Mutagenesis of wap domain-containing proteins from the black tiger shrimp Penaeus monodon

Abstract

The whey acidic protein (WAP) domain in various proteins has diverse biological functions including antimicrobial and antiproteinase activities. The crustinPm1, SWDPm2 and PmDWDall contain WAP domain but have different activities both antimicrobial and antiproteinase activities. In this study, the P₁ or P₁´ or both and the N-terminal glycine-rich and cysteine-rich regions of crustinPm1 were mutated and deleted. Substitution of P₁ from Pro to Leu and P₁´ from Pro to either Leu, His or Metdid not make the protein inhibitory against subtilisin, trypsin, chymotrypsin and elastase while the rcrustinPm1_WAP exhibited the strongest subtilisin inhibition followed by rcrustinPm1_delC and rcrustinPm1_WAP_LM. The rSWDPm2_E30R and rSWDPm2_E30P were as active the subtilisin inhibitor as the wildtype. The rcrustinPm1_WAP did not exhibit antimicrobial activity against Staphylococcus aureus while the rcrustinPm1_delG and rcrustinPm1_delC exhibited lower antimicrobial activity than the rcrustinPm1 indicating that the glycine-rich and cysteine-rich regions were important for antimicrobial activity and probablyone of the reasons that crustinPm1 is proteinase inhibitory inactive. The rPmDWD was not active against all four serine proteinases while its mutant rPmDWD_F70R was active against the subtilisin. The PmDWD was up-regulated in lymphoid tissue upon leg amputation and WSSV infection. The rPmDWD could inhibit the proteinase activities in lymphoid lysate from leg amputated and WSSV infected shrimp as well. In addition, it inhibited the crude proteinases from Bacillus subtilis.