Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis

Godahewa, G.I.,Perera, N.C.N.,Bathige, S.D.N.K.,Nam, B.H.,Noh, J.K.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.55 No.-

<P>The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277 amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CID. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451 bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12 h p.i.), Streptococcus iniae (12 h p.i.), rock bream iridovirus (RBIV) (6-12 h p.i.), and poly I:C (6 h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Molecular characterization and expression analysis of B cell activating factor from rock bream (Oplegnathus fasciatus)

Godahewa, G.I.,Perera, N.C.N.,Umasuthan, N.,Wan, Q.,Whang, I.,Lee, J. Pergamon Press ; Elsevier Science 2016 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.55 No.-

<P>B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) ligand family. BAFF has been shown to induce survival and proliferation of lymphocytes. We characterized the gene encoding BAFF (RbBAFF) in rock bream (Oplegnathus fasciatus), and attempted to determine its biological functions upon immune responses. In silk analysis of RbBAFF demonstrated the presence of common TNF ligand family features, including a TNF domain, a D-E loop, and three cysteine residues that are crucial for trimer formation. Amino acid sequence alignment confirmed that RbBAFF and its homologs were conserved at secondary and tertiary levels. Transcriptional analysis indicated that RbBAFF mRNAs were ubiquitously expressed in wide array of tissues. The higher levels of constitutive expression were observed in the kidney, head kidney and spleen, suggesting an important physiological relationship with lymphocytes. Under pathological conditions, RbBAFF mRNA levels were significantly elevated. The role of RbBAFF in lymphocyte survival and proliferation was confirmed by MIT assays and flow cytometry. Recombinant RbBAFF protein (10 mu g/mL) was able to prolong the survival and/or enhance the proliferation of rock bream lymphocytes by approximately 30%. Transcription of IL-10 and NF kappa B-1 was significantly stimulated by RbBAFF. Our findings provide further information regarding fish BAFF gene and its role in adaptive immunity. (C) 2015 Elsevier Ltd. All rights reserved.</P>

Antioxidative properties and structural features of atypical 2-Cys peroxiredoxin from <i>Sebastes schlegelii</i>

Godahewa, G.I.,Perera, N.C.N.,Nam, Bo-Hye,Lee, Jehee Elsevier 2018 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.82 No.-

<P><B>Abstract</B></P> <P>Atypical 2-Cys peroxiredoxin (Prx5) is an antioxidant protein that exerts its antioxidant function by detoxifying different reactive oxygen species (ROS). Here, we identified mitochondrial Prx5 from rockfish (SsPrx5) and described its specific structural and functional characteristics. The open reading frame (ORF) of SsPrx5 (570 bp) was translated into a 190-amino acid polypeptide that contained a mitochondrial targeting sequence (MTS), thioredoxin 2 domain, two Prx-specific signature motifs, and three conserved cysteine residues. Sequence comparison indicated that the SsPrx5 protein sequence shared greatest identity with teleost orthologs, where the phylogenetic results showed an evolutionary position within the fish Prx5. The coding sequence of SsPrx5 was scattered in six exons as found in other vertebrates. Additionally, the potent antioxidant functions of recombinantly expressed SsPrx5 protein was demonstrated by insulin reduction and extracellular H<SUB>2</SUB>O<SUB>2</SUB> scavenging both <I>in vitro</I> and <I>in vivo</I>. Quantitative real time PCR (qPCR) detected ubiquitous mRNA expression of <I>SsPrx5</I> in healthy rockfish tissues, with remarkable expression observed in gill, liver, and reproductive tissues. Prompt transcription of <I>SsPrx5</I> was shown in the immune-stimulated gill and liver tissues against <I>Streptococcus iniae</I> and lipopolysaccharide injection. Taken together, present results suggest the indispensable role of SsPrx5 in the rockfish antioxidant defense system against oxidative stresses and its role in maintaining redox balance upon pathogen invasion.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Atypical 2-Cys peroxiredoxin homolog was identified from <I>Sebastes schlegelii</I> (SsPrx5). </LI> <LI> SsPrx5 genomic structure was consisted with six exons and five introns. </LI> <LI> Extracellular H<SUB>2</SUB>O<SUB>2</SUB> scavenge of rSsPrx5 was observed in <I>in vitro</I> and <I>in vivo</I> assays. </LI> <LI> <I>SsPrx5</I> transcripts were ubiquitously detected in fourteen different tissues. </LI> <LI> Time course bacterial challenge showed the transcriptional modulation of <I>SsPrx5</I>. </LI> </UL> </P>

Insights into the multifunctional role of natural killer enhancing factor-A (NKEF-A/Prx1) in big-belly seahorse (<i>Hippocampus abdominalis</i>): DNA protection, insulin reduction, H₂O₂ scavenging, and immune modulation activity

Godahewa, G.I.,Perera, N.C.N.,Lee, Jehee Elsevier 2018 Gene Vol.642 No.-

<P><B>Abstract</B></P> <P>Natural killer enhancing factor A (NKEF-A), also known as peroxiredoxin 1 (Prx1), is a well-known antioxidant involved in innate immunity. Although NKEF-A/Prx1 has been studied in different fish species, the present study broadens the knowledge of <I>NKEF</I>-<I>A</I> gene in terms of molecular structure, function, and immune responses in fish species. <I>Hippocampus abdominalis</I> NKEF-A (<I>HaNKEF</I>-<I>A</I>) cDNA encoded a putative protein of 198 amino acids containing a thioredoxin_2 domain, VCP motifs, and three conserved cysteine residues including peroxidatic and resolving cysteines. Amino acid sequence comparison and phylogenetic breakdown showed the higher sequence identity and closer evolutionary position of <I>HaNKEF</I>-<I>A</I> to those of other fish counterparts. A recombinant protein of <I>HaNKEF</I>-<I>A</I> was shown to i) protect supercoiled DNA against mixed catalyzed oxidation, ii) reduce insulin disulfide bonds, and iii) scavenge extracellular H<SUB>2</SUB>O<SUB>2</SUB>. Results of in vitro assays demonstrated the concentration dependent antioxidant function of recombinant <I>HaNKEF</I>-<I>A</I>. In addition, qPCR assessments revealed that the <I>HaNKEF</I>-<I>A</I> transcripts were constitutively expressed in fourteen tissues with the highest expression in liver. As an innate immune response, <I>HaNKEF</I>-<I>A</I> transcripts were up-regulated in liver post injection of LPS, <I>Edwardsiella tarda</I>, <I>Streptococcus iniae</I>, and polyinosinic-polycytidylic acid. Thus, <I>HaNKEF</I>-<I>A</I> can safeguards big-belly seahorse from oxidative damage and pathogenic infections. This study provides insight into the functions of NKEF-A/Prx1 in fish species.</P> <P><B>Highlights</B></P> <P> <UL> <LI> NKEF-A homolog was identified from big-belly seahorse (<I>HaNKEF</I>-<I>A</I>). </LI> <LI> rHaNKEF-A protect the supercoiled DNA from ROS-provoked DNA damage. </LI> <LI> rHaNKEF-A diminish the intracellular ROS in cells under oxidative stress. </LI> <LI> <I>HaNKEF</I>-<I>A</I> transcripts were highly expressed in liver and modulated by pathogenic infection. </LI> </UL> </P>

Mitochondrial peroxiredoxin 3 (Prx3) from rock bream (<i>Oplegnathus fasciatus</i>): Immune responses and role of recombinant Prx3 in protecting cells from hydrogen peroxide induced oxidative stress

Godahewa, G.I.,Kim, Yucheol,Dananjaya, S.H.S.,Jayasooriya, R.G.P.T.,Noh, Jae Koo,Lee, Jehee,De Zoysa, Mahanama Elsevier 2015 Fish & shellfish immunology Vol.43 No.1

<P><B>Abstract</B></P> <P>Pathogenic infections and environmental factors cause a variety of stresses in fish including oxidative stress by rapid elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Transcriptional activation and expression of antioxidant enzymes are essential for reducing the oxidative stress. In this study, we present the molecular characterization, immune responses and ROS scavenging activity of mitochondrial peroxiredoxin 3 from <I>Oplegnathus fasciatus</I> (<I>RbPrx3</I>). Coding sequence (CDS) of <I>RbPrx3</I> contains 248 amino acids polypeptide which consists of highly conserved peroxiredoxin super family domain and two cysteine residues. Pairwise sequence comparison revealed that <I>RbPrx3</I> has the greatest identity (94.8%) to <I>Sparus aurata</I> Prx3. Transcriptional analysis of <I>RbPrx3</I> indicated the ubiquitously expressed mRNA in wide array of organs showing the highest expression in the liver of rock bream. Upon immune challenge of <I>Edwardsiella tarda</I>, <I>Streptococcus iniae</I>, rock bream iridovirus (RBIV) and lipopolysaccharide (LPS), <I>RbPrx3</I> mRNA level was up-regulated in immunocompetent liver tissues compared to unchallenged fish. Purified recombinant RbPrx3 treated THP-1 cells showed higher survival rate against H<SUB>2</SUB>O<SUB>2</SUB> induced oxidative stress and significantly reduced the level of intracellular ROS. Overall results from our study suggest that RbPrx3 may be involved in broader functions such as regulating oxidative stresses by scavenging ROS and activating immune responses in rock bream.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PRx3 is important for antioxidant defense and redox signaling. </LI> <LI> RbPrx3 is categorized into 2-Cys Prx. </LI> <LI> Constitutive expression of RbPrx3 is shown in wider array of rock bream tissues. </LI> <LI> Immune challenge activates the transcription of Prx3 in liver. </LI> <LI> Recombinant Prx6 can protect cells from oxidative stress by reducing H<SUB>2</SUB>O<SUB>2</SUB>. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Characterization of rock bream (Oplegnathus fasciatus) complement components C1r and C1s in terms of molecular aspects, genomic modulation, and immune responsive transcriptional profiles following bacterial and viral pathogen exposure

Godahewa, G.I.,Bathige, S.D.N.K.,Herath, H.M.L.P.B.,Noh, J.K.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.46 No.2

The complement components C1r and C1s play a crucial role in innate immunity via activation of the classical complement cascade system. As initiators of the pathogen-induced signaling cascade, C1r and C1s modulate innate immunity. In order to understand the immune responses of teleost C1r and C1s, Oplegnathus fasciatus C1r and C1s genes (OfC1r and OfC1s) were identified and characterized. The genomic sequence of OfC1r was enclosed with thirteen exons that represented a putative peptide with 704 amino acids (aa), whereas eleven exons of OfC1s represented a 691 aa polypeptide. In addition, genomic analysis revealed that both OfC1r and OfC1s were located on a single chromosome. These putative polypeptides were composed of two CUB domains, an EGF domain, two CCP domains, and a catalytically active serine protease domain. Phylogenetic analysis of C1r and C1s showed that OfC1r and OfC1s were evolutionary close to the orthologs of Pundamilia nyererei (identity = 73.4%) and Oryzias latipes (identity = 58.0%), respectively. Based on the results of quantitative real-time qPCR analysis, OfC1r and OfC1s transcripts were detected in all the eleven different tissues, with higher levels of OfC1r in blood and OfC1s in liver. The putative roles of OfC1r and OfC1s in response to pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and virus (rock bream iridovirus, RBIV) were investigated in liver and head kidney tissues. The transcription of OfC1r and OfC1s was found to be significantly upregulated in response to pathogenic bacterial and viral infections. Overall findings of the present study demonstrate the potential immune responses of OfC1r and OfC1s against invading microbial pathogens and the activation of classical signaling cascade in rock bream.

A cysteine protease (cathepsin Z) from disk abalone, <i>Haliotis discus discus</i>: Genomic characterization and transcriptional profiling during bacterial infections

Godahewa, G.I.,Perera, N.C.N.,Lee, Sukkyoung,Kim, Myoung-Jin,Lee, Jehee Elsevier 2017 Gene Vol.627 No.-

<P><B>Abstract</B></P> <P>Cathepsin Z (CTSZ) is lysosomal cysteine protease of the papain superfamily. It participates in the host immune defense <I>via</I> phagocytosis, signal transduction, cell-cell communication, proliferation, and migration of immune cells such as monocytes, macrophages, and dendritic cells. Hence, CTSZ is also acknowledged as an acute-phase protein in host immunity. In this study, we sought to identify the CTSZ homolog from disk abalone (AbCTSZ) and characterize it at the molecular, genomic, and transcriptional levels. <I>AbCTSZ</I> encodes a protein with 318 amino acids and a molecular mass of 36kDa. The structure of AbCTSZ reveals amino acid sequences that are characteristic of the signal sequence, pro-peptide, peptidase-C1 papain family cysteine protease domain, mini-loop, HIP motif, <I>N</I>-linked glycosylation sites, active sites, and conserved Cys residues. A pairwise comparison revealed that AbCTSZ shared the highest amino acid homology with its molluscan counterpart from <I>Crassostrea gigas</I>. A multiple alignment analysis revealed the conservation of functionally crucial elements of AbCTSZ, and a phylogenetic study further confirmed a proximal evolutionary relationship with its invertebrate counterparts. Further, an analysis of <I>AbCTSZ</I> genomic structure revealed seven exons separated by six introns, which differs from that of its vertebrate counterparts. Quantitative real time PCR (qPCR) detected the transcripts of <I>AbCTSZ</I> in early developmental stages and in eight different tissues. Higher levels of <I>AbCTSZ</I> transcripts were found in trochophore, gill, and hemocytes, highlighting its importance in the early development and immunity of disk abalone. In addition, we found that viable bacteria (<I>Vibrio parahaemolyticus</I> and <I>Listeria monocytogenes</I>) and bacterial lipopolysaccharides significantly modulated <I>AbCTSZ</I> transcription. Collectively, these lines of evidences suggest that <I>AbCTSZ</I> plays an indispensable role in the innate immunity of disk abalone.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CTSZ homolog was identified from disk abalone (<I>AbCTSZ</I>). </LI> <LI> <I>AbCTSZ</I> gDNA sequence was consisted with seven exons. </LI> <LI> Higher levels of <I>AbCTSZ</I> transcripts were detected in trochophore, gill and hemocyte. </LI> <LI> <I>AbCTSZ</I> is crucial in abalone early development and innate immunity. </LI> </UL> </P>

Two carboxypeptidase counterparts from rock bream (Oplegnathus fasciatus): Molecular characterization, genomic arrangement and immune responses upon pathogenic stresses

Godahewa, G.I.,Wickramaarachchi, W.D.N.,Whang, I.,Bathige, S.D.N.K.,Lim, B.S.,Choi, C.Y.,De Zoysa, M.,Noh, J.K.,Lee, J. Elsevier 2014 Veterinary immunology and immunopathology Vol. No.

Carboxypeptidases (CPs) are proteases that hydrolyze C-terminal peptide bonds. They are involved in regulating the complement system of the immune system. Here, we report the molecular characterization and immune response of two carboxypeptidases, named carboxypeptidase A (Rb-CPA) and carboxypeptidase N1 (Rb-CPN1), from rock bream. The genomic sequence of Rb-CPA contains 12 exons interrupted by 11 introns, while the genomic sequence of Rb-CPN1 has 9 exons and 8 introns. The cDNA sequence of Rb-CPA encodes a 421-amino-acid (AA) polypeptide (48kDa), and the cDNA of Rb-CPN1 encodes a 448-AA polypeptide (51kDa). The amino acid sequences of Rb-CPA and Rb-CPN1 were found to harbor two characteristic Zn-binding signature domains and a peptidase-M14 Zn carboxypeptidase site. Pairwise analysis revealed that Rb-CPA and Rb-CPN1 had the highest identity with the corresponding proteins from Anoplopoma fimbria (87.6%) and Dicentrarchus labrax (96.9%), respectively. qPCR results indicated that Rb-CPA and Rb-CPN1 were constitutively expressed mainly in the kidney, heart, liver, and head kidney. Both genes were transcriptionally regulated in the liver upon challenge with pathogenic bacteria (Streptococcus iniae, Edwardsiella tarda), rock bream iridovirus (RBIV), and the immune modulators polyinosinic:polycytidylic acid and lipopolysaccharide. Taken together, our findings suggest that Rb-CPA and Rb-CPN1 have immune-related functions in rock bream.

Characterization of a 1-cysteine peroxiredoxin from big-belly seahorse (Hippocampus abdominalis); insights into host antioxidant defense, molecular profiling and its expressional response to septic conditions

Godahewa, G.I.,Perera, N.C.N.,Elvitigala, D.A.S.,Jayasooriya, R.G.P.T.,Kim, G.Y.,Lee, J. Academic Press 2016 Fish & Shellfish Immunology Vol.57 No.-

1-cysteine peroxiredoxin (Prx6) is an antioxidant enzyme that protects cells by detoxifying multiple peroxide species. This study aimed to describe molecular features, functional assessments and potential immune responses of Prx6 identified from the big-belly seahorse, Hippocampus abdominalis (HaPrx6). The complete ORF (666 bp) of HaPrx6 encodes a polypeptide (24 kDa) of 222 amino acids, and harbors a prominent peroxiredoxin super-family domain, a peroxidatic catalytic center, and a peroxidatic cysteine. The deduced amino acid sequence of HaPrx6 shares a relatively high amino acid sequence similarity and close evolutionary relationship with Oplegnathus fasciatus Prx6. The purified recombinant HaPrx6 protein (rHaPrx6) was shown to protect plasmid DNA in the Metal Catalyzed Oxidation (MCO) assay and, together with 1,4-Dithiothreitol (DTT), protected human leukemia THP-1 cells from extracellular H<SUB>2</SUB>O<SUB>2</SUB>-mediated cell death. In addition, quantitative real-time PCR revealed that HaPrx6 mRNA was constitutively expressed in 14 different tissues, with the highest expression observed in liver tissue. Inductive transcriptional responses were observed in liver and kidney tissues of fish after treating them with bacterial stimuli, including LPS, Edwardsiella tarda, and Streptococcus iniae. These results suggest that HaPrx6 may play an important role in the immune response of the big-belly seahorse against microbial infection. Collectively, these findings provide structural and functional insights into HaPrx6.

Analysis of complete genome and pathogenicity studies of the spring viremia of carp virus isolated from common carp (<i>Cyprinus carpio carpio</i>) and largemouth bass (<i>Micropterus salmoides</i>): An indication of SVC disease threat in Korea

Godahewa, G.I.,Lee, Seongdo,Kim, Jeongeun,Perera, N.C.N.,Kim, Myoung-Jin,Kwon, Mun Gyeong,Jee, Bo Young,Hwang, Seong Don,Lee, Jehee ELSEVIER 2018 VIRUS RESEARCH Vol.255 No.-

<P><B>Abstract</B></P> <P>A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea.</P> <P><B>Highlights</B></P> <P> <UL> <LI> SVCV isolation from common carp and largemouth bass. </LI> <LI> Complete genome analysis revealed 11,034 bp nucleotides in length. </LI> <LI> Phylogenetic analysis revealed the Korean SVCV were clustered within the Asian clade. </LI> <LI> Virus transmission studies showed that the highest virulence is at 15 °C. </LI> <LI> Pathogenicity of SVCV was high in injection method than the other methods. </LI> </UL> </P>