Comparative Evaluation of Two Superoxide Dismutases from Redlip Mullet, Liza haematocheila

Abstract

In the enzymatic antioxidant system, superoxide dismutases (SODs) are the most important metalloenzymes; they are involved in dismutation of toxic superoxide anions (O 2 into hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ). Superoxide dismutases (SODs) can be classified into four groups based on the metal residue that binds to the active site: manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/ZnSOD), iron superoxide dismutase (FeSOD) and nickel superoxide dismutase (NiSOD). Both CuZnSOD and MnSOD are the main SODs which presented in the cytoplasm and mitochondria of cells in the animals, respectively, that help to protect the cells from the adverse effects of excess ROS. In this study, Insilico analysis including multiple sequence alignments, 3D structures, homology analysis and evolutionary relationship were assessed to identify the structural features of mullet CuZnSOD (MuCuZnSOD) and MnSOD (MuMnSOD) using different bioinformatics tools. Transcriptional tissue expression was performed to evaluate the MuCuZnSOD and MuMnSOD expression in 12 different healthy tissues, whereas temporal mRNA expression analysis was conducted using different pathogenic stimulants such as lipopolysaccharide, Lactococcus garvieae, and polyinosinic-polycytidylic acid (poly I:C) with blood and liver. Functional characteristics of two SODs were investigated by conventional xanthine oxidase assay and bacterial assay. Also, the peroxidation function of MuCuZnSOD was demonstrated by the cell viability (MTT) assay in the presence of HCO 3 ¯ ions. The coding sequence of MuCuZnSOD was contained 465 bp and MuMnSOD was possessed 684 bp. The predicted molecular weight of the encoded protein of the MuCuZnSOD was 15.86 kDa, and its isoelectric point was 5.68 while, the MuMnSOD molecular weight was 25.39 kDa, and its isoelectric point was 8.37. Both MuCuZnSODnd MuMnSOD were detected as intracellular proteins due to the absence of signal peptides in their amino acid sequences. Results of multiple sequence alignments revealed that the MuCuZnSOD contained CuZnSOD domain, Copper/Zinc superoxide dismutase signature 1 (SOD_CU_ZN_1) and Copper/Zinc superoxide dismutase signature 2 (SOD_CU_ZN_2), and MuMnSOD contained iron/manganese superoxide dismutase C-terminal domain (SOD_Fe_C domain), iron/manganese superoxide dismutase N-terminal domain (SOD_Fe_N domain) and the manganese and iron superoxide dismutase signature (Mn/Fe SOD) were highly conserved among their analyzed orthologs. The pairwise alignment results showed that the highest identity was provided by the ( % identity) whereas, MuCuZnSOD gene evolutionary related to the Moreover, pairwise alignment revealed that the protein sequence matched to Larimichthys crocea with a sequence identity of 95.2%. Phylogenetic tree analysis showed that the MuMnSOD was included in the category of teleosts. The 3D structures of the MuCuZnSOD homo-dimer and MuMnSOD homo-tetramer were identified by the template of the crystal structure of human superoxide dismutase I with 68.83 % identity and human mitochondrial manganese superoxide dismutase with 84.34 % identity, respectively. Quantitative real-time PCR showed that the highest MuCuZnSOD and MuMnSOD mRNA expressions were in blood cells. Blood is the main oxygen transporter in the whole body, and it may be the main tissue which subjected to oxidative stress conditions due to direct exposure to the different ROS. The highest expression of MuCuZnSOD can be observed at 48 h post injection of poly I:C in the liver and 24 h post-injection in blood. After L. garvieae injection, the MuCuZnSOD expression was highly up-regulated at 24 h in both liver and blood tissues. The HighestMuCuZnSOD expression with LPS was observed at 72 h post injection in liver tissue and 6 h in blood. Furthermore, MuMnSOD was highly expressed at 24 h and 48 h postinjection of L. garvieae in blood and liver whereas, the highest MuMnSOD mRNA expression level was observed 24 h and 6 h post-injection with LPS in the liver and blood, respectively. However, the modulation patterns of both SODs against pathogenic stimulants in the blood and liver were different. Therefore, it could be suggested that mRNA expression of MuMnSOD might be tissue-specific in the presence of different immune stimulants. The optimum temperature for XOD activity of both recombinant SODs was found to be 25 °C whereas, optimum pH levels for the activity of MuCuZnSOD and MuMnSOD were pH 9 and pH 7, respectively. Relative XOD activity was significantly increased with the dose of rMuMnSOD, revealing its dose dependency. The activity of rMuCuZnSOD and rMuMnSOD was highly inhibited by potassium cyanide (KCN) and N-N'-diethyl-dithiocarbamate (DDC). Also, the MTT assay revealed that rMuCuZnSOD protein has affected to enhance the cell viability by the peroxidation activity in the presence of HCO 3 ¯ ions. Moreover, results obtained from the antibacterial assay explained that both recombinant proteins reduce the growth of grampositive and negative bacteria. Results of the present study suggest that MuCuZnSOD and MuMnSOD act as antioxidant enzymes and participate in the immune response in red lip mullet.