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

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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.
Author(s)
Don Manuwelge Kalana Prabhath Sirisena
Issued Date
2019
Awarded Date
2019. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/common/orgView/000000009016
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
Lee, Je Hee
Table Of Contents
SUMMARY . i
ACKNOWLEDGEMENT iv
LIST OF FIGURES viii
LIST OF TABLES . ix
1 INTRODUCTION . 1
1.1 Red lip mullet, Liza haematocheila as a cultured fish . 1
1.2 Reactive oxygen species, oxidative stress and antioxidants 2
1.3 Superoxide dismutase 4
1.4 Objectives of the study 7
2 MATERIALS AND METHODS . 8
2.1 Experimental fish rearing and tissue collection 8
2.2 Construction of cDNA database of redlip mullet 8
2.3 Immune challenge experiment for redlip mullet . 9
2.4 Isolation of RNA and cDNA synthesis. 9
2.5 Relative mRNA expression 9
2.6 In silico analysis 10
2.7 Preparation of the expression vector pMAL-c5X 11
2.8 Overexpression and purification of recombinant proteins . 12
2.9 Functional assays for rMuCuZnSOD and rMuMnSOD 13
2.9.1 Xanthine/xanthine oxidase (XOD) assay. 13
2.9.2 Biochemical properties of rMuCuZnSOD and rMuMnSOD 13
2.9.3 Peroxidation activity of rMuCuZnSOD by cell viability assay 14
2.9.4 Antibacterial activity of rMuCuZnSOD and rMuMnSOD . 14
2.10 Statistical analysis 15
3 RESULTS AND DISCUSSION . 16
3.1 Structural features of MuCuZnSOD and MuMnSOD sequences 16
3.2 Analysis of homology and evolutionary relationship 21
3.3 Characterization of tertiary structure of MuCuZnSOD and MuMnSOD . 25
3.4 Analysis of transcriptional tissue expression 26
3.5 Temporal transcriptional expression profiles of MuCuZnSOD and MuMnSOD 29
3.6 Bacterial overexpression and recombinant protein purification 34
3.7 Analysis of the antioxidant functions of recombinant SODs. 35
3.7.1 Determination of antioxidant ability of rMuCuZnSOD and rMuMnSOD at different pH values . 36
3.7.2 Determination of antioxidant activity of rMuCuZnSOD and rMuMnSOD at different temperatures 37
3.7.3 Determination of antioxidant ability of rMuCuZnSOD and rMuMnSOD at different dosages 39
3.7.4 Determination of the effect of inhibitory factors on rMuCuZnSOD and rMuMnSOD. 40
3.8 Determination of Peroxidation activity of rMuCuZnSOD 41
3.9 Determination of antibacterial activity of rMuCuZnSOD and rMuMnSOD 43
4 CONCLUSION . 46
5 REFERENCES 47
Degree
Master
Publisher
제주대학교 대학원
Citation
Don Manuwelge Kalana Prabhath Sirisena. (2019). Comparative Evaluation of Two Superoxide Dismutases from Redlip Mullet, Liza haematocheila
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General Graduate School > Marine Life Sciences
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