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

Abstract

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. © 2012 Elsevier Ltd.