title: Chymase inhibitor-sensitive synthesis of endothelin-1 (1-31) by recombinant mouse mast cell protease 4 and human chymase.
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.
authors: Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: walid.semaan@usherbrooke.ca.
authors: Walid
authors: W
authors: Semaan
authors: Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: louisane.desbiens@usherbrooke.ca.
authors: Louisane
authors: L
authors: Desbiens
authors: Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: martin.houde@usherbrooke.ca.
authors: Martin
authors: M
authors: Houde
authors: Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: julie.labonte@usherbrooke.ca.
authors: Julie
authors: J
authors: Labonté
authors: Phenoswitch Bioscience Inc., 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: hugo.gagnon@psbinc.ca.
authors: Hugo
authors: H
authors: Gagnon
authors: Biomedical Computation Center, Osaka Medical College, 2-7 Daigakumachi, Takatsuki 569-0801, Osaka Prefecture, Japan. Electronic address: center@art.osaka-med.ac.jp.
authors: Daisuke
authors: D
authors: Yamamoto
authors: Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka Prefecture, Japan. Electronic address: pha010@art.osaka-med.ac.jp.
authors: Shinji
authors: S
authors: Takai
authors: Department of Medicine, Brigham and Women's Hospital, Harvard University, 75 Francis St, Boston, MA 02115, United States. Electronic address: tlaidlaw@partners.org.
authors: Tanya
authors: T
authors: Laidlaw
authors: Department of Anatomy and Cell Biology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: ghassan.bkaily@usherbrooke.ca.
authors: Ghassan
authors: G
authors: Bkaily
authors: Division of Cardiology, McGill University, 1650 Avenue Cedar, Montreal, QC, Canada H3G 1A4. Electronic address: adel.giaid@mcgill.ca.
authors: Adel
authors: A
authors: Schwertani
authors: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Anatomi och fysiologi, Biokemi, Box 575, BMC B9 plan4, Dag Hammarskjölds v-Husarg, 751 23 and Uppsala University, Department of Medical Biochemistry and Microbiology, Uppsala, Sweden. Electronic address: gunnar.pejler@slu.se.
authors: Gunnar
authors: G
authors: Pejler
authors: Department of Surgery, Division of Urology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: christine.levesque@usherbrooke.ca.
authors: Christine
authors: C
authors: Levesque
authors: Department of Surgery, Division of Urology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: roxane.desjardins@usherbrooke.ca.
authors: Roxane
authors: R
authors: Desjardins
authors: Department of Surgery, Division of Urology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: robert.day@usherbrooke.ca.
authors: Robert
authors: R
authors: Day
authors: Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: labpdj@usherbrooke.ca.
authors: Pedro
authors: P
authors: D'Orléans-Juste
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D053818:Chymases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019332:Endothelin-1 / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000138:chemical synthesis*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D019332:Endothelin-1; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; C114932:endothelin-1 (1-31); D012697:Serine Endopeptidases; C066932:mast cell protease 4; D053818:Chymases
keywords: Chymase; In silico analysis; Mass spectrometry; Radiotelemetry; Recombinant enzymes