Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the ‘aggrecanase’ and the MMP sites

Abstract

The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704–709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor ‘aggrecanase ’ catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269–20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the ‘aggrecanase ’ site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS …) and BC-14 (anti-FFGV …) to distinguish between ‘aggrecanase ’ and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong ‘aggrecanase ’ activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for ‘aggrecanase ’ activity in vivo.