The N-terminal glycine-rich and cysteine-rich regions are essential for antimicrobial activity of crustinPm1 from the black tiger shrimp Penaeus monodon

An antimicrobial protein crustinPm1 from Penaeus monodon is a WAP domain-containing protein with an antimicrobial activity against Gram-positive bacteria but does not have antiproteinase activity. The lack of antiproteinase is speculated to be due to the P1′ Met and/or the length of spacing between the conserved Cys2 and Cys3 while the antimicrobial activity may be due to the N-terminal Gly-rich and Cys-rich regions. In this study, the P1–P1′ and the N-terminal Gly-rich and Cys-rich regions of crustinPm1 were mutated by amino acid substitution or deletion. Substitutions of P1–P1′ from Pro–Pro to Leu–Leu, Leu–His, Leu–Met, Leu–Ala and P1′ from Pro to Met did not make the protein inhibitory to subtilisin, trypsin, chymotrypsin and elastase. The mutations at P1–P1′ positions in rcrustinPm1 had no effect on antibacterial activity. The WAP domain mutant with both Gly-rich and Cys-rich regions deleted did not exhibit antibacterial activity against Staphylococcus aureus while the deletion mutants of either Gly-rich or Cys-rich regions exhibited lower antibacterial activity than the wild type crustinPm1. Therefore, both Gly-rich and Cys-rich regions attached to a WAP domain are essential for efficient antibacterial activity of crustinPm1.