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J. Biol. Chem., Vol. 283, Issue 15, 10068-10078, April 11, 2008

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

From the Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, 641-12, Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan

The extracellular domain of β-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[Abstract/Free Full Text] ) 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 Ala⁸⁸ and Gly⁹⁴ in the non-prime side and Tyr¹⁴⁵ and Thr¹⁴⁶ 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 Pro¹⁰ of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr³ 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.

Received for publication, November 20, 2007 , and in revised form, December 26, 2007.

^* This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data, Fig. S1, and Tables SI and SII.

¹ To whom correspondence should be addressed: Div. of Cell Biology, Kihara Inst. for Biological Research, Yokohama City University, 641-12, Maiokacho, Totsuka-ku, Yokohama 244-0813, Japan. Tel.: 81-45-820-1905; Fax: 81-45-820-1901; E-mail: shigashi{at}yokohama-cu.ac.jp.

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