Dithiol Glutaredoxin Systems and Their Functional Significance in Teleost Immune Responses; Deciphering Roles of Glutaredoxin 1 and 2 in Redox Homeostasis of Big Belly Seahorse (Hippocampus abdominalis)
- Abstract
- Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. In this study, we characterized glutaredoxin 1 (HaGrx1) and glutaredoxin2 (HaGrx2) from big-belly seahorse, Hippocampus abdominalis. In-silico analysis showed that HaGrx1 contained the classical glutaredoxin 1 structure with a CSYC thioredoxin active site motif. HaGrx2 possess Glutaredoxin 2 structure with CPYC active site. According to multiple sequence alignment and phylogenetic reconstruction, HaGrx1 and HaGrx2 presented the highest homology to the Grx1, and Grx2 ortholog from Hippocampus comes respectively. Transcriptional studies demonstrated the ubiquitous distribution of HaGrx1 and HaGrx2 transcripts in all the seahorse tissues tested. HaGrx1 represented the highest expression in muscles whereas HaGrx2 highly expressed in brain and skin. Significant modulation (p < 0.05) of HaGrx1 and HaGrx2 transcripts were observed in blood as well as in liver upon stimulation with pathogen-associated molecular patterns and - hydroxyethyl disulfide reduction assay confirmed the antioxidant activity of recombinant HaGrx1. Further, dehydroascorbate reduction and insulin disulfide reduction assays revealed the oxidoreductase activity of HaGrx1 and HaGrx2. HaGrx1 utilized 1,4-dithiothreitol, L-cysteine, 2- mercaptoethanol, and reduced L-glutathione as reducing agent with different dehydroascorbate reduction activity levels. HaGrx2 represented 84% activity that of human glutaredoxin1. Further, HaGrx2 exhibit antiapoptotic activity against H2O2 induced oxidative stress. Altogether, our results suggested a vital role of HaGrx1 and HaGrx2 in redox homeostasis as well as the host innate immune defense system.
- Issued Date
- 2019
- Awarded Date
- 2019. 8
- Type
- Dissertation
- Affiliation
- 제주대학교 대학원
- Department
- 대학원 해양생명과학과
- Advisor
- Lee, Je Hee
- Table Of Contents
- SUMMARY V
ACKNOWLEDGMENT VI
List of Figures VII
List of Tables . VIII
BACKGROUND 1
1.1. Big-belly Seahorse (Hippocampus abdominalis) 2
1.2. Diseases in seahorse 3
1.3. Oxidative stress . 4
1.4. Antioxidant system against oxidative stress 4
1.5. Glutaredoxin 5
1.6. Dithiol glutaredoxin system 6
1.7. Deglutathionylation and functions of dithiol glutaredoxin . 6
1.8. Hypothesis and objectives of the present study. 7
CHAPTER 1 . 9
2.1. Introduction . 10
2.2. Materials and Methods 12
2.2.1. Identification of Grx1 from H. abdominalis . 12
2.2.2. Bioinformatic analysis of HaGrx1 sequences 12
2.2.3. Sample collection for tissue-specific distribution and temporal expression analysis 13
2.2.4. RNA extraction and cDNA synthesis . 14
2.2.5. Quantitative real-time PCR (qPCR) analysis . 14
2.2.6. Statistical analyses of qPCR data . 15
2.2.7. Recombinant plasmid construction 15
2.2.8. Overexpression and purification of HaGrx1 fusion protein . 16
2.2.9. Functional assays 17
2.2.9.1. Dehydroascorbic (DHA) reductase assay 17
2.2.9.2. Relative DHAR activity of HaGrx1 with different thiol compounds 17-Hydroxyethyl Disulfide (HED) and reduction assay . 18
2.2.9.4. Turbidimetric assay of insulin disulfide reduction 18
2.3. Results . 18
2.3.1. Sequence analysis of HaGrx1 18
2.3.2. Tissue distribution and immune challenge . 22
2.3.3. Expression and purification of rHaGrx1 24
2.3.4. Functional studies . 25
2.3.4.1. Dehydroascorbic (DHA) reduction assay 25
2.3.4.2. Relative DHAR activity of HaGrx1 with different thiol compounds 25
2.3.4.3. HED reduction assay . 27
2.3.4.4. Turbidimetric assay of insulin disulfide reduction 27
2.4 Discussion 29
CHAPTER 2 . 37
3.1. Introduction . 38
3.2. Materials and methods 39
3.2.1. Identification and bioinformatic analysis of Grx2 sequence from Hippocampus abdominalis 39
3.2.2. Acclimatization of big- belly seahorses, tissue sampling and immune stimulation . 40
3.2.3. RNA isolation and cDNA synthesis . 41
3.2.4. The mRNA expression profiling by Quantitative real-time PCR (qPCR) analysis . 41
3.2.5. Recombinant plasmid construction 42
3.2.6. Overexpression and purification of recombinant HaGrx2 . 42
3.2.7. Functional assays 43
3.2.7.1. HaGrx2 deglutathionylation activity corresponds to the Human Grx1 . 43
3.2.7.2. Oxidoreductase activity of HaGrx2 . 43
3.2.7.3. Cell culture and transfection of HaGrx2 . 44
3.2.7.4. Cell viability assay upon H 2 O 2 45
3.3. Results . 45
3.3.1. Bioinformatic analysis of HaGrx2 . 45
3.3.2. Spatial and temporal expression profile of HaGrx2 . 49
3.3.3. Overexpression and purification of rHaGrx2 as MBP fusion protein 51
3.3.4 Functional studies 52
3.3.4.1. The deglutathionylation activity of rHaGrx2 correspond to the human Grx1 52
3.3.4.2. Oxidoreductase activity of rHaGrx2 . 52
3.3.4.3. Cytoprotective activity upon oxidative stress . 54
3.4. Discussion . 54
References 62
- Degree
- Master
- Publisher
- 제주대학교 대학원
- Citation
- Welivitiye Kankanamge Malithi Omeka. (2019). Dithiol Glutaredoxin Systems and Their Functional Significance in Teleost Immune Responses; Deciphering Roles of Glutaredoxin 1 and 2 in Redox Homeostasis of Big Belly Seahorse (Hippocampus abdominalis)
- Appears in Collections:
- General Graduate School > Marine Life Sciences
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