Format

Send to

Choose Destination
Biochem Pharmacol. 2015 Mar 15;94(2):91-100. doi: 10.1016/j.bcp.2015.02.001. Epub 2015 Feb 7.

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

Author information

1
Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: walid.semaan@usherbrooke.ca.
2
Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: louisane.desbiens@usherbrooke.ca.
3
Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: martin.houde@usherbrooke.ca.
4
Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: julie.labonte@usherbrooke.ca.
5
Phenoswitch Bioscience Inc., 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: hugo.gagnon@psbinc.ca.
6
Biomedical Computation Center, Osaka Medical College, 2-7 Daigakumachi, Takatsuki 569-0801, Osaka Prefecture, Japan. Electronic address: center@art.osaka-med.ac.jp.
7
Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka Prefecture, Japan. Electronic address: pha010@art.osaka-med.ac.jp.
8
Department of Medicine, Brigham and Women's Hospital, Harvard University, 75 Francis St, Boston, MA 02115, United States. Electronic address: tlaidlaw@partners.org.
9
Department of Anatomy and Cell Biology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: ghassan.bkaily@usherbrooke.ca.
10
Division of Cardiology, McGill University, 1650 Avenue Cedar, Montreal, QC, Canada H3G 1A4. Electronic address: adel.giaid@mcgill.ca.
11
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.
12
Department of Surgery, Division of Urology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: christine.levesque@usherbrooke.ca.
13
Department of Surgery, Division of Urology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: roxane.desjardins@usherbrooke.ca.
14
Department of Surgery, Division of Urology, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address: robert.day@usherbrooke.ca.
15
Department of Pharmacology, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Electronic address: labpdj@usherbrooke.ca.

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.

KEYWORDS:

Chymase; In silico analysis; Mass spectrometry; Radiotelemetry; Recombinant enzymes

PMID:
25667044
DOI:
10.1016/j.bcp.2015.02.001
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center