A novel prophenoloxidase, hemocyanin encoded copper containing active enzyme from prawn: gene characterization.

The copper containing prophenoloxidase enzyme plays a crucial role in the defense system of arthropods, especially crustaceans and insects. In this study, we have reported a full length cDNA of prophenoloxidase identified from the constructed cDNA library of freshwater prawn Macrobrachium rosenbergii by genome sequence FLX technology. The identified full length M. rosenbergii prophenoloxidase (MrProPO) consists of 3378 base pairs (bp) with an open reading frame (ORF) of 2099 bp. This ORF encoded a polypeptide of 700 amino acids (aa) with an estimated molecular mass of 80 kDa and a predicted isoelectric point (pI) of 6.7. The motif analysis of MrProPO shows two copper binding sites (CuA and CuB) along with hemocyanin signatures and a thiol-ester like motif. MrProPO exhibited the maximum similarity (97%) with ProPO from Macrobrachium nipponense and is closely clustered with other crustacean ProPO in the phylogenetic tree. Bioinformatics analysis suggests that MrProPO is a member of the prophenoloxidase family, due to the conserved domains, motifs and similarity with other known ProPOs. The 3D structural analysis of MrProPO reveals that it has more random coils, moderate α-helices, few extended β-sheets and a very few β-turns. Among the 700 aa of MrProPO, 355 (50.71%), 206 (29.43%), 110 (15.71%) and 29 (4.14%) amino acids are responsible for random coils, α-helices, extended β-sheets and β-turns respectively. The gene expression results indicate MrProPO is widely distributed in all the tissues studied, but significantly (P<0.05) highest expression was observed in hepatopancreas. The relative expression of mRNA was quantified in hepatopancreas after being infected with virus [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacteria (Aeromonas hydrophila and Vibrio harveyi) using real-time PCR. MrProPO mRNA transcription significantly (P<0.05) increased at 24h post injection (p.i.) with subsequent decrease at 48 h p.i. in both viral and bacterial infected prawns. The highest enzyme activity was observed in hepatopancreas, which was also significantly higher (P<0.05) than detected in other tissues. Similar to gene expression results, the enzyme activity reached the peak at 24h p.i. and then the activity started decreasing. Overall results indicate that MrProPO is very likely to participate in the acute response against pathogen entry in prawns.