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.