title: Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the 'aggrecanase' and the MMP sites. Characterization of MT1-MMP catabolic activities on the interglobular domain of aggrecan.
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.
authors.affiliation: Biomedical Research, Hoechst Marion Roussel Deutschland GmbH, Building H 528, Rheingaustrasse 190, D-65174 Wiesbaden, Federal Republic of Germany.
authors.forename: F H
authors.initials: FH
authors.lastname: Büttner
authors.forename: C E
authors.initials: CE
authors.lastname: Hughes
authors.forename: D
authors.initials: D
authors.lastname: Margerie
authors.forename: A
authors.initials: A
authors.lastname: Lichte
authors.forename: H
authors.initials: H
authors.lastname: Tschesche
authors.forename: B
authors.initials: B
authors.lastname: Caterson
authors.forename: E
authors.initials: E
authors.lastname: Bartnik
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001859:Bone Neoplasms / Q000473:pathology; D002384:Catalysis; D002813:Chondrosarcoma / Q000473:pathology; D017364:Collagenases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D006801:Humans; D037181:Lectins, C-Type; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020784:Matrix Metalloproteinase 8; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: C508321:Acan protein, rat; D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D017364:Collagenases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020784:Matrix Metalloproteinase 8; C083641:aggrecanase