title: 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 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.
authors.affiliation: Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden.
authors.forename: Ulrika
authors.initials: U
authors.lastname: Karlson
authors.forename: Gunnar
authors.initials: G
authors.lastname: Pejler
authors.forename: Bianca
authors.initials: B
authors.lastname: Tomasini-Johansson
authors.forename: Lars
authors.initials: L
authors.lastname: Hellman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053818:Chymases; D006801:Humans; D066298:In Vitro Techniques; D008407:Mast Cells / Q000201:enzymology*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D019151:Peptide Library; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; D053818:Chymases