Kinoshita T, Sato H, Okada A, Ohuchi E, Imai K, Okada Y, Seiki M
Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo 108, Japan.
J Biol Chem. 1998 Jun 26;273(26):16098-103. doi: 10.1074/jbc.273.26.16098.
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.
膜型1基质金属蛋白酶（MT1-MMP）/MMP-14是细胞表面前明胶酶A（proGelA）/前MMP-2的激活剂。然而，存在一个矛盾的现象，即金属蛋白酶组织抑制剂-2（TIMP-2）作为MT1-MMP的抑制剂，却是表达MT1-MMP的细胞激活proGelA所必需的。在本研究中，将在C末端具有FLAG标签序列的截短型MT1-MMP（MT1-F）固定在琼脂糖珠（MT1-F/B）上，用于分析TIMP-2的作用。TIMP-2以剂量依赖性方式抑制MT1-F/B对合成肽底物的蛋白水解活性。相反，TIMP-2在低浓度时促进MT1-F/B对proGelA的加工，而在高浓度时抑制该过程。TIMP-2通过形成三聚体复合物促进proGelA与珠上的MT1-F结合，随后对proGelA进行加工。在仍有未占据的MT1-F的条件下观察到TIMP-2的刺激作用。因此，三元复合物被认为是一种将底物浓缩到珠表面并将其呈现给相邻游离MT1-F的方式。
