ADAMTS4 Cleaves at the Aggrecanase Site (Glu373-Ala374) and Secondarily at the Matrix Metalloproteinase Site (Asn341-Phe342) in the Aggrecan Interglobular Domain

Abstract Two major proteolytic cleavages, one at NITEGE373↓A374RGSVI and the other at VDIPEN341↓F342FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu373-Ala374 site is cleavedin vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn341-Phe342, is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE373 and G1-VDIPEN341 in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu373-Ala374 site, but also, albeit slowly and secondarily, at the Asn341-Phe342 site. Cleavage at the Asn341-Phe342 site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu373-Ala374 site and secondarily generates G1-VDIPEN341 by removal of the Phe342–Glu373 peptide from G1-NITEGE373. These results show that G1-VDIPEN341 is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.