MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE (MT1-MMP) CLEAVES THE RECOMBINANT AGGRECAN SUBSTRATE RAGG1(MUT) AT THE AGGRECANASE AND THE MMP SITES - CHARACTERIZATION OF MT1-MMP CATABOLIC ACTIVITIES ON THE INTERGLOBULAR DOMAIN OF AGGRECAN

The recent detection of membrane type 1 matrix metallo-proteinase (MT1 -MMP) expression in human articular cartilage [Buttner, Chubinskaya, M argerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arth ritis Rheum. 40, 704-709] prompted our investigation of MT1-MMP's cata bolic activity within the interglobular domain of aggrecan. For these studies we used rAAg1(mut), a mutated form of the recombinant fusion p rotein (rAgg1) that has been used as a substrate to monitor 'aggrecana se' catabolism in vitro [Hughes, Buttner, Eidenmuller, Caterson and Ba rtnik (1997) J. Biol. Chem. 272, 20269-20274]. The rAgg1 was mutated ( G(332) to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycopro tein products. This mutation yielded a homogeneous recombinant product . Studies in vitro with retinoic acid-stimulated rat chondrosarcoma ce lls indicated that the rAgg1(mut) substrate was cleaved at the 'aggrec anase' site equivalent to Glu(373)-Ala(374) (human aggrecan sequence e numeration) in its interglobular domain sequence segment. The differen tial catabolic activities of the recombinant catalytic domain (ed) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compare d by using this rAgg1(mut) as substrate. Coomassie staining of rAAg1(m ut) catabolites separated by SDS/PAGE showed similar patterns of degra dation with all three recombinant enzymes. However, comparative immuno detection analysis, with neoepitope antibodies BC-3 (anti-ARGS...) and BC-14 (anti-FFGV...) to distinguish between 'aggrecanase' and MMP-gen erated catabolites, indicated that the catalytic domain of MT1-MMP exh ibited 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 site s within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for 'aggrecanase' activity in vi vo.