MOLECULAR INSIGHTS AND FUNCTIONAL ANALYSIS OF MANGANESE SUPEROXIDE DISMUTASE IN REDLIP MULLET Liza haematocheilus
Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant enzyme which is called as metalloenzyme. The main function of this enzyme is dismutation of the toxic superoxide anion (O2-) into less toxic hydrogen peroxide (H2O2) and oxygen (O2). Structural analysis of mullet MnSOD (MuMnSOD) was practiced by using different bioinformatics web tools. Pairwise alignment results revealed that protein sequence matched to Larimichthys crocea with a 95.2% sequence identity and 98.8% similarity. Results of the phylogenetic analysis of MuMnSOD showed a close relationship with Fundulus heteroclitus and Austrofundulus limnaeus MnSODs. Multiple sequence alignment showed that the SOD Fe-N domain, SOD Fe-C domain, and Mn/Fe SOD signature were highly conserved among the other examined MnSOD orthologs. Hence, results of multiple sequence alignment suggested that the catalytic function of MnSOD may well conserve among its orthologs. Quantitative real-time PCR exhibited, the highest MuMnSOD mRNA expression in blood followed by liver, kidney, heart and muscle tissue among 12 different tissues from healthy mullet fish. Since blood is a main tissue which transports oxygen into host cells, generation of reactive oxygen species (ROS) in the blood is greater than the other organs. Therefore, the mullet antioxidative defense system may activate and produce more MuMnSOD transcripts against the formed O2- in blood. Hence, expression level of the MnSOD might be high in order to combat these ROSs. Highest MuMnSOD expression was observed against Lactococcus garvieae at 6 hours post infection in head kidney. L. garvieae is a serious pathogenic bacterium which coursed haemorrhagic septicemia in mullet fish. When pathogens attacked immune tissues, ROS generation is increased due to the respiratory burst. Hence, antioxidant defense mechanism of mullet may activate and expression of MuMnSOD might be enhanced due to high ROS level. Xanthine oxidase assay (XOD assay) revealed the ROS-scavenging ability of purified recombinant protein (rMuMnSOD). The optimum temperature and the pH for XOD activity were 25˚C and pH 7 respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. However, the activity of rMuMnSOD was inhibited by potassium cyanide (KCN). It might be due to binding of CN¯ ions into active sites of the MnSOD and replaced the Mn+ ions. Inclusively, results of the present study revealed that MuMnSOD act as an antioxidant enzyme and as an immune-related substance in the mullet fish.