title: Analysis of tissue inhibitor of metalloproteinases-2 effect on pro-matrix metalloproteinase-2 activation by membrane-type 1 matrix metalloproteinase using baculovirus/insect-cell expression system.
abstract: Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14) is known to activate pro-matrix metalloproteinase-2 (pro-MMP-2; progelatinase A) on the cell surface. To analyse the tissue inhibitor of metalloproteinases-2 (TIMP-2) effect on activation of pro-MMP-2 by MT1-MMP, we have expressed the full-size MT1-MMP (fMT1-MMP) and a transmembrane (TM)-domain-deleted soluble MT1-MMP (sMT1-MMP) in the baculovirus/Sf9 (Spodoptera frugiperda 9) insect-cell system, where neither endogenous gelatinolytic MMPs nor TIMP-2 are expressed. Both fMT1-MMP and sMT1-MMP expressed in the expression system were found not to contain the pro-domain and were able to activate the TIMP-2-free pro-MMP-2. Both in the insect cells and in vitro, activation of pro-MMP-2 by fMT1-MMP was enhanced at low concentrations of TIMP-2 and inhibited by its higher concentrations. The maximal enhancing effect was detected at 0.05 molar fraction of TIMP-2/fMT1-MMP. In contrast, activation of pro-MMP-2 by sMT1-MMP was dose-dependently inhibited by TIMP-2. These results demonstrate that the TM domain of MT1-MMP is not required for the ability to activate pro-MMP-2, but is required for the enhancing effect of TIMP-2 on pro-MMP-2 activation by recruiting pro-MMP-2 to the MT1-MMP-TIMP-2 complex as a cell-surface pro-MMP-2 receptor. Moreover, our data strongly suggest that the pro-domain of MT1-MMP is not required for the TIMP-2-mediated enhancing effect on pro-MMP-2 activation. In addition, the pro-MMP-2 in the MT1-MMP-TIMP-2-pro-MMP-2 ternary complex was not activated without external activator, but readily by addition of sMT1-MMP. This result demonstrates that MT1-MMP free of TIMP-2 would be the enzyme responsible for activation of the pro-MMP-2 in the ternary complex under physiological conditions.
authors.affiliation: Department of Biochemistry, College of Science, and Bioproducts Research Center, Yonsei University, Seoul 120-749, Korea.
authors.forename: Y
authors.initials: Y
authors.lastname: Jo
authors.forename: J
authors.initials: J
authors.lastname: Yeon
authors.forename: H J
authors.initials: HJ
authors.lastname: Kim
authors.forename: S T
authors.initials: ST
authors.lastname: Lee
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000235:genetics / Q000821:virology; D053511:Matrix Metalloproteinase 14; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D004792:Enzyme Precursors; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14