Identification of a region of β-amyloid precursor protein essential for its gelatinase A inhibitory activity

Abstract

Because β-amyloid precursor protein (APP) has the abilities both to interact with extracellular matrix and to inhibit gelatinase A activity, this molecule is assumed to play a regulatory role in the gelatinase A-catalyzed degradation of extracellular matrix. To determine a region of APP essential for the inhibitory activity, we prepared various derivatives of APP. Functional analyses of proteolytic fragments of soluble APP (sAPP) and glutathione S-transferase fusion proteins, which contain various COOH-terminal parts of sAPP, showed that a site containing residues 579-601 of APP770 is essential for the inhibitory activity. Moreover, a synthetic decapeptide containing the ISYGNDALMP sequence corresponding to residues 586-595 of APP770 had a gelatinase A inhibitory activity slightly higher than that of sAPP. Studies of deletion, of the NH2- and COOH-terminal residues and alanine replacement of internal residues of the decapeptide further revealed that Tyr588, Asp591, and Leu593 of APP mainly stabilize the interaction between gelatinase A and the inhibitor. We also found that the residues of Ile586, Met594, and Pro595 modestly contribute to the inhibitory activity. The APP-derived decapeptide efficiently inhibited the activity of gelatinase A (IC50 = 30 nM), whereas its inhibitory activity toward membrane type 1 matrix metalloproteinase was much weaker (IC50 = 2 μM). The decapeptide had poor inhibitory activity toward gelatinase B, matrilysin, and stromelysin (IC50 > 10 αM). The APP-derived inhibitor formed a complex with active gelatinase A but not with progelatinase A, and the complex formation was prevented completely by a hydroxamate-based synthetic inhibitor. Therefore, the decapeptide region of APP is likely an active site-directed inhibitor that has high selectivity toward gelatinase A.