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Umasuthan, Navaneethaiyer,Whang, Ilson,Saranya Revathy, Kasthuri,Oh, Myung-Joo,Jung, Sung-Ju,Choi, Cheol Young,Lee, Jeong-Ho,Noh, Jae Koo,Lee, Jehee Elsevier 2012 FISH AND SHELLFISH IMMUNOLOGY Vol.32 No.5
<P><B>Abstract</B></P><P>Angiotensinogen (AGT) is the precursor of the renin-angiotensin system and contributes to osmoregulation, acute-phase and immune responses. A full-length cDNA of the <I>AGT</I> (2004 bp with a 1389 bp coding region) was isolated from rock bream (Rb), <I>Oplegnathus fasciatus</I>. The encoded polypeptide of 463 amino acids had a predicted molecular mass of 51.6 kDa. <I>RbAGT</I> possessed a deduced signal peptide of 22 residues upstream of a putative angiotensin I sequence (<SUP>23</SUP>NRVYVHPFHL<SUP>32</SUP>). <I>RbAGT</I> possessed a specific domain profile and a signature motif which are characteristics of the serpin family. Sequence homology and phylogenetic analysis indicated that <I>RbAGT</I> was evolutionarily closest to AGT of <I>Rhabdosargus sarba</I>. The mRNA expression profile of <I>RbAGT</I> was determined by quantitative RT-PCR and it demonstrated a constitutive and tissue-specific expression with the highest transcript level in the liver. Significantly up-regulated <I>RbAGT</I> expression was elicited by systemic injection of a lipopolysaccharide, rock bream iridovirus (RBIV) and bacteria (<I>Edwardsiella tarda</I> and <I>Streptococcus iniae</I>), revealing its pathogen inducibility. <I>RbAGT</I> manifested a down-regulated response to systemic injury, contemporaneously with two other serpins, protease nexin-1 (<I>PN-</I>1), and heparin cofactor II (<I>HCII</I>). In addition, a synchronized expression pattern was elicited by <I>RbAGT</I> and <I>RbTNF-α</I> in response to injury, suggesting that TNF-α might be a potential modulator of <I>AGT</I> transcription.</P> <P><B>Graphical abstract</B></P><P><ce:figure id='dfig1'></ce:figure></P><P><B>Highlights</B></P><P>► Molecular characterization of angiotensinogen from rock bream (<I>RbAGT</I>). ► Tissue-specific transcriptional profile of <I>RbAGT.</I> ► Response of hepatic <I>RbAGT</I> against LPS, bacteria and iridovirus. ► Temporal expression of hematic <I>RbAGT</I> upon injury. ► Expressional relationship between <I>RbAGT</I>, <I>HCII</I>, <I>PN-1</I> and <I>TNF-α</I>.</P>
Umasuthan, Navaneethaiyer,Bathige, S.D.N.K.,Whang, Ilson,Lim, Bong-Soo,Choi, Cheol Young,Lee, Jehee Elsevier 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.43 No.2
<P><B>Abstract</B></P> <P>As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This study investigates the molecular and expressional profiles of an <I>IRAK4-like</I> homolog from <I>Oplegnathus fasciatus</I> (<I>OfIRAK4</I>). The <I>OfIRAK4</I> gene (8.2?kb) was structured with eleven exons and ten introns. A putative coding sequence (1395bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4 protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain (PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initiation site revealed the presence of putative regulatory elements, including NF-κB-binding sites, which are possibly involved in transcriptional control of <I>OfIRAK4</I>. Quantitative real-time PCR (qPCR) was employed to assess the transcriptional expression of <I>OfIRAK4</I> in different juvenile tissues and post-injection of different immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected with highest level in liver. <I>In?vivo</I> flagellin (FLA) challenge significantly intensified its mRNA levels in intestine, liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expression was also determined in liver and head kidney of animals challenged with potent immunogens (LPS and poly I:C) and pathogens (<I>Edwardsiella tarda</I> and <I>Streptococcus iniae</I> and rock bream iridovirus (RBIV)). Taken together, these data implicate that <I>OfIRAK4</I> might be engaged in antibacterial and antiviral immunity in rock bream.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Rock bream <I>IRAK4</I> (<I>OfIRAK4</I>) had 11 exons encoding its protein with a bipartite domain structure. </LI> <LI> Teleost IRAK4s are distinct from tetrapod IRAK4s in terms of gene structure and phylogeny. </LI> <LI> Liver was the primary site of <I>OfIRAK4</I>'s transcription. </LI> <LI> <I>OfIRAK4</I>'s putative role in flagellin sensing was evidenced from its transcriptional response. </LI> <LI> Immunogenic PAMPs/pathogens induced <I>OfIRAK4</I>'s expression implied its association with immunity. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
Umasuthan, Navaneethaiyer,Bathige, S.D.N.K.,Thulasitha, William Shanthakumar,Jayasooriya, R.G.P.T.,Shin, Younhee,Lee, Jehee Elsevier 2017 Fish & shellfish immunology Vol.62 No.-
<P><B>Abstract</B></P> <P>Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, <I>Oplegnathus fasciatus.</I> Both <I>Oftlr5m</I> (6.7 kb) and <I>Ofmyd88</I> (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the <I>tlr5m</I> gene has evolved with extensive rearrangement; whereas, the <I>myd88</I> gene has maintained a stable structure throughout the evolution. Inspection of 5′ flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected <I>Oftlr5m</I> mRNA in eleven tissues with the highest abundance in liver. <I>In vivo</I> flagellin administration strongly induced the transcripts of both <I>Oftlr5m</I> and <I>Ofmyd88</I> in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with <I>Oftlr5m</I> and <I>Ofmyd88</I> demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> genes in rock bream display quinquepartite structure. </LI> <LI> While gene structure of <I>tlr5m</I> is evolved with rearrangements, <I>myd88</I> is preserved. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> showed similar tissue mRNA profile with highest level in liver. </LI> <LI> <I>Oftlr5m</I> and <I>Ofmyd88</I> were induced by ultrapure flagellin in gill and head kidney. </LI> <LI> They individually and synergetically activated NF-κB upon flagellin-stimulation. </LI> </UL> </P>