TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads.
Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface. However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP. In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2. The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner. In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations. TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA. A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available. Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F.
Kinoshita
T
T
Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo 108, Japan.
Sato
H
H
Okada
A
A
Ohuchi
E
E
Imai
K
K
Okada
Y
Y
Seiki
M
M
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli; D018093:Gelatinases / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*; D014162:Transfection
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D012685:Sepharose; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase