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Tharuka, M.D. Neranjan,Priyathilaka, Thanthrige Thiunuwan,Yang, Hyerim,Pavithiran, Amirthalingam,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.86 No.-
<P><B>Abstract</B></P> <P>Viperin is recognized as an antiviral protein that is stimulated by interferon, viral exposures, and other pathogenic molecules in vertebrate. In this study, a viperin homolog in the Big-belly seahorse (<I>Hippocampus abdominalis</I>; HaVip) was functionally characterized to determine its subcellular localization, expression pattern, and antiviral activity <I>in vitro</I>. The <I>HaVip</I> coding sequence encodes a 348 amino acid polypeptide with predicted molecular weight of 38.48 kDa. Sequence analysis revealed that HaVip comprises three main domains: the N-terminal amphipathic α-helix, a radical S-adenosyl-<SMALL>L</SMALL>-methionine (SAM) domain, and a conserved C-terminal domain. Transfected GFP-tagged HaVip protein was found to localize to the endoplasmic reticulum (ER). Overexpressed-HaVip in FHM cells was found to significantly reduce viral capsid gene expression in VHSV infection <I>in vitro</I>. Under normal physiological conditions, <I>HaVip</I> expression was ubiquitously detected in all 14 examined tissues of the seahorse, with the highest expression observed in the heart, followed by skin and blood. <I>In vivo</I> studies showed that <I>HaV</I>ip was rapidly and predominantly upregulated in blood, kidney, and intestinal tissue upon poly (I:C) stimulus. LPS and <I>Streptococus iniae</I> challenges caused a significant increase in expression of <I>HaVip</I> in all the analyzed tissues. The obtained results suggest that HaVip is involved in the immune system of the seahorse, triggering antiviral and antibacterial responses, upon viral and bacterial pathogenic infections.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Identified and characterized viperin from big-belly seahorse. </LI> <LI> The HaVip gene was ubiquitously expressed in unchallenged-fish tissues. </LI> <LI> Modulated HaVip transcription pattern revealed its contribution in the immune response. </LI> <LI> Overexpression of HaVip in FHM cells showed the antiviral activity. </LI> </UL> </P>
M.D. Neranjan Tharuka,Hyerim Yang,Jehee Lee 제주대학교 해양과학연구소 2020 해양과환경연구소 연구논문집 Vol.44 No.-
Interferon regulatory factors (IRFs) are among the most important transcription mediators and have multiple biological functions, such as antiviral and antimicrobial defense, cell differentiation, immune modulation, and apoptosis. Three IRF family members (HaIRF4-like, HaIRF6, and HaIRF8) of the big belly seahorse (Hippocampus abdominalis) were molecularly and functionally characterized at the sequence and transcriptional level. The coding sequences of HaIRF4-like, HaIRF6, and HaIRF8 were 1214, 1485, and 1266 bp in length, encoding proteins of size 46.21, 55.32, and 47.56 kDa, respectively. Potential viral transcription and replication was detected against VHSV infection using qPCR in HaIRFs-transfected FHM cells. IRFs significantly reduced viral gene expression at 24 h and 48 h post infection and the expression of interferon-stimulated genes (ISGs) was modulated at transcriptional level upon HaIRF overexpression in FHM cells. Subcellular HaIRF localization was observed using GFP-tagged expression vectors in FHM cells. HaIRF4-like and HaIRF8 were localized to the nucleus, whereas HaIRF6 was observed in the cytoplasm. All three IRFs were ubiquitously expressed in all analyzed tissues of the big belly seahorse. The mRNA expression of IRF4-like, IRF6, and IRF8 increased significantly post injection in the blood and gills following LPS, poly (I:C), and Streptococcus iniae challenge. These findings demonstrate that seahorse IRFs are involved in host defense mechanisms against immune stimulants and HaIRFs induce interferon and ISGs which trigger antiviral activity against viral infections in the host.
Rajamanthrilage Kasun Madusanka,M.D. Neranjan Tharuka,D.S. Liyanage,D.M.K.P. Sirisena,Jehee Lee 제주대학교 해양과학연구소 2020 해양과환경연구소 연구논문집 Vol.44 No.-
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.
Rajamanthrilage Kasun Madusanka,M.D. Neranjan Tharuka,W.S.P. Madhuranga,Seongdo Lee,Jehee Lee 제주대학교 해양과학연구소 2020 해양과환경연구소 연구논문집 Vol.44 No.-
Peroxiredoxins are a group of thiol-specific antioxidant proteins that take six isoforms in vertebrates and allow the innate immune system to sense and detoxify reactive oxygen species. In this study, we identified and characterized the perxiredoxin-1 (SsPrdx1) cDNA sequence from the rockfish, Sebastes schlegelii. In silico analysis revealed that SsPrdx1 contained a 594 bp long open reading frame (ORF) encoding a protein of 198 amino acids, with a predicted molecular weight and theoretical isoelectric point of 21.97 kDa and 6.30, respectively. The SsPrdx1 gene comprised six exons linked by five introns, while peroxiredoxin signature motifs were found in the highly conserved third, fourth, and fifth exons. Phylogenetic analysis and sequence alignment suggested that SsPrdx1 is evolutionarily conserved and that its most closely related counterpart is Salarias fasciatus. Recombinant SsPrdx1 (rSsPrdx1) displayed supercoiled DNA protection and insulin disulfide reduction activities in a concentration-dependent manner, while cells transiently transfected with pcDNA3.1 (+)/SsPrdx1 exhibited significant cytoprotective effects under oxidative stress and wound healing activity. SsPrdx1 transcripts were constitutively expressed under normal physiological conditions, with the highest expression observed in the blood. Moreover, SsPrdx1 expression increased in the blood, spleen, and liver following immune provocation by LPS, poly I:C, and Streptococcus iniae injection. Thus, this study provides insights into the role of SsPrdx1 in rockfish immune protection.
W.S.P. Madhuranga,M.D. Neranjan Tharuka,J.C. Harasgama,Hyukjae Kwon,Qiang Wan,Jehee Lee 제주대학교 해양과학연구소 2021 해양과환경연구소 연구논문집 Vol.45 No.-
Interferon-induced protein 35 kDa (IFP35) has been demonstrated to play important roles in antiviral defense, inflammatory response and cancer progression. However, its precise function in teleost fish remains to be elucidated. Herein, we functionally characterized the rock bream (Oplegnathus fasciatus) IFP35 (OfIFP35) to understand its expression pattern, subcellular localization, antiviral activity, and regulation of downstream genes. OfIFP35 consists of an 1107 bp open reading frame encoding 368 amino acids, including two N-mycinteractor (Nmi)/IFP35 domains (NIDs). The predicted molecular weight of OfIFP35 was 42 kDa, with a theoretical isoelectric point (pI) of 5.10. Evolutionary conservation of IFP35 was analyzed using multiple, pairwise alignments and phylogenetic tree analysis. OfIFP35 in rock bream was found to be highest expressed in the gills. Immune challenges with iridovirus, polyinosinic:polycytidylic acid, lipopolysaccharide, and live bacteria (Streptococcus iniae and Edwardsiella tarda) significantly upregulated its mRNA expression in gill and liver tissues of the rock bream. GFP-tagged OfIFP35 was localized in the cytoplasm of FHM cells, and its overexpression significantly suppressed VHSV transcription in vitro. Moreover, the analysis of downstream gene expression revealed that OfIFP35 could activate the type I interferon pathway. Collectively, these findings indicate that OfIFP35 is important for the immune system of rock bream as it promotes defense responses during viral infections.