Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile.
Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.
Karlson
Ulrika
U
Department of Cell and Molecular Biology, Uppsala University, The Biomedical Center, SE-751 24 Uppsala, Sweden.
Pejler
Gunnar
G
Froman
Gunnar
G
Hellman
Lars
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D053818:Chymases; D017931:DNA Primers; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C066932:mast cell protease 4; D053818:Chymases