A homolog of teleostean signal transducer and activator of transcription 3 (STAT3) from rock bream, <i>Oplegnathus fasciatus</i>: Structural insights, transcriptional modulation, and subcellular localization

Bathige, S.D.N.K.,Thulasitha, William Shanthakumar,Umasuthan, Navaneethaiyer,Jayasinghe, J.D.H.E.,Wan, Qiang,Nam, Bo-Hye,Lee, Jehee Elsevier 2017 Veterinary immunology and immunopathology Vol.186 No.-

<P><B>Abstract</B></P> <P>Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors in the Janus kinase (JAK)/STAT signaling pathway, and it was previously considered as acute phase response factor. A number of interleukins (ILs) such as IL-5, IL-6, IL-9, IL-10, IL-12, and IL-22 are known to be involved in activation of STAT3. In addition, various growth factors and pathogenic or oxidative stresses mediate the activation of a wide range of functions via STAT3. In this study, a STAT3 homolog was identified and functionally characterized from rock bream (RbSTAT3), <I>Oplegnathus fasciatus</I>. <I>In silico</I> characterization revealed that the RbSTAT3 amino acid sequence shares highly conserved common domain architectural features including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, and Src homology 2 (SH2) domains. In addition, a fairly conserved transcriptional activation domain (TAD) was located at the C-terminus. Comparison of RbSTAT3 with other counterparts revealed higher identities (>90%) with fish orthologs. The genomic sequence of <I>RbSTAT3</I> was obtained from a bacterial artificial chromosome (BAC) library, and was identified as a multi-exonic gene (24 exons), as found in other vertebrates. Genomic structural comparison and phylogenetic studies have showed that the evolutionary routes of teleostean and non-teleostean vertebrates were distinct. Quantitative real time PCR (qPCR) analysis revealed that the spatial distribution of <I>RbSTAT3</I> mRNA expression was ubiquitous and highly detectable in blood, heart, and liver tissues. Transcriptional modulation of <I>RbSTAT3</I> was examined in blood and liver tissues after challenges with bacteria (<I>Edwardsiella tarda</I> and <I>Streptococcus iniae</I>), rock bream irido virus (RBIV), and immune stimulants (LPS and poly (I:C)). Significant changes in <I>RbSTAT3</I> transcription were also observed in response to tissue injury. In addition, the transcriptional up-regulation of <I>RbSTAT3</I> was detected in rock bream heart cells upon recombinant rock bream IL-10 (rRbIL-10) treatment. Subcellular localization and nuclear translocation of rock bream STAT3 following poly (I:C) treatment were also demonstrated. Taken together, the results of the current study provide important evidence for potential roles of rock bream STAT3 in the immune system and wound healing processes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Identification of STAT3 (RbSTAT3) ortholog from rock bream. </LI> <LI> Genomic structure and putative promoter analysis of <I>RbSTAT3</I>. </LI> <LI> Expression profile (<I>In vivo</I>) of <I>RbSTAT3</I> upon pathogenic and PAMP stress, and tissue injury. </LI> <LI> Expression profile (<I>In vitro</I>) of <I>RbSTAT3</I> in response to rock bream IL-10. </LI> <LI> Subcellular localization of RbSTAT3. </LI> </UL> </P>

An invertebrate signal transducer and activator of transcription 5 (STAT5) ortholog from the disk abalone, Haliotis discus discus: Genomic structure, early developmental expression, and immune responses to bacterial and viral stresses

Bathige, S.D.N.K.,Umasuthan, N.,Park, H.C.,Lee, J. Pergamon Press ; Elsevier Science 2016 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.56 No.-

<P>Signal transducer and activator of transcription (STAT) family members are key signaling molecules that transduce cellular responses from the cell membrane to the nucleus upon Janus kinase (JAK) activation. Although seven STAT members have been reported in mammals, very limited information on STAT genes in molluscans is available. In this study, we identified and characterized a STAT paralog that is homologous to STAT5 from the disk abalone, Haliotis discus discus, and designated as AbSTAT5. Comparison of the deduced amino acid sequence for AbSTAT5 (790 amino acids) with other counterparts revealed conserved residues important for functions and typical domain regions, including the N-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, and Src homology 2 (SH2) domains as mammalian counterparts. Analysis of STAT phylogeny revealed that AbSTAT5 was clustered with the molluscan subgroup in STAT5 Glade with distinct evolution. According to the genomic structure of AbSTAT5, the coding sequence was distributed into 20 exons with 19 introns. Immunologically essential transcription factor-binding sites, such as GATA-1, HNF, SP1, C/EBP, Oct-1, AP1, c-Jun, and Sox-2, were predicted at the 5'-proximal region of AbSTAT5. Expression of AbSTAT5 mRNA was detected in different stages of embryonic development and observed at considerably higher levels in the morula and late veliger stages. Tissue-specific expressional studies revealed that the highest level of AbSTAT5 transcripts was detected in hemocytes, followed by gill tissues. Temporal expressions of AbSTAT5 were analyzed upon live bacterial (Vibrio parahemolyticus and Listeria monocytogenes), viral (viral hemorrhagic septicemia virus), and pathogen-associated molecular pattern (lipopolysaccharides and Poly I:C) stimulations, and significant elevations indicated immune modulation. These results suggest that AbSTAT5 may be involved in maintaining innate immune responses from developmental to adult stages in the disk abalone. Further, this study provides a basis for structural and functional exploration of STAT members in the invertebrate JAK/STAT signaling pathway. (C) 2015 Elsevier Ltd. All rights reserved.</P>

Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens

Bathige, S.D.N.K.,Umasuthan, N.,Jayasinghe, J.D.H.E.,Godahewa, G.I.,Park, H.C.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.56 No.-

<P>The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qD0 consisted of 807, 1305, and 660 bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different response profiles. The results of this study suggest that AbC1qDCs are involved in immune responses against invading bacterial pathogens. (C) 2016 Published by Elsevier Ltd.</P>

Antibacterial activity and immune responses of a molluscan macrophage expressed gene-1 from disk abalone, Haliotis discus discus

Bathige, S.D.N.K.,Umasuthan, N.,Whang, I.,Lim, B.S.,Won, S.H.,Lee, J. Academic Press 2014 FISH AND SHELLFISH IMMUNOLOGY Vol.39 No.2

The membrane-attack complex/perforin (MACPF) domain-containing proteins play an important role in the innate immune response against invading microbial pathogens. In the current study, a member of the MACPF domain-containing proteins, macrophage expressed gene-1 (MPEG1) encoding 730 amino acids with the theoretical molecular mass of 79.6 kDa and an isoelectric point (pI) of 6.49 was characterized from disk abalone Haliotis discus discus (AbMPEG1). We found that the characteristic MACPF domain (Val<SUP>131</SUP>-Tyr<SUP>348</SUP>) and transmembrane segment (Ala<SUP>669</SUP>-Ile<SUP>691</SUP>) of AbMPEG1 are located in the N- and C-terminal ends of the protein, respectively. Ortholog comparison revealed that AbMPEG1 has the highest sequence identity with its pink abalone counterpart, while sequences identities of greater than 90% were observed with MPEG1 members from other abalone species. Likewise, the furin cleavage site KRRRK was highly conserved in all abalone species, but not in other species investigated. We identified an intron-less genomic sequence within disk abalone AbMPEG1, which was similar to other mammalian, avian, and reptilian counterparts. Transcription factor binding sites, which are important for immune responses, were identified in the 5'-flanking region of AbMPEG1. qPCR revealed AbMPEG1 transcripts are present in every tissues examined, with the highest expression level occurring in mantle tissue. Significant up-regulation of AbMPEG1 transcript levels was observed in hemocytes and gill tissues following challenges with pathogens (Vibrio parahemolyticus, Listeria monocytogenes and viral hemorrhagic septicemia virus) as well as pathogen-associated molecular patterns (PAMPs: lipopolysaccharides and poly I:C immunostimulant). Finally, the antibacterial activity of the MACPF domain was characterized against Gram-negative and -positive bacteria using a recombinant peptide. Taken together, these results indicate that the biological significance of the AbMPEG1 gene includes a role in protecting disk abalone through the ability of AbMPEG1 to initiate an innate immune response upon pathogen invasion.

Genomic characterization, expression analysis, and antimicrobial function of a glyrichin homologue from rock bream, Oplegnathus fasciatus

Kasthuri, S.R.,Wan, Q.,Umasuthan, N.,Bathige, S.D.N.K.,Lim, B.S.,Jung, H.B.,Lee, J.,Whang, I. Academic Press 2013 Fish & shellfish immunology Vol.35 No.5

Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at <SUP>22</SUP>GFMMGFAVGMAAGAMFGTFSCLR<SUP>44</SUP>. Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.

A TELEOST SPECIFIC GLUTATHIONE S-TRANSFERASE FAMILY PROTEIN FROM BLACK ROCKFISH

J.D.H.E. Jayasinghe,S.D.N.K. Bathige,Jehee Lee 한국수산과학회 양식분과 2015 한국수산과학회 양식분과 학술대회 Vol.2015 No.5

A C1 inhibitor ortholog from rock bream (Oplegnathus fasciatus): Molecular perspectives of a central regulator in terms of its genomic arrangement, transcriptional profiles and anti-protease activities of recombinant peptide

Umasuthan, N.,Bathige, S.D.N.K.,Revathy, K.S.,Wickramaarachchi, W.D.N.,Wan, Q.,Whang, I.,Kim, E.,Park, M.A.,Park, H.C.,Lee, J. Pergamon Press ; Elsevier Science 2014 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.42 No.2

C1 inhibitor (C1Inh), a member of serpin superfamily, is a crucial regulator of the activation of various plasmatic cascades associated with immunity and inflammation. This study describes the identification and characterization of a C1Inh gene from rock bream Oplegnathus fasciatus (OfC1Inh) at structural, expressional and functional levels. The cDNA-(2245bp) and corresponding gDNA-sequences (5.2kbp) of OfC1Inh were isolated from rock bream transcriptome- and BAC-libraries, respectively. Predicted amino acid sequence of OfC1Inh revealed a two-domain architecture composed of an N-terminal region with two Ig-like domains and a C-terminal region with a serpin domain. Tertiary model of OfC1Inh disclosed its active site topology. In the multi-exonic genomic arrangement of OfC1Inh, it consisted of eleven exons disjoined by ten introns as observed in few other fish homologs. Our comparative analysis indicated that the teleostean C1Inhs were distinct from their non-teleostean vertebrate counterparts in terms of their (1) extended N-terminal domains, (2) evolutionary divergence and (3) exon-intron distribution. The OfC1Inh had a TATA-deficient promoter with a putative initiator element, and two tandemly arranged downstream promoter elements. Several components associated with the immune and inflammatory transcriptional activation were also predicted to exist in 5' flanking region of OfC1Inh. The exclusive mRNA levels in liver and moderate levels in extra-hepatic tissues intimated the diversified importance of OfC1Inh in rock bream physiology. We also provide an evidence for the involvement of OfC1Inh in immune balance, based on its modulated transcription upon different PAMP (lipopolysaccharide and poly I:C)- or pathogen (Streptococcus iniae and rock bream irido virus)-challenges. A recombinantly expressed fusion protein [(r)OfC1Inh] was employed in demonstrating the anti-protease function of OfC1Inh. The (r)OfC1Inh exhibited detectable inhibitory activity against C1 esterase and thrombin, where the anti-C1 esterase role was shown to be potentiated by heparin. Taken together, the results of this study provide the first line of evidence for the possible involvement of a teleostean C1Inh in fish immunity, based on its expressional response(s) and inhibitory properties against two enzymes involved in biological cascades.

Two duplicated chicken-type lysozyme genes in disc abalone Haliotis discus discus: Molecular aspects in relevance to structure, genomic organization, mRNA expression and bacteriolytic function

Umasuthan, N.,Bathige, S.D.N.K.,Kasthuri, S.R.,Wan, Q.,Whang, I.,Lee, J. Academic Press 2013 FISH AND SHELLFISH IMMUNOLOGY Vol.35 No.2

Lysozymes are crucial antibacterial proteins that are associated with catalytic cleavage of peptidoglycan and subsequent bacteriolysis. The present study describes the identification of two lysozyme genes from disc abalone Haliotis discus discus and their characterization at sequence-, genomic-, transcriptional- and functional-levels. Two cDNAs and BAC clones bearing lysozyme genes were isolated from abalone transcriptome and BAC genomic libraries, respectively and sequences were determined. Corresponding deduced amino acid sequences harbored a chicken-type lysozyme (LysC) family profile and exhibited conserved characteristics of LysC family members including active residues (Glu and Asp) and GS(S/T)DYGIFQINS motif suggested that they are LysC counterparts in disc abalone and designated as abLysC1 and abLysC2. While abLysC1 represented the homolog recently reported in Ezo abalone [1], abLysC2 shared significant identity with LysC homologs. Unlike other vertebrate LysCs, coding sequence of abLysCs were distributed within five exons interrupted by four introns. Both abLysCs revealed a broader mRNA distribution with highest levels in mantle (abLysC1) and hepatopancreas (abLysC2) suggesting their likely main role in defense and digestion, respectively. Investigation of temporal transcriptional profiles post-LPS and -pathogen challenges revealed induced-responses of abLysCs in gills and hemocytes. The in vitro muramidase activity of purified recombinant (r) abLysCs proteins was evaluated, and findings indicated that they are active in acidic pH range (3.5-6.5) and over a broad temperature range (20-60 <SUP>o</SUP>C) and influenced by ionic strength. When the antibacterial spectra of (r)abLysCs were examined, they displayed differential activities against both Gram positive and Gram negative strains providing evidence for their involvement in bacteriolytic function in abalone physiology.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus)

Umasuthan, N.,Bathige, S.D.N.K.,Noh, J.K.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.47 No.1

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38β (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (~15.6 kb) and OfMAPK14 (~13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.

Transcriptional profiling, molecular cloning, and functional analysis of C1 inhibitor, the main regulator of the complement system in black rockfish, <i>Sebastes schlegelii</i>

Nilojan, Jehanathan,Bathige, S.D.N.K.,Thulasitha, W.S.,Kwon, Hyukjae,Jung, Sumi,Kim, Myoung-Jin,Nam, Bo-Hye,Lee, Jehee Elsevier 2018 FISH AND SHELLFISH IMMUNOLOGY Vol.75 No.-

<P><B>Abstract</B></P> <P>C1-inhibitor (C1inh) plays a crucial role in assuring homeostasis and is the central regulator of the complement activation involved in immunity and inflammation. A C1-inhibitor gene from <I>Sebastes schlegelii</I> was identified and designated as <I>SsC1inh</I>. The identified genomic DNA and cDNA sequences were 6837 bp and 2161 bp, respectively. The genomic DNA possessed 11 exons, interrupted by 10 introns. The amino acid sequence possessed two immunoglobulin-like domains and a serpin domain. Multiple sequence alignment revealed that the serpin domain of SsC1inh was highly conserved among analyzed species where the two immunoglobulin-like domains showed divergence. The distinctiveness of teleost C1inh from other homologs was indicated by the phylogenetic analysis, genomic DNA organization, and their extended N-terminal amino acid sequences. Under normal physiological conditions, <I>SsC1inh</I> mRNA was most expressed in the liver, followed by the gills. The involvement of SsC1inh in homeostasis was demonstrated by modulated transcription profiles in the liver and spleen upon pathogenic stress by different immune stimulants. The protease inhibitory potential of recombinant SsC1inh (rSsC1inh) and the potentiation effect of heparin on rSsC1inh was demonstrated against C1esterase and thrombin. For the first time, the anti-protease activity of the teleost C1inh against its natural substrates C1r and C1s was proved in this study. The protease assay conducted with recombinant black rockfish C1r and C1s proteins in the presence or absence of rSsC1inh showed that the activities of both proteases were significantly diminished by rSsC1inh. Taken together, results from the present study indicate that SsC1inh actively plays a significant role in maintaining homeostasis in the immune system of black rock fish.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A C1 inhibitor gene was identified from Black rockfish with serpin features. </LI> <LI> Genomic DNA was made up of 11 exons and 10 introns. </LI> <LI> Modulated transcriptional patterns were observed after immune stimulation. </LI> <LI> Antiprotease activity of SsC1inh is enhanced by the addition of heparin. </LI> <LI> SsC1inh significantly diminished the activity of SsC1r and SsC1s. </LI> </UL> </P>