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Research paper

Molecular profiling and functional delineation of peroxiredoxin 3 (HaPrx3) from the big-belly seahorses (Hippocampus abdominalis) and understanding their immunological responses

원문정보

Anushka Vidurangi Samaraweera, M.D. Neranjan Tharuka, Thanthrige Thiunuwan Priyathilaka, Hyerim Yang, Sukkyoung Lee, Jehee Lee

제주대학교 해양과학연구소 해양과학연구소 연구논문집 제45권 2021.12 pp.77-91
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초록

영어

Peroxiredoxins (Prxs) are ubiquitously expressed antioxidant proteins that can protect aerobic organisms from oxidative stress. Here, we characterized the HaPrx3 homolog at the molecular level from big-belly seahorse (Hippocampus abdominalis) and analyzed its functional activities. The coding sequence of HaPrx3 consists of 726 bp, which encodes 241 amino acids. The predicted molecular weight and theoretical isoelectric point (pI) of HaPrx3 was 26.20 kDa and 7.04, respectively. Multiple sequence alignments revealed that the arrangements of domains, catalytic triads, dimers, and decamer interfaces of HaPrx3 were conserved among Prx sequences of other organisms. According to the phylogenetic analysis, HaPrx3 is clustered with the teleost Prx3 subclade. The highest transcript level of HaPrx3 was detected in the ovary tissue among fourteen healthy fish tissues. The mRNA levels of HaPrx3 in blood and liver tissues were significantly (P < 0.05) upregulated in response to lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), Edwardsiella tarda, and Streptococcus iniae, suggesting its involvement in immune responses. Under functional properties, insulin disulfide reduction assay confirmed the oxidoreductase activity of recombinant HaPrx3. A cell viability assay and Hoechst staining indicated cell survival ability and reduction of apoptotic activity, respectively. Moreover, a peroxidase activity assay verified peroxidase activity, while a metal-catalyzed oxidation (MCO) assay indicated the DNA protection ability of HaPrx3. Collectively, it is concluded that HaPrx3 may play a significant role in oxidative stress and immune responses against pathogenic infections in big-belly seahorses.

목차

ABSTRACT
1. Introduction
2. Materials and methods
2.1. Rearing experimental seahorses, the challenge experiment, and tissue sampling
2.2. Total RNA extraction and cDNA synthesis
2.3. Identification of Prx3 sequence and bioinformatics analysis
2.4. Spatial and temporal mRNA expression of HaPrx3
2.5. Construction of recombinant plasmid pMAL-c5X/HaPrx3
2.6. Protein expression and purification of recombinant HaPrx3 (rHaPrx3) fusion protein
2.7. Functional analysis of rHaPrx3
2.8. Statistical analysis
3. Results
3.1. Sequence analysis, protein models, alignment, and phylogenetic relationship of HaPrx3
3.2. Spatial transcriptional analysis of HaPrx3
3.3. Temporal expression of HaPrx3 after immune challenge
3.4. Expression and purification of recombinant HaPrx3
4. Discussion
References

키워드

저자정보

  • Anushka Vidurangi Samaraweera Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea
  • M.D. Neranjan Tharuka Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea
  • Thanthrige Thiunuwan Priyathilaka Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA
  • Hyerim Yang Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea
  • Sukkyoung Lee Department of Marine Life Sciences & Fish Vaccine Research Center, Jeju National University, 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, Marine Science Institute, Jeju National University, Jeju Self-Governing Province 63333, Republic of Korea

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