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

Authors: Karlson, U  Pejler, G  Tomasini-Johansson, B  Hellman, L 
Citation: Karlson U, etal., J Biol Chem. 2003 Oct 10;278(41):39625-31. Epub 2003 Aug 4.
Pubmed: (View Article at PubMed) PMID:12900423
DOI: Full-text: DOI:10.1074/jbc.M301512200

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 His6 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.

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CRRD Object Information
CRRD ID: 5128475
Created: 2011-03-04
Species: All species
Last Modified: 2011-03-04
Status: ACTIVE



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RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.