ROLE OF MELANIZATION INHIBITION PROTEIN IN REGULATION OF PROPHENOLOXIDASE SYSTEM OF BLACK TIGER SHRIMP Penaeus monodon

Abstract

Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. The proPO system produces the reactive intermediates and melanin to fight against invading microorganism. However, the proPO system must be tightly regulated to avoid the excess production of their components such as melanin that can damage host cells. In this study, a melanization inhibition protein from black tiger shrimp Penaeus monodon, named PmMIP, which has been previously identified from P. monodon EST Database and 5’ and 3’ Rapid Amplification of cDNA Ends (RACE), was further characterized. The open reading frame of PmMIP contains 957 bp encoding a protein of 318 amino acid residues with a predicted molecular mass of 34.75 kDa and pI 5.5. The sequence analysis by BLASTX revealed that PmMIP exhibits 80% similarity to PlMIP from crayfish Pacifastacus leniusculus. The amino acid sequence analysis of PmMIP shows a fibrinogen-related domain and aspartic-rich region. PmMIP transcript was highly expressed in various shrimp tissues such as gills, hepatopancreas, intestine, lymphoid organ, and muscle. The expression of PmMIP in gill was down-regulated after Vibrio harveyi infection and PmMIP protein in plasma was decreased at 6 h and recovered at 24 h post infection. Double-stranded RNA mediated gene silencing of PmMIP could suppress both PmMIP transcriptional and translational levels and resulted in an increase in hemolymph phenoloxidase activity and proteinase activity. Furthermore, the recombinant PmMIP (rPmMIP) protein was successfully expressed in Escherichia coli system and purified by nickel-NTA affinity chromatography. rPmMIP was found to inhibit hemolymph PO activity. These results suggested that PmMIP was involved in the proPO system by acting as a negative regulator and interfering hemolymph proteinase function.