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Glutathione-S-transferase alpha-4 in Hippocampus abdominalis (big-belly seahorse): Molecular characterization, antioxidant properties, and its potent immune response

초록

영어

Glutathione-S-transferase (GST) is a key enzyme in the phase-II detoxification process and is a biomarker of oxidative stress. In this study, we analyzed the molecular, biochemical, and antioxidant properties of GST alpha-4 from Hippocampus abdominalis (HaGSTA-4). Also, the spatial and temporal expression of HaGSTA-4 upon immune challenge with abiotic and biotic stimulants were evaluated. The HaGSTA-4 ORF encodes 223 amino acids with a molecular weight of 25.7 kDa, and an estimated isoelectric point (pI) of 8.47. It consists of the GST_C superfamily and thioredoxin-like superfamily domain. The phylogenetic tree revealed that HaGSTA-4 is evolutionarily conserved with its GST alpha class counterparts. From pairwise alignment, the highest values of identity (78.5%) and similarity (85.7%) were with Parambassis ranga GSTA-4. Protein rHaGSTA-4 exhibited the highest conjugation activity towards 1-chloro-2,4-dinitrobenzene (CDNB) at pH 7 and 20 ◦C. A disk diffusion assay showed that rHaGSTA-4 significantly protects cells from the stress of exposure to ROS inducers such as CuSO4, CdCl2, and ZnCl2. Furthermore, overexpressed HaGSTA-4 defended cells against oxidative stress caused by H2O2; evidence of selenium-independent peroxidase activity. From qPCR, the tissue-specific expression profile demonstrates that HaGSTA-4 is most highly expressed in the kidney, followed by the intestine and stomach, among fourteen different tissues extracted from healthy seahorses. The mRNA expression profile of HaGSTA-4 upon immune challenge varied depending on the tissue and the time after challenge. Altogether, this study suggests that HaGSTA-4 may be involved in protection against oxidative stress, in immune defense regulation, and xenobiotic metabolism.

목차

ABSTRACT
1. Introduction
2. Materials and methods
2.1. HaGSTA-4 sequence analysis
2.2. Rearing of seahorses and tissue collection
2.3. Immune challenge experiment with bacterial pathogens and immune stimulants
2.4. RNA isolation and cDNA synthesis
2.5. Quantitative real-time PCR (qPCR) for analyzing the spatial and temporal expression of HaGSTA-4
2.6. Preparation of recombinant HaGSTA-4 plasmid constructs
2.7. Recombinant protein (rHaGSTA-4-MBP) expression and purification
2.8. The HaGSTA-4 - pcDNA™3.1(+) vector construction, cell culture, and transfection
2.9. Functional characterization of rHaGSTA-4
2.10. Statistical analysis
3. Results
3.1. HaGSTA-4 sequence analysis
3.2. Tissue-expression pattern of HaGSTA-4
3.3. Temporal expression of HaGSTA-4 after immune challenge
3.4. Induction and purification of rHaGSTA-4
3.5. Specific activities and kinetics of recombinant HaGSTA-4
3.6. Effect of environmental variables (pH and temperature) on rHaGSTA-4 activity
3.7. The antioxidant activity of rHaGSTA-4
3.8. Cell protective role of HaGSTA-4 against H2O2 toxicity
4. Discussion
5. Conclusion
References

저자정보

  • Kishanthini Nadarajapillai Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, 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
  • Sarithaa Sellaththurai 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
  • Taehyug Jeong 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
  • Sukkyoung 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
  • 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

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