Rodent alpha-chymases are elastase-like proteases.
Although the alpha-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent alpha-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent alpha-chymases have unique biological activity different from the chymases of other species.
Kunori
Yuichi
Y
TEIJIN Institute for Biomedical Research, Hino, Tokyo, Japan. y.kinori@teijin.co
Koizumi
Masahiro
M
Masegi
Tsukio
T
Kasai
Hidenori
H
Kawabata
Hiroshi
H
Yamazaki
Yuzo
Y
Fukamizu
Akiyoshi
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D053818:Chymases; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000378:metabolism; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; D053818:Chymases