMTS-4 towards aggrecan, whilst the proteolytic activity remains intact. Cleavages releasing the ancillary domains of ADAMTS' may therefore alter the catalytic activity of these enzymes against proteoglycans and also nonglycosylated polypeptides. More information is required about potential substrates for the processed forms of ADAMTS-4.