PROPERTIES AND FUNCTIONS OF PROPHENOLOXIDASE COMPONENTS IN CRUSTACEANS

Abstract

Melanization is a major defense mechanism in invertebrate that responds to the pathogens. In this study, we investigated the function of two serine proteinase homologues (PmMasSPH1 and PmMasSPH2) from black tiger shrimp Penaeus monodon. Sequence analysis revealed that PmMasSPH1 and PmMasSPH2 exhibited high sequence similarity to freshwater crayfish Pacifastacus leniusculus PlSPHs with the conserved clip-domain and serine proteinase-like domain at the N-terminus and C-terminus, respectively. The dsRNA-mediated gene suppression of PmMasSPH1 and PmMasSPH2 showed the significant decrease in hemolymph PO activity by 66.5% and 63.7%, respectively and a significant increase in the hemolymph bacterial count as compared with the control. In addition, the PmMasSPH1 suppression resulted in a decrease in the expression of antimicrobial peptide genes (PenmonPEN3, crustinPm1, and Crus-likePm) suggesting the cross-talk between the proPO system and antimicrobial peptide synthesis pathway. The co-immunoprecipitation of PmMasSPHs and PmPPAEs indicated the protein-protein interaction between PmMasSPH1 and PmPPAE2. PmMasSPHs exhibit binding ability to peptidoglycan (PGN), Gram-positive bacteria cell wall component, suggesting that PmMasSPH1 and PmMasSPH2 likely bind to PGN and activate the proPO system.

This study also investigated the cleavage of the proPO gene of freshwater crayfish P. leniusculus by caspases-1 and the proPO activating enzymes (ppA) and the roles of the cleaved fragments in bacterial clearance and antimicrobial activity. These fragments include PlproPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the N-terminal fragments cleaved by caspase-1. The injection of the cleaved peptides along with Escherichia coli, PlproPO-ppA, PlproPO-casp1 and PlproPO-casp2, showed significantly lower bacterial counts compared to the control (bacterial injection alone). PlproPO-ppA displayed the antimicrobial activity in the in vitro experiment, however, proPO-casp1 and -2 did not show the sign of activity. The viability assay indicated that the viability of agglutinated E. coli was affected by the recombinant proPO-ppA fragment. These findings suggest a new function of proPO in the P. leniusculus immune response.