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 P(1)' 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 P(1)-P(1)' and the N-terminal Gly-rich and Cys-rich regions of crustinPm1 were mutated by amino acid substitution or deletion. Substitutions of P(1)-P(1)' from Pro-Pro to Leu-Leu, Leu-His, Leu-Met, Leu-Ala and P(1)' from Pro to Met did not make the protein inhibitory to subtilisin, trypsin, chymotrypsin and elastase. The mutations at P(1)-P(1)' 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.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Penaeidae/genetics , Penaeidae/metabolism , Peptide Hydrolases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Amino Acid Motifs , Amino Acid Substitution , Animals , Cloning, Molecular , Cysteine/genetics , Gene Expression Profiling , Glycine/genetics , Mutant Proteins/genetics , Mutant Proteins/metabolism , Penaeidae/microbiology , Protein Structure, Tertiary , Sequence Alignment , Staphylococcus aureus/physiologyABSTRACT
A homolog of mammalian secretory leucocyte proteinase inhibitor or SLPI known as a double WAP domain (DWD) protein has been found in penaeid shrimp and believed to play an important role in innate immune system of the shrimp. The PmDWD identified from the Penaeus monodon EST database was investigated for its expression under pathogen infection. Infections by Vibrio harveyi and white spot syndrome virus (WSSV) up-regulated the expression of the PmDWD, which was peaked at about 24 h post infection and, then, subsided to more or less normal level. The PmDWD was expressed in various tissues of normal, 24-h WSSV-injected and leg-amputated shrimp, predominantly in the hemocytes. The expression was dramatically increased in lymphoid organ upon WSSV infection and leg amputation. The recombinant PmDWD (rPmDWD) was not active against the commercial proteinases: trypsin, chymotrypsin, elastase and subtilisin while its mutant rPmDWD_F70R was active against the subtilisin. By using agar diffusion assay, the rPmDWD inhibited the crude proteinases from lymphoid organs of leg-amputated and WSSV-infected shrimp. It inhibited the crude proteinases from Bacillus subtilis as well. Unlike the mammalian SLPIs, the rPmDWD had no antimicrobial activity against various bacteria.
