Interaction of α2‐Macroglobulin with Matrix Metalloproteinases and Its Use for Identification of Their Active Forms a

The activities of matrix metalloproteinases (MMPs) are regulated by two major groups of endogenous inhibitors: (1) tissue inhibitors of metalloproteinases (TIMPs) secreted from a number of cell types and (2) a general plasma proteinase inhibitor, a2-macroglobulin (a2M) and its related proteins. TIMPs probably control MMP activities immediately around the cell in the tissues, whereas a2M functions as a regulator of MMPs in body fluids, especially in the inflammatory sites. More than 95% of anticollagenase activity in plasma is due to a2M.I Human a2M is a tetrameric glycoprotein of 725 kDa. It consists of four identical subunits of 185 kDa that are linked in pairs by disulfide bonds and assembled non~ovalently.~.~ The inhibition mechanism of a2M is unique. It inhibits almost all endopeptidases regardless of their spe~ificities.~,~ The binding of a2M and a proteinase is initiated by proteolytic attack of the enzyme on the particular locus, so-called bait region, located near the middle of the subunit. The cleavage of the bait region triggers a conformational change in the a2M molecule that in turn entraps the enzyme without blocking the active site of the enzyme! The enzyme within the complex remains free to hydrolyze low molecular mass substrates but is restricted from reactions with large protein substrates. Inactive proteinases do not bind to azM.4 Another molecular feature of a2M is that the inhibitor contains an unusual intrachain P-cysteinyl-y-glutamyl th io le~ter .~ .~ Upon proteolysis of the bait region of azM, the thioester bond becomes immediately available for nucleophilic attack by the proteinase, and a large portion of the trapped enzymes are covalently linked to the a2M s u b ~ n i t . ~ . ~ The rate of complex formation with a2M differs among proteinases. In general, proteinases with limited specificity react slowly with a2M. We therefore investigated the rate of interaction of a2M with MMP-1 (interstitial collagenase), which readily digests the triple helical region of interstitial collagens I, 11, and 111, but has limited activity on other proteins. Kinetic studies indicated that ci2M is about 150 times better substrate for MMP-1 than collagen I, indicating it is a major inhibitor of collagenase activity? We also show that the unique trapping reaction of a2M that occurs only with active enzymes is useful for identification of catlytically active forms of MMPs. This feature was used to investigate the activation processes of the proh4MP-2 (progelatinase AFTIMP-2 and the proMMP-9 (progelatinase B)-TIMP-l complexes upon treatment with 4-aminophenyl mercuric acetate (APh4A) or trypsin.