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

Buttner FH, Hughes CE, Margerie D, Lichte A, Tschesche H, Caterson B, Bartnik E (1998)
BIOCHEMICAL JOURNAL 333: 159-165.

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Abstract
The recent detection of membrane type 1 matrix metallo-proteinase (MT1-MMP) expression in human articular cartilage [Buttner, 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 rAAg1(mut), a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor 'aggrecanase' catabolism in vitro [Hughes, Buttner, Eidenmuller, Caterson and Bartnik (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 glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1(mut) substrate was cleaved at the 'aggrecanase' site equivalent to Glu(373)-Ala(374) (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (ed) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1(mut) as substrate. Coomassie staining of rAAg1(mut) 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.
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Buttner FH, Hughes CE, Margerie D, et al. 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. BIOCHEMICAL JOURNAL. 1998;333:159-165.
Buttner, F. H., Hughes, C. E., Margerie, D., Lichte, A., Tschesche, H., Caterson, B., & Bartnik, E. (1998). 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. BIOCHEMICAL JOURNAL, 333, 159-165.
Buttner, F. H., Hughes, C. E., Margerie, D., Lichte, A., Tschesche, H., Caterson, B., and Bartnik, E. (1998). 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. BIOCHEMICAL JOURNAL 333, 159-165.
Buttner, F.H., et al., 1998. 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. BIOCHEMICAL JOURNAL, 333, p 159-165.
F.H. Buttner, et al., “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”, BIOCHEMICAL JOURNAL, vol. 333, 1998, pp. 159-165.
Buttner, F.H., Hughes, C.E., Margerie, D., Lichte, A., Tschesche, H., Caterson, B., Bartnik, E.: 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. BIOCHEMICAL JOURNAL. 333, 159-165 (1998).
Buttner, FH, Hughes, CE, Margerie, D, Lichte, A, Tschesche, Harald, Caterson, B, and Bartnik, E. “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”. BIOCHEMICAL JOURNAL 333 (1998): 159-165.
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