Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity

Abstract

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His(6) purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala- Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1( Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/ Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.

Published in

Journal of Biological Chemistry
2003, volume: 278, number: 41, pages: 39625-39631
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

SLU Authors

• Pejler, Gunnar

  • Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences
    

Associated SLU-program

Future Animal Health and Welfare (until Jan 2017)

UKÄ Subject classification

Biochemistry and Molecular Biology

Publication identifier

• DOI: https://doi.org/10.1074/jbc.M301512200

Permanent link to this page (URI)

https://res.slu.se/id/publ/66600