Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

Shouichi Higashi, Kaoru Miyazaki
2008 Journal of Biological Chemistry  
The extracellular domain of ␤-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study (Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020 -14028) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction
more » ... t correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala 88 and Gly 94 in the non-prime side and Tyr 145 and Thr 146 in the prime side of the substrate-binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro 10 of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr 3 of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate-binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor. . 2 The abbreviations used are: MMP, matrix metalloproteinase; APP, ␤-amyloid precursor protein; ECM, extracellular matrix; TIMP(s), tissue inhibitor(s) of metalloproteinases; MT1-MMP, membrane-type 1 MMP; APP-IP, APP-derived inhibitory peptide; GST, glutathione S-transferase; APMA, p-aminophenyl mercuric acetate; APMSF, p-amidinophenyl methanesulfonyl fluoride hydrochloride; BSA, bovine serum albumin.
doi:10.1074/jbc.m709509200 pmid:18238779 fatcat:nwn7um6savauvckr6qwndo47ri