Identification of amino acid residues of the matrix metalloproteinase-2 essential for its selective inhibition by beta-amyloid precursor protein-derived inhibitor.
The extracellular domain of beta-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 that 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.
Higashi
Shouichi
S
Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, Japan. shigashi@yokohama-cu.ac.jp
Miyazaki
Kaoru
K
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics
D016564:Amyloid beta-Protein Precursor; D061965:Matrix Metalloproteinase Inhibitors; D009842:Oligopeptides; D011480:Protease Inhibitors; C060932:MMP7 protein, human; D020783:Matrix Metalloproteinase 7; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9