Earticle

초록

영어

Glutaredoxins are a group of heat stable oxidoreductases ubiquitously found in prokaryotes and eukaryotes. They are widely known for GSH (glutathione)-dependent protein disulfide reduction and cellular redox homeostasis. This study was performed to identify and characterize rockfish (Sebastes schlegelii) glutaredoxin 1 (SsGrx1) at molecular, transcriptional, and functional levels. The coding sequence of SsGrx1 was 318 bp in length and encoded a protein containing 106 amino acids. The molecular weight and theoretical isoelectric point of the putative SsGrx1 protein were 11.6 kDa and 6.71 kDa, respectively. The amino acid sequence of SsGrx1 comprised a CPYC redox active motif surrounded by several conserved GSH binding sites. The modeled protein structure was found to consist of five α-helices and four β-sheets, similar to human Grx1. SsGrx1 showed a tissue specific expression in all the tissues tested, with the highest expression in the kidney. Immune stimulation by lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (polyI:C), and Streptococcus iniae (S. iniae) could significantly modulate the SsGrx1 expression pattern in the blood and gills. Analysis of its subcellular localization disclosed that SsGrx1 was prominently localized in the cytosol. Recombinant SsGrx1 (rSsGrx1) exhibited significant activity in insulin disulfide reduction assay and HED (β-Hydroxyethyl Disulfide) assay. Furthermore, transient overexpression of SsGrx1 in FHM (fathead minnow) cells significantly enhanced cell survival upon H2O2-induced apoptosis. Collectively, our findings strongly suggest that SsGrx1 plays a crucial role in providing rockfish immune protection against pathogens and oxidative stress.

목차

ABSTRACT
1. Introduction
2. Methodology
2.1. Experimental fish, RNA extraction and cDNA library construction
2.2. Sequence characterization and in-silico analysis of SsGrx1
2.3. Spatial distribution analysis and tissue sampling
2.4. Fish challenge and tissue collection
2.5. RNA purification and quantitative polymerase chain reaction (qPCR)
2.6. Construction of recombinant plasmids
2.7. Fusion protein expression and purification
2.8. Subcellular localization of SsGrx1
2.9. Oxidoreductase activity assay using insulin-disulfide reduction
2.10. HED (β-hydroxyethyl disulfide) assay
2.11. Cell culture, transient transfection and anti-apoptotic assay
3. Results
3.1. Sequence characteristics, homology and phylogenetic relationships
3.2. Tertiary structure of SsGrx1
3.3. Spatial and temporal expression patterns of SsGrx1
3.4. Subcellular localization of SsGrx1
3.5. Expression and purification of rSsGrx1
3.6. Insulin disulfide reduction assay
3.7. HED assay
3.8. Anti-apoptotic properties of SsGrx1 in-vitro
4. Discussion
5. Conclusion
Declaration of Competing Interest
Acknowledgements
Appendix A. Supplementary data
References

키워드

저자

• Rajamanthrilage Kasun Madusanka [ Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea, Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea ]
• M.D. Neranjan Tharuka [ Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea, Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea ]
• D.S. Liyanage [ Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea, Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea ]
• D.M.K.P. Sirisena [ Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea, Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea ]
• Jehee Lee [ Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea, Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea ] Corresponding Author

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  해양과학연구소 연구논문집 [Bulletin of the Marine Science Institute]

• 간기

  폐간

• pISSN

  1225-5734

• 수록기간

  1977~2021

• 십진분류

  KDC 454 DDC 551