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Herath, H.M.L.P.B.,Wickramasinghe, P.D.S.U.,Bathige, S.D.N.K.,Jayasooriya, R.G.P.T.,Kim, Gi-Young,Park, Myoung Ae,Kim, Chul,Lee, Jehee Elsevier 2017 FISH AND SHELLFISH IMMUNOLOGY Vol.60 No.-
<P><B>Abstract</B></P> <P>Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H<SUB>2</SUB>O<SUB>2</SUB>, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H<SUB>2</SUB>O<SUB>2</SUB>. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, <I>Haliotis discus discus</I>. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171–246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu<SUP>+2</SUP> and Cd<SUP>+2</SUP> ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, <I>AbGSR</I> was found to be ubiquitously distributed in different types of abalone tissues. <I>AbGSR</I> mRNA expression was significantly upregulated in response to three immune challenges: <I>Vibrio parahaemolyticus</I>, <I>Listeria monocytogenes</I>, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We identified a glutathione reductase homolog (AbGSR) from disk abalone. </LI> <LI> AbGSR resembled functionally important domain architecture of GSR family. </LI> <LI> Recombinant AbGSR confirmed its biochemical properties via enzymatic assays. </LI> <LI> First functional antioxidant properties assessment of a molluscan GSR. </LI> <LI> <I>AbGSR</I> expression was modulated upon induced pathogen stress in gill and hemocytes. </LI> </UL> </P>
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>
H.M.L.P.B Herath,THANTHRIGETHIUNUWAN PRIYATHILAKA,Don Anushka Sandaruwan Elvitigala,Navaneethaiyer Umasuthan,이제희 한국수산과학회 2015 Fisheries and Aquatic Sciences Vol.18 No.3
The follistatin (FST) gene encodes a monomeric glycoprotein that plays a role in binding and inhibiting the functions of members of the transforming growth factor (TGF)-β superfamily. Thus, FST facilitates a wide variety of functions, ranging from muscle growth, to inflammation and immunity. In this study, we sought to characterize an FST counterpart, RbFST, which was identified from rock bream Oplegnathus fasciatus. The RbFST cDNA sequence (2,419 bp) contains a 933-bp open reading frame (ORF) that encodes a putative amino acid sequence for RbFST (35 kDa). The putative amino acid sequence contains a Kazal-type serine protease inhibitor domain (51-98 residues) and an EF-hand, calcium-binding domain (191-226 residues). Additionally, this sequence shares a high identity (98.7%) with the Siniperca chuatsi FST sequence, with which it also has the closest evolutionary relationship according to a phylogenetic study. Omnipresent distribution of RbFST transcripts were detected in the gill, liver, spleen, head kidney, kidney, skin, muscle, heart, brain, and intestine of healthy animals, with significantly higher expression levels in the heart, followed by the liver tissue. Under pathogenic stress caused by two bacterial pathogens, Streptococcus iniae and Edwardsiella tarda, RbFST transcription was found to be significantly up-regulated. Altogether, our findings suggest the putative role of RbFST in immune related responses against pathogenic infections, further prefiguring its significance in rock bream physiology.
Herath, H.M.L.P.B.,Elvitigala, D.A.S.,Godahewa, G.I.,Umasuthan, N.,Whang, I.,Noh, J.K.,Lee, J. Elsevier/North-Holland 2016 Gene Vol.575 No.2
Interleukin 1β (IL-1β) and interleukin 8 (IL-8) are two major pro-inflammatory cytokines which play a central role in initiation of inflammatory responses against bacterial- and viral-infections. IL-1β is a member of the interleukin 1 family proteins and IL-8 is classified as a CXC-chemokine. In the current study, putative IL-1β and IL-8 counterparts were identified from a black rockfish transcriptomic database and designated as RfIL-1β and RfIL-8. The RfIL-1β cDNA sequence consists of 1140 nucleotides with a 759bp open reading frame (ORF) which encodes a 252 amino acid (aa) protein, whereas the RfIL-8 cDNA sequence (898bp) harbors a 300bp ORF encoding a 99 aa protein. Furthermore, the RfIL-1β aa sequence contains an IL-1 super family-like domain and an N-terminal IL-1 super family propeptide, while the amino acid sequence of RfIL-8 consists of a typical chemokine-CXC domain. Analysis of sequenced BAC clones containing RfIL-1β and RfIL-8 showed each gene to contain 4 exons interrupted by 3 introns. Pairwise comparison and phylogeny analysis of these cytokine sequences clearly revealed their closer relationship with other corresponding members of teleosts compared to birds and mammals. Constitutive differences in RfIL-1β and RfIL-8 mRNA expression were detected in a tissue-specific manner with the highest expression of each mRNA in spleen tissue. Two immune challenge experiments were conducted with Streptococcus iniae and polyinosinic:polycytidylic acid (poly I:C; a viral double stranded RNA mimic), and transcripts were quantified in spleen and peripheral blood cells. Significantly increased RfIL-1β and RfIL8 transcript levels were detected with almost similar profile patterns, further suggesting a putative involvement of these pro-inflammatory cytokines in the rockfish immunity.
Herath, H.M.L.P.B,Priyathilaka, Thanthrige Thiunuwan,Elvitigala, Don Anushka Sandaruwan,Umasuthan, Navaneethaiyer,Lee, Jehee The Korean Society of Fisheries and Aquatic Scienc 2015 Fisheries and Aquatic Sciences Vol.18 No.3
The follistatin (FST) gene encodes a monomeric glycoprotein that plays a role in binding and inhibiting the functions of members of the transforming growth factor (TGF)-${\beta}$ superfamily. Thus, FST facilitates a wide variety of functions, ranging from muscle growth, to inflammation and immunity. In this study, we sought to characterize an FST counterpart, RbFST, which was identified from rock bream Oplegnathus fasciatus. The RbFST cDNA sequence (2,419 bp) contains a 933-bp open reading frame (ORF) that encodes a putative amino acid sequence for RbFST (35 kDa). The putative amino acid sequence contains a Kazal-type serine protease inhibitor domain (51-98 residues) and an EF-hand, calcium-binding domain (191-226 residues). Additionally, this sequence shares a high identity (98.7%) with the Siniperca chuatsi FST sequence, with which it also has the closest evolutionary relationship according to a phylogenetic study. Omnipresent distribution of RbFST transcripts were detected in the gill, liver, spleen, head kidney, kidney, skin, muscle, heart, brain, and intestine of healthy animals, with significantly higher expression levels in the heart, followed by the liver tissue. Under pathogenic stress caused by two bacterial pathogens, Streptococcus iniae and Edwardsiella tarda, RbFST transcription was found to be significantly up-regulated. Altogether, our findings suggest the putative role of RbFST in immune related responses against pathogenic infections, further prefiguring its significance in rock bream physiology.