Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

Sammanfattning

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.

Publicerad i

Journal of Biological Chemistry
2002, volym: 277, nummer: 21, sidor: 18579-18585
Utgivare: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

SLU författare

• Pejler, Gunnar

  • Institutionen för veterinärmedicinsk kemi, Sveriges lantbruksuniversitet
    

Associerade SLU-program

Framtidens djurhälsa och djurvälfärd (tom Jan 2017)

UKÄ forskningsämne

Biokemi
Molekylärbiologi

Publikationens identifierare

• DOI: https://doi.org/10.1074/jbc.M110356200

Permanent länk till denna sida (URI)

https://res.slu.se/id/publ/66602