TIMP-2 Promotes Activation of Progelatinase A by Membrane-type 1 Matrix Metalloproteinase Immobilized on Agarose Beads

Yasunori Okada,Eiko Ohuchi,Kazushi Imai,Hiroshi Sato,Takeshi Kinoshita,Motoharu Seiki,Akiko Okada

doi:10.1074/jbc.273.26.16098

Abstract

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.

Highlights

Membrane-type 1 matrix metalloproteinase (MT1MMP)/Matrix metalloproteinases (MMPs)-14 is the activator of progelatinase A/proMMP-2 on the cell surface
It was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for progelatinase A (proGelA) activation by the cells expressing MT1-MMP
ProGelA had been reported to be activated by an unknown MMP-like activity on the surface of cancer and fibroblastic cells (10 –15), and we identified MT1-MMP as such an activator on the cell surface [16, 17]

Summary

Introduction

Membrane-type 1 matrix metalloproteinase (MT1MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface. 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. MMPs are produced as a zymogen (proMMP) that needs proteolytic activation by eliminating the N-terminal propeptide for the enzymes to function [7]. TIMP-2 mediates formation of a trimolecular complex by binding both proGelA and MT1-MMP, its relevance to proGelA processing on the cell surface was not clear, because the catalytic function of MT1-MMP in the complex is inhibited by the TIMP-2. Unlike the cell-mediated activation, the in vitro reaction with recombinant enzymes did not require TIMP-2 for proGelA activation. The difference may be caused by the HLD of MT1-MMP which was deleted in the recombinant enzymes or by the transmembrane domain that links the enzyme on the cell surface, or some important factors included in the crude preparations were missing in the assay

Methods

Results

Conclusion