Identification of a region of beta-amyloid precursor protein essential for its gelatinase A inhibitory activity.
Because beta-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 APP(770) is essential for the inhibitory activity. Moreover, a synthetic decapeptide containing the ISYGNDALMP sequence corresponding to residues 586-595 of APP(770) had a gelatinase A inhibitory activity slightly higher than that of sAPP. Studies of deletion of the NH(2)- and COOH-terminal residues and alanine replacement of internal residues of the decapeptide further revealed that Tyr(588), Asp(591), and Leu(593) of APP mainly stabilize the interaction between gelatinase A and the inhibitor. We also found that the residues of Ile(586), Met(594), and Pro(595) modestly contribute to the inhibitory activity. The APP-derived decapeptide efficiently inhibited the activity of gelatinase A (IC(50) = 30 nm), whereas its inhibitory activity toward membrane type 1 matrix metalloproteinase was much weaker (IC(50) = 2 microm). The decapeptide had poor inhibitory activity toward gelatinase B, matrilysin, and stromelysin (IC(50) > 10 microm). 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.
Higashi
Shouichi
S
Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Japan. shigashi@yokohama-cu.ac.jp
Miyazaki
Kaoru
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000502:physiology*; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005780:Gelatin / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011498:Protein Precursors; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D016229:Amyloid beta-Peptides; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D005780:Gelatin; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; D014357:Trypsin; D013917:Thrombin; C036813:lysyl endopeptidase; D019278:Matrix Metalloproteinase 3; D020783:Matrix Metalloproteinase 7; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1