Characterization of a nucleotide-oligomerization domain (NOD) like receptor C5 (NLRC5) subfamily member from black rockfish (Sebastes schlegelii), portraying its transcriptional responses against immune stimulants

Elvitigala, Don Anushka Sandaruwan,Thulasitha, William Shanthakumar,Lee, Jehee Springer-Verlag 2016 Genes & Genomics Vol.38 No.3

<P>Nucleotide-oligomerization domain like receptors (NLRs) are recently identified group of pattern recognition receptors which involve in sensing broad range of pathogen associated molecular patterns or damage associated molecular patterns to trigger corresponding immune responses in host cells. In this study, we identified and characterized a NLRC5 family member from a previously established black rockfish cDNA database, designating as 'RfNLRC5'. The complete open reading frame of RfNLRC5 consists of 5808 bp which encodes for a protein of 1936 amino acids with the predicted molecular mass of 213 kDa. Intriguingly, RfNLRC5 harbored only two typical domain signatures of NLR superfamily, namely NACHT domain and LRRs. However, it was phylogenetically closely related to the telostan counterparts. As expected, RfNLRC5 shared significant sequence compatibility with its teleostan counterparts, eminently with that of large yellow croaker. As detected by our qPCR assay, RfNLRC5 was universally distributed in tissues examined, albeit with different levels. Therein, more pronounced expression levels were detected in blood cells and spleen tissues. After treating the na < ve fish with immune stimulants; lipopolysaccharides and Polyinosinic:polycytidylic acid (poly I:C), RfNLRC5 mRNA expression in blood cells and spleen tissues was found to modulate significantly with notable inductive responses. Collectively, our results in this study hint a potential role of RfNLRC5 in host innate immune responses against bacterial or viral infections.</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>

First report on the gastropod proapoptotic AIF3 counterpart from disk abalone (Haliotis discus discus) deciphering its transcriptional modulation by induced pathogenic stress

Elvitigala, D.A.S.,Jayasooriya, R.G.P.T.,whang, I.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.47 No.2

Apoptosis inducing factor (AIF) is a flavoprotein that is involved in oxidative phosphorylation and induces apoptosis in eukaryotic cells. There are three isozymes of AIF that have been identified to date, designated as AIF1, AIF2, and AIF3; the human AIF3 is also known as an AIF-like protein (AIFL). This study aimed to identify and characterize a homologue of AIF3 from disk abalone (AbAIF3) that belongs to the phylum Mollusca. The open reading frame (ORF) of AbAIF3 is 1749 base pairs (bp) in length and encodes a protein of 583 amino acids, with a predicted molecular mass of 63.14 kDa. Based on our in-silico analysis, the AbAIF3 protein harbored the typical domain architecture as that of the known AIF family proteins, consisting of N-terminal Rieske and pyridine nucleotide-disulphide oxidoreductase domain. Comparative protein sequence analysis confirmed that AbAIF3 is a homolog of AIF3. Moreover, our phylogenetic analysis revealed that AbAIF3 had a close evolutionary relationship with the molluscan counterparts. Interestingly, AbAIF3 was shown to induce apoptosis in HEK293T cells using transfection assays followed by flow cytometric analysis. In addition, we found that AbAIF3 mRNA expression was ubiquitous in physiologically important tissues, and significantly modulated upon experimental immune stimulations in hemocytes. Collectively, our study illustrates the indispensable role of AbAIF3 in inducing apoptosis in disk abalones, which in turn might be involved in hosts' immune defense mechanisms against microbial infections.

Molecular insights of the first gastropod TLR counterpart from disk abalone (Haliotis discus discus), revealing its transcriptional modulation under pathogenic stress

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Nam, B.H.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.35 No.2

Toll-like receptors (TLRs) are well-characterized pattern recognition receptors of innate immunity, known to induce immune responses against the pathogens by interacting with evolutionarily conserved pathogen-associated molecular patterns (PAMPs). In this study, a novel TLR homolog from disk abalone (Haliotis discus discus) was identified and characterized at molecular level. The open reading frame (ORF) of AbTLR is 3804 bp in length and encodes a 1268 amino acid peptide with a calculated molecular mass of 143.5 kDa. The deduced protein shows typical TLR domain architecture, with leucine rich repeats (LRR) and the toll-interleukin receptor (TIR) domain. Phylogenetic analysis revealed a close evolutionary relationship for AbTLR to its invertebrate counterparts, with close clustering to the molluscan homologs. Quantitative real-time PCR detected ubiquitous transcription of AbTLR in healthy tissues, but with highest levels in hemocytes. Differential transcriptional modulation of AbTLR was observed in abalone hemocytes and gills upon immune challenge, whereby Vibrio parahaemolyticus and purified lipopolysaccharide (LPS) enhanced the transcript level prominently. In addition, the viral hemorrhagic septicemia virus induced AbTLR transcription in hemocytes and gills, representing the first evidence of viral-induced immune response in mollusks to date. Collectively, our findings support a putative role for AbTLR in abalone antiviral and antibacterial defense.

Molecular profile and functional characterization of the ferritin H subunit from rock bream (Oplegnathus fasciatus), revealing its putative role in host antioxidant and immune defense

Elvitigala, D.A.S.,Priyathilaka, T.T.,Lim, B.S.,Whang, I.,Yeo, S.Y.,Choi, C.Y.,Lee, J. Pergamon Press ; Elsevier Science 2014 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.47 No.1

Ferritins are iron binding proteins made out of 24 subunits, involved in iron homeostasis and metabolism in cellular environments. Here, we sought to identify and functionally characterize a one type of subunits of ferritin (ferritin H-like subunit) from rock bream (Oplegnathus fasciatus; RbFerH). The complete coding sequence of RbFerH was 531bp in length, encoding a 177-amino acid protein with a predicted molecular mass of 20.8kDa. The deduced protein structure possessed the domain architecture characteristic of known ferritin H subunits, including metal ligands for iron binding, a ferroxidase center, and two iron-binding region signatures. As expected, the 5' untranslated region of the RbFerH cDNA sequence contained a putative iron response element region, a characteristic regulatory element involved in its translation. The RbFerH gene comprised 5 exons and 4 introns spanning a 4195bp region. Overexpressed recombinant RbFerH protein demonstrated prominent Fe(II) ion depriving activity, bacteriostatic properties, and protective effects against oxidative double-stranded DNA damage. Using quantitative polymerase chain reaction (qPCR), we found that RbFerH was expressed ubiquitously in the majority of physiologically important tissues in rock bream. A greater abundance of the mRNA transcripts were detected in blood and liver tissues. Upon administering different microbial pathogens and pathogen-derived mitogens, RbFerH transcription was markedly elevated in the blood of rock bream. Taken together, our findings suggest that RbFerH acts as a potent iron sequestrator in rock bream and may actively participate in antimicrobial as well as antioxidative defense.

A teleostean counterpart of ferritin M subunit from rock bream (Oplegnathus fasciatus): An active constituent in iron chelation and DNA protection against oxidative damage, with a modulated expression upon pathogen stress

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Oh, M.J.,Jung, S.J.,Park, C.J.,Lee, J. Academic Press 2013 FISH AND SHELLFISH IMMUNOLOGY Vol.35 No.5

Ferritins are biological iron chelators that can sequestrate excess iron to maintain iron homeostasis in the body. Ferritins basically consist of 2 types of subunits, designated as H and L. However, another new subunit, ferritin ''M'' which possesses characteristic features of both the H and L subunits, was recently identified in lower vertebrates, mostly in fish. In this study, a ferritin M-like subunit from rock bream (Oplegnathus fasciatus) (RbFerM) was characterized at the molecular level, and its transcriptional profile was analyzed in healthy fish, as well as in pathogen- and mitogen-stimulated fish. Furthermore, its functional properties were evaluated using the recombinant protein. The complete coding sequence of RbFerM was 528 bp in length, encoding a 176-amino acid peptide with a calculated molecular mass of 20 kDa. In silico analysis of RbFerM revealed that it has features similar to both the mammalian ferritin subunits, H and L. Phylogenetic analysis depicted the higher evolutionary proximity of RbFerM with its fish counterparts. Quantitative real time polymerase chain reaction (PCR) analysis detected a ubiquitous transcriptional profile of RbFerM in selected tissues of rock bream, in which more pronounced expression was observed in blood and liver tissues. Significant transcriptional inductions of RbFerM were detected in liver tissues upon lipopolysaccharides (LPS), Edwardsiella tarda, Streptococcus iniae, and rock bream irido virus (RBIV) exposures in time-course immune-challenge experiments. The purified recombinant protein of RbFerM demonstrated detectable iron chelating activity that varied with the temperature. Moreover, the recombinant RbFerM rendered a detectable protection effect against iron (II) and H<SUB>2</SUB>O<SUB>2</SUB>-mediated DNA damage.

Molecular cloning, expression and functional characterization of a teleostan cytokine-induced apoptosis inhibitor from rock bream (Oplegnathus fasciatus)

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Yeo, S.Y.,Choi, C.Y.,Noh, J.K.,Lee, J. Pergamon Press ; Elsevier Science 2015 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.52 No.1

Apoptosis plays a key role in the physiology of multicellular organisms and is regulated by different promoting and inhibitory mechanisms. Cytokine-induced apoptotic inhibitor (CIAPI) was recently identified as a key factor involved in apoptosis inhibition in higher vertebrate lineages. However, most of the CIAPIs of lower vertebrate species are yet to be characterized. Herein, we molecularly characterized a teleostan counterpart of CIAPI from rock bream (Oplegnathus fasciatus), designating as RbCIAPI. The complete coding region of RbCIAPI was consisted of 942 nucleotides encoding a protein of 313 amino acids with a predicted molecular mass of ~33@?kDa. RbCIAPI gene exhibited a multi-exonic architecture, consisting 9 exons interrupted by 8 introns. Protein sequence analysis revealed that RbCIAPI shares significant homology with known CIAPI counterparts, and phylogenetic reconstruction confirmed its closer evolutionary relationship with its fish counterparts. Ubiquitous spatial distribution of RbCIAPI was detected in our quantitative real time polymerase chain reaction (qPCR) analysis, where more prominent expression levels were observed in the blood and liver tissues. Moreover, the RbCIAPI basal transcription level was found to be modulated by different bacterial and viral stimuli, which could be plausibly supported by our previous observations on the transcriptional modulation of the caspase 3 counterpart of rock bream (Rbcasp3) in response to the same stimuli. In addition, our in vitro functional assay demonstrated that recombinant RbCIAPI could detectably inhibit the proteolysis activity of recombinant Rbcasp3. Collectively, our preliminary results suggest that RbCIAPI may play an anti-apoptotic role in rock bream physiology, likely by inhibiting the caspase-dependent apoptosis pathway. Therefore, RbCIAPI potentially plays an important role in host immunity by regulating the apoptosis process under pathogenic stress.

Caspase 3 from rock bream (Oplegnathus fasciatus): Genomic characterization and transcriptional profiling upon bacterial and viral inductions

Elvitigala, D.A.S.,Whang, I.,Premachandra, H.K.A.,Umasuthan, N.,Oh, M.J.,Jung, S.J.,Yeo, S.Y.,Lim, B.S.,Lee, J.H.,Park, H.C.,Lee, J. Academic Press 2012 Fish & Shellfish Immunology Vol.33 No.1

Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. In this study, caspase 3 was identified (Rbcasp3) and characterized from rock bream (Oplegnathus fasciatus). The full-length cDNA of Rbcasp3 is 2683 bp and contains an open reading frame of 849 bp, which encodes a 283 amino acid protein with a calculated molecular mass of 31.2 kDa and isoelectric point of 6.31. The amino acid sequence resembles the conventional caspase 3 domain architecture, including crucial amino acid residues in the catalytic site and binding pocket. The genomic length of Rbcasp3 is 7529 bp, and encompasses six exons interrupted by five introns. Phylogenetic analysis affirmed that Rbcasp3 represents a complex group in fish that has been shaped by gene duplication and diversification. Many putative transcription factor binding sites were identified in the predicted promoter region of Rbcasp3, including immune factor- and cancer signal-inducible sites. Rbcasp3, excluding the pro-domain, was expressed in Escherichia coli. The recombinant protein showed a detectable activity against the mammalian caspase 3/7-specific substrate DEVD-pNA, indicating a functional role in physiology. Quantitative real time PCR assay detected Rbcasp3 expression in all examined tissues, but with high abundance in blood, liver and brain. Transcriptional profiling of rock bream liver tissue revealed that challenge with lipopolysaccharides (LPS) caused prolonged up-regulation of Rbcasp3 mRNA whereas, Edwardsiella tarda (E. tarda) stimulated a late-phase significant transcriptional response. Rock bream iridovirus (RBIV) up-regulated Rbcasp3 transcription significantly at late-phase, however polyinosinic-polycytidylic acid (poly(I:C)) induced Rbcasp3 significantly at early-phase. Our findings suggest that Rbcasp3 functions as a cysteine-aspartate-specific protease and contributes to immune responses against bacterial and viral infections.

Molecular profile and expressional modulation of a Toll like receptor-1 homolog from rock bream (Oplegnathus fasciatus)

Elvitigala, Don Anushka Sandaruwan,Premachandra, H. K. A.,Yeo, Sang-Yeob,Choi, Cheol Young,Whang, Ilson,Lee, Jehee Springer-Verlag 2015 Genes & Genomics Vol.37 No.5

Marine teleost ortholog of catalase from rock bream (Oplegnathus fasciatus): Molecular perspectives from genomic organization to enzymatic behavior with respect to its potent antioxidant properties

Elvitigala, D.A.S.,Premachandra, H.K.A.,Whang, I.,Priyathilaka, T.T.,Kim, E.,Lim, B.S.,Jung, H.B.,Yeo, S.Y.,Park, H.C.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.35 No.4

Catalases are well known antioxidant enzymes that can mainly dismutate hydrogen peroxide into water and oxygen in order to prevent oxidative stress. The complete genomic DNA (gDNA) sequence of the catalase gene from rock bream (Oplegnathus fasciatus) was identified from our custom-constructed BAC genomic DNA library and designated as RbCat. RbCat consists of 13 exons, separated by 12 introns, within a 13,722-bp gDNA sequence. The complete cDNA sequence (3303 bp) of RbCat is comprised of a 1581-bp coding region, encoding a peptide of 527 amino acids (aa) in length, with a predicted molecular mass of 60 kDa and a theoretical isoelectric point of 8.34. The anticipated promoter region of RbCat contains several transcription factor-binding sites, including sites that bind with immune- and antioxidant-responsive signaling molecules, suggesting its substantial transcriptional regulation. RbCat resembles the typical catalase family signature, i.e., it is composed of the catalase proximal active site motif along with a catalase proximal heme-ligand signature motif and shares great homology with its fish counterparts. According to multiple sequence alignment, functionally important amino acids present in RbCat were thoroughly conserved among its vertebrate counterparts. Phylogenetic analysis revealed that RbCat evolved from a vertebrate origin, and further positioned it in the fish clade. Recombinant RbCat had noticeable peroxidase activity against its substrate, hydrogen peroxide, in a dose-dependent manner. However, it demonstrated substantial peroxidase activity within a broad range of temperatures and pH values. Constitutive RbCat mRNA expression of different magnitudes was detected in a tissue-specific manner, suggesting its diverse role in physiology with respect to the tissue type. Moreover, immune challenge experiments using Edwardsiella tarda and rock bream iridovirus (RBIV) as live pathogens and polyinosinic:polycytidylic acid and lipopolysaccharide as mitogens revealed that the transcription of RbCat can be modulated by immune stimulation. Collectively, the results obtained in this study suggest that RbCat can function as a potent antioxidant enzyme in rock bream and may play a role in post-immune responses with respect to its peroxidase activity.