Biochemical Pharmacology 2015-03-15

Chymase inhibitor-sensitive synthesis of endothelin-1 (1-31) by recombinant mouse mast cell protease 4 and human chymase.

Walid Semaan, Louisane Desbiens, Martin Houde, Julie Labonté, Hugo Gagnon, Daisuke Yamamoto, Shinji Takai, Tanya Laidlaw, Ghassan Bkaily, Adel Schwertani, Gunnar Pejler, Christine Levesque, Roxane Desjardins, Robert Day, Pedro D'Orléans-Juste

Index: Biochem. Pharmacol. 94(2) , 91-100, (2015)

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Abstract

Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1-31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas. The specificity of these activities was assessed with the chymase inhibitor TY-51469 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylic acid). For similar affinities, rmMCP-4 showed a higher activity toward the fluorogenic substrate and a higher ability to process Big ET-1 as compared to recombinant CMA1 (chymase activity (kcat/KM in μM(-1)s(-1)): 2.29 × 10(-4)vs. 6.41 × 10(-6); ET-1 (1-31) production: 2.19 × 10(-3)vs. 6.57 × 10(-5)), and both of these activities of mouse and human chymase were sensitive to TY-51469. Furthermore, extracts from mouse peritoneal mast cells, LUVA cells and human aorta homogenates contained processing activities toward the fluorogenic chymase substrate as well as Big ET-1, all of which were sensitive to TY-51469. Finally, the pressor responses to Big ET-1 but not to ET-1 were significantly reduced in conscious and free moving mMCP-4 KO mice when compared to wild type congeners. Our results suggest that both mouse and human chymases have potent ET-1 (1-31)-producing abilities, with the murine isoform being more efficient.Copyright © 2015 Elsevier Inc. All rights reserved.