Abstract
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.
Highlights
Among the Matrix metalloproteinases (MMPs) family, MMP-2 and MMP-9 are critical in the invasion of tumor cells across basement membranes, because of their strong activity against type IV collagen, a major component of basement membranes [11,12,13]
Only the catalytic domain of MMP-2 is likely sufficient for the selective interactivity toward 3163v, this protease did not interfere with the action with amyloid precursor protein (APP)-IP
We explored the amino acid residues of MMP-2 that correlate with the selective inhibition by APP-IP and found that residues Ala88, Gly94, Leu115, Tyr145, and Thr146 in the catalytic domain of MMP-2 were involved in the selectivity at least in comparison with the corresponding residues of MMP-7 and those of MMP-9
Summary
Among the MMP family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are critical in the invasion of tumor cells across basement membranes, because of their strong activity against type IV collagen, a major component of basement membranes [11,12,13]. The activity of MMP9-cat-FLAG was inhibited by APP-IP with a very high IC50 value (250 M), suggesting that the catalytic domain contains structural elements unfavorable for its interaction with APP-IP.
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