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Comparative analysis of selected superoxide dismutases (SODs) from big belly seahorse (Hippocampus abdominalis) and black rockfish (Sebastes schlegelii); revealing their putative significance in host antioxidant defense system

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Abstract
Cellular redox processes such as oxidative phosphorylation may lead to excess electrons in solution. As cells have reasonable oxygen concentrations, superoxide radical (O2•−) can be rapidly formed by attachment of the electron. Superoxide radical is not a particularly strong reductant or oxidant, so it is rather unreactive with the amino acids that comprise the protein backbone, with the notable exceptions of the sulfur-containing amino acids, cysteine and methionine. It is, however, very reactive with some transition metal complexes and the corresponding aquated ions, particularly copper, iron and manganese. Superoxide dismutases (SODs) are enzymes that function to catalytically convert O2•− to oxygen (O2) and hydrogen peroxide (H2O2). Based on the metal co-factor they harbor, SODs can be classified into four groups: iron SOD (FeSOD), manganese SOD (MnSOD), copper-zinc SOD (CuZnSOD), and nickel SOD (NiSOD). The evolutionary reason for the separation of SODs with different metal requirements is probably related to the different availability of soluble transition metal compounds in the biosphere in relation to the O2 content of the atmosphere in different geological eras.
Two intracellular types of SODs are known in mammalian cells, a mitochondrial, tetrameric manganese-containing enzyme (Mn-SOD) and a cytosolic, dimeric copper/ zinc-containing enzyme (Cu/Zn-SOD). Although these two SODs catalyze the same reaction, they have quite distinct structures and appear to be unrelated in terms of their evolution. SODs in primates have been studied mainly in humans, and to a much lesser extent in nonhuman primates. SODs have been found to be important for the long life-span of primates. The overall mechanism by which SODs function has been called a "ping-pong" mechanism as it involves the sequential reduction and oxidation of the metal center, with the concomitant oxidation and reduction of superoxide radicals at virtually diffusion controlled rates that generally include a pH range where the rate is unchanging.
The main role of SODs in all aerobic organisms is to neutralize the O2•− produced in the cytosol, mitochondria and endoplasmic reticulum of cells. However, the SOD can also have a pro-oxidant effect because the dissociation of the O2•− produces H2O2, which is toxic to cells. It is to remove this dangerous H2O2 that the presence of others antioxidant systems, such as CAT and GPx enzymes, becomes necessary.
In this study, four SOD genes including the CuZnSOD and MnSOD, each from big belly seahorse and rockfish have been identified and characterized at molecular and functional level. This report is divided into three main chapters based on the two different species and metal cofactors.
In chapter I, seahorse CuZnSOD and rockfish CuZnSOD were characterized at molecular level while analyzing their functional characteristics features and transcriptional modulation under pathological conditions. The complete cDNA sequences of ShCuZnSOD and RfCuZnSOD were consisted of 842 bp and 853 bp, respectively. Their putative polypeptide sequences were 154 aa (15.94 kDa) and 154 aa (16.04 kDa), respectively. Subsequently, the identified sequences were characterized using various bioinformatics tools, while comparing the sequences with other known similitudes. Both ShCuZnSOD and RfCuZnSOD shared similar domain architecture including putative N-glycosylation sites, a typical Cu/Zn_SOD domain, two signature motifs and three cysteine residues. ShCuZnSOD and RfCuZnSOD shared highest identity with Siniperca chuatsi (87.7%) and Lates calcarifer (87 %), respectively. Phylogenetic analysis revealed that both ShCuZnSOD and RfCuZnSOD were tightly clustered with the fish clade. The antioxidant function of ShCuZnSOD and RfCuZnSOD in the antioxidant system was evaluated via the xanthine/XOD assay. The highest activity of rShCuZnSOD was observed at pH 9; where the optimum pH for the rRfCuZnSOD was pH 8. The highest activity was recorded at 25 °C for both rCuZnSODs. According to the results the SOD activity was increased with the increasing concentration of both rCuZnSODs. rMBP did not have a significant impact on antioxidant activity. KCN, DDC, and NaN3 showed significant effects on the relative activity of both rCuZnSODs but EDTA had no effect. The peroxidation function of both rShCuZnSOD and rRfCuZnSOD were assessed by investigating cell viability using an MTT assay. Cell viability increased in the presence of HCO3- and rCuZnSODs in a dose dependent manner; the highest percentages were observed in the 100 μg/mL of rCuZnSODs, which resulted in ~73% increase in rShCuZnSOD and ~ 68% in rRfCuZnSOD. Extracellular H2O2 scavenging activity of rShCuZnSOD and rRfCuZnSOD in the presence of HCO3-, and the level of intracellular H2O2 in THP-1 cells were measured by flow cytometry. Intracellular ROS levels in the cells fell drastically after 100 μg/mL of rCuZnSOD (both rShCuZnSOD and rRFCuZnSOD) although the cells were exposed to oxidative stress by H2O2. The mRNA both ShCuZnSOD and RfCuZnSOD were significantly expressed in blood. In addition, both ShCuZnSOD and RfCuZnSOD were transcriptionally responded to immune challenges.
Chapter II enlightened the molecular characteristics and transcriptional properties of ShMnSOD and RfMnSOD. The complete cDNA sequence of ShMnSOD and RfMnSOD consisted of 1021 bp and 1061 bp, repectively. Two conservative domains including; Iron/Manganese SOD, C-terminal domain and Iron/Manganese SOD (SOD Fe-C domain), N-terminal domain (SOD Fe-N domain) were detected via the motif scan analyzer from both ShMnSOD and RfMnSOD. Clustal W pairwise alignment revealed the highest identity and similarity of ShMnSOD with Opleganathus fasciatus MnSOD (91.6% and 94.2% respectively) and RfMnSOD with Oplegnathus faciatus (97.3% and 99.6% repectively). The highest activity of rShMnSOD in scavenging superoxide radicals was observed at pH 9. Contritely, the highest SOD activity of rRfMnSOD was observed at pH 8. The optimum temperature for its SOD activity of both rMnSODs was recorded at 25 ºC. Moreover, the activity of both rMnSODs increased while increasing the concentration of rMnSODs revealing their dose dependency. The highest inhibition was observed with the incubation of KCN and followed by NaN3 for both rShMnSOD and rRfMnSOD. A constitutive expression of ShMnOSD and RfMnSOD with variable levels was observed in all fourteen tissues examined in the tissue specific expressional analysis. The highest expression was observed in the ovary and then followed by heart and brain in ShMnSOD. However, the highest expression of RfMnSOD was observed in blood and followed by ovary and skin. Additionally, both ShMnSOD and RfMnSOD mRNA were showed significant inductions against to immune stimulants used in the experiment. Third chapter describes the featured structural and functional variations of CuZnSOD and MnSOD of two different species.
Author(s)
나라헨피타게나디샤니
Issued Date
2017
Awarded Date
2017. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007997
Alternative Author(s)
Perera, Narahenpitage Chathurika Nadeeshani
Affiliation
제주대학교 일반대학원
Department
대학원 해양생명과학과
Advisor
이제희
Table Of Contents
Acknowledgement iv
Summary . vi
Table of Content x
List of Figures xiii
List of Tables xiv
1. Introduction 1
1.1 General introduction . 1
1.2 Reactive oxygen species and Antioxidants 1
1.3 Superoxide dismutases (SODs) 2
1.4 Seahorse and Rockfish 3
1.5 Aims of this work . 4
2. Methodology 5
2.1 Construction of cDNA databases . 5
2.2 In silico profiling 5
2.3 Preparation of the recombinant plasmid constructs 6
2.4 Over expression and purification of recombinant plasmids . 7
2.5 In vitro XOD/XO assay 8
2.5.1 Effect of pH 9
2.5.2 Effect of temperature . 9
2.5.3 Effect of concentration on activity . 9
2.5.4 Effect of inhibitors . 9
2.6 Peroxidation function analysis 10
2.6.1 Cell viability assay . 10
2.6.2 ROS scavenging assay . 10
2.7 Animal husbandry. 11
2.7.1 Seahorse rearing and tissue isolation . 11
2.7.2 Rockfish rearing and tissue isolation . 11
2.8 Immune challenge experiment 12
2.8.1 Seahorse . 12
2.8.2 Rockfish . 12
2.9 Total RNA extraction and first strand cDNA synthesis . 13
2.10 Quantitative real-time PCR (qPCR) . 13
2.11 Statistical analysis. 14
3. Chapter I . Identification and molecular characterization of ShCuZnSOD and RfCuZnSOD while deciphering their roles in host acute phase response 16
3.1 In silico analysis of CuZnSODs . 16
3.1.1 Delineation of sequence features and domain architecture 16
3.1.2 Homology analysis. 19
3.1.3 Phylogenetic reconstruction . 21
3.1.4 Tertiary structural characterization 24
3.2 Antioxidant activity analysis of rCuZnSODs . 25
3.2.1 Protein expression and purification . 25
3.2.2 SOD assay with the effect of pH 26
3.2.3 SOD assay with the effect of temperature . 27
3.2.4 Dose dependent antioxidant activity 28
3.2.5 Effect of inhibitors . 29
3.3 Peroxidation function of rCuZnSOD 29
3.3.1 Effect on cell viability 29
3.3.2 Extracellular ROS scavenging ability 31
3.4 Expression analysis of CuZnSODs . 34
3.4.1 Spatial mRNA expression 34
3.4.2 Temporal mRNA expression . 36
4. Chapter II Molecular characterization of ShMnSOD and RfMnSOD while portraying their antioxidant functions 37
4.1 In silico analysis of MnSODs . 37
4.1.1 Delineation of sequence features and domain architecture 37
4.1.1 Homology analysis. 39
4.1.2 Phylogenetic reconstruction . 42
4.1.3 Tertiary structure characterization . 43
4.2 Antioxidant activity analysis of rMnSODs 44
4.2.1 SOD assay with the effect of pH 44
4.2.2 SOD assay with the effect of temperature . 45
4.2.3 Dose dependent antioxidant activity 45
4.2.4 Effect of inhibitors . 46
4.3 Expression analysis of MnSODs. 47
4.3.1 Spatial mRNA expression 47
4.3.2 Temporal mRNA expression . 48
5. Chapter III Comparative analysis on structural and functional features of two different SODs; including CuZnSOD and MnSOD 50
Conclusions 53
Reference 54
Degree
Master
Publisher
제주대학교 일반대학원
Citation
나라헨피타게나디샤니. (2017). Comparative analysis of selected superoxide dismutases (SODs) from big belly seahorse (Hippocampus abdominalis) and black rockfish (Sebastes schlegelii); revealing their putative significance in host antioxidant defense system
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General Graduate School > Marine Life Sciences
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