Two distinct CXC chemokine receptors (CXCR3 and CXCR4) from the big-belly seahorse <i>Hippocampus abdominalis</i>: Molecular perspectives and immune defensive role upon pathogenic stress

Priyathilaka, Thanthrige Thiunuwan,Oh, Minyoung,Bathige, S.D.N.K.,De Zoysa, Mahanama,Lee, Jehee Elsevier 2017 FISH AND SHELLFISH IMMUNOLOGY Vol.65 No.-

<P><B>Abstract</B></P> <P>CXC chemokine receptor 3 (CXCR3) and 4 (CXCR4) are members of the seven transmembrane G protein coupled receptor family, involved in pivotal physiological functions. In this study, seahorse <I>CXCR3</I> and <I>CXCR4</I> (designated as <I>HaCXCR3</I> and <I>HaCXCR4</I>) cDNA sequences were identified from the transcriptome library and subsequently molecularly characterized. <I>HaCXCR3</I> and <I>HaCXCR4</I> encoded 363 and 373 amino acid long polypeptides, respectively. The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors, including seven transmembrane domains and a G protein coupled receptors family 1 profile with characteristic DRY motifs. Amino acid sequence comparison and phylogenetic analysis of these two CXC chemokine receptors revealed a close relationship to their corresponding teleost counterparts. Quantitative real time PCR analysis revealed that <I>HaCXCR3</I> and <I>HaCXCR4</I> were ubiquitously expressed in all the tested tissues, with highest expression levels in blood cells. The seahorse blood cells and kidney <I>HaCXCR3</I> and <I>HaCXCR4</I> mRNA expressions were differently modulated when challenged with <I>Edwardsiella tarda</I>, <I>Streptococcus iniae</I>, lipopolysaccharide, and polyinosinic:polycytidylic acid, confirming their involvement in post immune responses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Full length coding sequences of CXC chemokine receptor 3 and 4 from Seahorse (HaCXCR3 and HaCXCR4) were identified. </LI> <LI> The HaCXCR3 and HaCXCR4 deduced proteins have typical structural features of chemokine receptors. </LI> <LI> The <I>HaCXCR3</I> and <I>HaCXCR4</I> mRNA was significantly up-regulated upon live pathogens and PAMPs in blood and kidney. </LI> </UL> </P>

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system

Priyathilaka, T.T.,Bathige, S.D.N.K.,Herath, H.M.L.P.B.,Lee, S.,Lee, J. Academic Press 2017 FISH AND SHELLFISH IMMUNOLOGY Vol.69 No.-

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS)]. Collectively, these findings suggest that AbCD63 and AbTSPAN33 are involved in innate immune responses in disk abalone during pathogenic stress.

Identification and molecular characterization of peroxiredoxin 6 from Japanese eel (Anguilla japonica) revealing its potent antioxidant properties and putative immune relevancy

Priyathilaka, T.T.,Kim, Y.,Udayantha, H.M.V.,Lee, S.,Herath, H.M.L.P.B.,Lakmal, H.H.C.,Elvitigala, D.A.S.,Umasuthan, N.,Godahewa, G.I.,Kang, S.I.,Jeong, H.B.,Kim, S.K.,Kim, D.J.,Lim, B.S. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.51 No.-

<P>Peroxiredoxins (Prdx) are thiol specific antioxidant enzymes that play a pivotal role in cellular oxidative stress by reducing toxic peroxide compounds into nontoxic products. In this study, we identified and characterized a peroxiredoxin 6 counterpart from Japanese eel (Anguilla japonica) (AjPrdx6) at molecular, transcriptional and protein level. The identified full-length coding sequence of AjPrdx6 (669 bp) coded for a polypeptide of 223 as residues (24.9 kDa). Deduced protein of AjPrdx6 showed analogy to characteristic structural features of 1-cysteine peroxiredoxin sub-family. According to the topology of the generated phylogenetic reconstruction AjPrdx6 showed closest evolutionary relationship with Salmo salar. As detected by Quantitative real time PCR (qPCR), AjPrdx6 mRNA was constitutively expressed in all the tissues examined. Upon the immune challenges with Edwardsiella tarda, lipopolysaccharides and polyinosinic:polycytidylic acid, expression of AjPrdx6 mRNA transcripts were significantly induced. The general functional properties of Prdx6 were confirmed using purified recombinant AjPrdx6 protein by deciphering its potent protective effects on cultured vero cells (kidney epithelial cell from an African green monkey) against H2O2-induced oxidative stress and protection against oxidative DNA damage elicited by mixed function oxidative (MFO) system. Altogether, our findings suggest that AjPrdx6 is a potent antioxidant protein in Japanese eels and its putative immune relevancy in pathogen stress mounted by live-bacteria or pathogen associated molecular patterns (PAMPs). (C) 2016 Elsevier Ltd. All rights reserved.</P>

Molecular identification and functional analysis of two variants of myeloid differentiation factor 88 (MyD88) from disk abalone (<i>Haliotis discus discus</i>)

Priyathilaka, Thanthrige Thiunuwan,Bathige, S.D.N.K.,Lee, Seongdo,Lee, Jehee Elsevier 2018 Developmental and comparative immunology Vol.79 No.-

<P><B>Abstract</B></P> <P>Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein of the Toll-like receptor (TLR)- and interleukin 1 receptor-mediated signaling pathways and is involved in a diverse array of inflammatory responses via NF-κB activation. In the present study, two MyD88 variants were identified from disk abalone (<I>Haliotis discus discus</I>) and designated AbMyD88-2 and AbMyD88-X. The deduced AbMyD88-2 and AbMyD88-X comprised 433 and 354 amino acids with predicted molecular masses of 48.85 kDa and 40.17 kDa, respectively. AbMyD88-2 and AbMyD88-X possessed typical MyD88 domain structural features including an N-terminal death domain (DD) and C-terminal toll interleukin 1 receptor (TIR) domain similar to those in mammals. Expression analysis of <I>AbMyD88-2</I> and <I>AbMyD88-X</I> mRNA at different early embryonic developmental stages of abalone by qPCR revealed that their constitutive expression at all developmental stages analyzed with the considerably higher values at the 16-cell (<I>AbMyD88-</I>2) and morula stages (<I>AbMyD88-X</I>). In unchallenged disk abalones, <I>AbMyD88-2</I> was highly expressed in muscles, while <I>AbMyD88-X</I> mRNA was predominantly transcribed in hemocytes. Moreover, <I>AbMyD88-2</I> and <I>AbMyD88-X</I> mRNA were differentially modulated in abalone hemocytes after a challenge with live bacteria (<I>Vibrio parahaemolyticus</I>, <I>Listeria monocytogenes</I>), virus (viral hemorrhagic septicemia virus), and pathogen-associated molecular patterns (lipopolysaccharides and Poly I:C). Overexpression of AbMyD88-2 and AbMyD88-X in HEK293T cells induced the activation of the NF-κB promoter. AbMyD88-2 and AbMyD88-X involvement in inflammatory responses was characterized by their overexpression in RAW264.7 murine macrophage cells. These results revealed comparatively higher NO (Nitric oxide) production, induction of inflammatory mediator genes (<I>iNOS</I> and <I>COX2</I>), and proinflammatory genes (<I>IL1β, IL6</I> and <I>TNFα</I>) expression in abalone MyD88s-overexpressing cells than in mock control in the presence or absence of LPS stimulation. Altogether, these results suggest that existence of a MyD88-dependent like signaling pathway in disk abalone and that both AbMyD88-2 and AbMyD88-X might be involved in innate immune and inflammatory responses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Two MyD88 isoforms were identified from Disk abalone (AbMyD88-2 and AbMyD88-X). </LI> <LI> AbMyD88-2 and AbMyD88-X possessed typical MyD88 domain structural features. </LI> <LI> Both MyD88 isoforms were constitutive expressed at early embryonic developmental stages of disk abalone. </LI> <LI> The mRNA expression of AbMyD88-2 and AbMyD88-X was differentially modulated upon immune stimulation in vivo. </LI> <LI> AbMyD88-2 and AbMyD88-X involved in inflammatory responses via NF-κB activation. </LI> </UL> </P>

Biochemical characterization and structural analysis of GST homologue form bigbelly seahorse (Hippocampus abdominalis)

Thanthrige Thiunuwan Priyathilaka,S.D.N.K. Bathige,Jehee Lee 한국수산해양기술학회(구 한국어업기술학회) 2016 한국수산해양기술학회 학술발표대회 Vol.2016 No.10

Evidences for existence of MyD88 dependent-like TLR signaling pathway in disk abalone (Haliotis discus discus)

Thanthrige Thiunuwan Priyathilaka,S.D.N.K. Bathige,Jehee Lee 한국수산해양기술학회(구 한국어업기술학회) 2017 한국수산해양기술학회 학술발표대회 Vol.2017 No.11

넙치 (Paralichthys olivaceus) vasa 유전자의 full-length cDNA 분리 및 생식소 특이적 발현

정형복,김유철,김효원,Thanthrige Thiunuwan Priyathilaka,이성도,Viraj Udayantha Herath Mudiyanselage,최재영,황일선,진창남,허윤성,서종표,임봉수 제주대학교 해양과환경연구소 2014 해양과환경연구소 연구논문집 Vol.38 No.-

Until recent, primordial germ cells(PGCs) are recognized only by morphological observation, such as their large size and low nucleocytoplasmic ratio. For the molecular analysis of the reproduction, it is important to identify a specific marker of germ cell development and differentiation. The VASA, which was first identified in Drosophila, is reported as a germ-line cell specific marker gene in animals. Many other researches verified its germ cell specific expression during embryogenesis and gametogenesis. VASA is a member of the DEAD(Asp-Glu-Ala-Asp) protein family of ATP-dependent RNA helicase, and plays a critical role in germ-line cell linage. Vasa is expected to be an useful molecular marker for identification of PGCs in reproduction researches of aquaculture species. In this study, we isolated the vasa cDNA, and surveyed its gonad-specific expression in Paralichthys olivaceus. The full-length cDNA of P. olivaceus vasa cDNA was isolated and deduced amino acid sequence was compared to those of the other teleosts. It was 2,461bp long, consisted in 646 amino acids in its ORF region, 175bp of 5’-UTR, and 345bp 3’-UTR. Flounder VASA contained conserved DEAD box, arginine-glycine rich region, and other domains were found. Flounder vasa expressed strongly in the testis and ovary, confirming its property as an gonad-specific marker. These result is expected to be an useful marker for flounder reproduction research and related aquaculture industry.

Two phospholipid scramblase 1–related proteins (PLSCR1like-a & -b) from <i>Liza haematocheila:</i> Molecular and transcriptional features and expression analysis after immune stimulation

Sandamalika, W.M. Gayashani,Priyathilaka, Thanthrige Thiunuwan,Nam, Bo-Hye,Lee, Jehee ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.87 No.-

<P><B>Abstract</B></P> <P>Phospholipid scramblases (PLSCRs) are a family of transmembrane proteins known to be responsible for Ca<SUP>2+</SUP>-mediated bidirectional phospholipid translocation in the plasma membrane. Apart from the scrambling activity of PLSCRs, recent studies revealed their diverse other roles, including antiviral defense, tumorigenesis, protein–DNA interactions, apoptosis regulation, and cell activation. Nonetheless, the biological and transcriptional functions of PLSCRs in fish have not been discovered to date. Therefore, in this study, two new members related to the PLSCR1 family were identified in the red lip mullet (<I>Liza haematocheila</I>) as <I>MuPLSCR1like-a</I> and <I>MuPLSCR1like-b</I>, and their characteristics were studied at molecular and transcriptional levels. Sequence analysis revealed that MuPLSCR1like-a and MuPLSCR1like-b are composed of 245 and 228 amino acid residues (aa) with the predicted molecular weights of 27.82 and 25.74 kDa, respectively. A constructed phylogenetic tree showed that MuPLSCR1like-a and MuPLSCR1like-b are clustered together with other known PLSCR1 and -2 orthologues, thus pointing to the relatedness to both PLSCR1 and PLSCR2 families. Two-dimensional (2D) and 3D graphical representations illustrated the well-known 12-stranded β-barrel structure of MuPLSCR1like-a and MuPLSCR1like-b with transmembrane orientation toward the phospholipid bilayer. In analysis of tissue-specific expression, the highest expression of <I>MuPLSCR1like-a</I> was observed in the intestine, whereas <I>MuPLSCR1like-b</I> was highly expressed in the brain, indicating isoform specificity. Of note, we found that the transcription of <I>MuPLSCR1like-a</I> and <I>MuPLSCR1like-b</I> was significantly upregulated when the fish were stimulated with poly(I:C), suggesting that such immune responses target viral infections. Overall, this study provides the first experimental insight into the characteristics and immune-system relevance of <I>PLSCR1</I>-related genes in red lip mullets.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>MuPLSCR1like-a</I> and <I>MuPLSCR1like-b</I> were identified from red lip mullet. </LI> <LI> Highest expression of <I>MuPLSCR1like-a</I> was observed in intestine. </LI> <LI> <I>MuPLSCR1like-b</I> was highly expressed in brain. </LI> <LI> Both genes were significantly upregulated after stimulated with poly(I:C). </LI> </UL> </P>

A teleostan homolog of catalase from black rockfish (<i>Sebastes schlegelii</i>): Insights into functional roles in host antioxidant defense and expressional responses to septic conditions

Elvitigala, Don Anushka Sandaruwan,Priyathilaka, Thanthrige Thiunuwan,Whang, Ilson,Nam, Bo-Hye,Lee, Jehee Elsevier 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.44 No.1

<P><B>Abstract</B></P> <P>Antioxidative defense renders a significant protection against environmental stress in organisms and maintains the correct redox balance in cells, thereby supporting proper immune function. Catalase is an indispensable antioxidant in organisms that detoxifies hydrogen peroxides produced in cellular environments. In this study, we sought to molecularly characterize a homolog of catalase (RfCat), identified from black rockfish (<I>Sebastes schlegelii</I>). <I>RfCat</I> consists of a 1581?bp coding region for a protein of 527 amino acids, with a predicted molecular weight of 60?kD. The protein sequence of RfCat harbored similar domain architecture to known catalases, containing a proximal active site signature and proximal heme ligand signature, and further sharing prominent homology with its teleostan counterparts. As affirmed by multiple sequence alignments, most of the functionally important residues were well conserved in RfCat. Furthermore, our phylogenetic analysis indicates its common vertebrate ancestral origin and a close evolutionary relationship with teleostan catalases. Recombinantly expressed RfCat demonstrated prominent peroxidase activity that varied with different substrate and protein concentrations, and protected against DNA damage. <I>RfCat</I> mRNA was ubiquitously expressed among different tissues examined, as detected by qPCR. In addition, <I>RfCat</I> mRNA expression was modulated in response to pathogenic stress elicited by <I>Streptococcus iniae</I> and poly I:C in blood and spleen tissues. Collectively, our findings indicate that RfCat may play an indispensable role in host response to oxidative stress and maintain a correct redox balance after a pathogen invasion.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Homolog of catalase was identified from black rockfish (RfCat) RfCat. </LI> <LI> RfCat resembled typical catalase domain architecture. </LI> <LI> Recombinant RfCat (rRfCat) showed detectable peroxidase activity. </LI> <LI> rRfCat could notably protect bacterial cells and DNA from oxidative damage. </LI> <LI> Transcriptional level of RfCat was modulated under pathogenic stress. </LI> </UL> </P>

Molecular and transcriptional insights into viperin protein from Big-belly seahorse (<i>Hippocampus abdominalis</i>), and its potential antiviral role

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>