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Umasuthan, N.,Saranya Revathy, K.,Lee, Y.,Whang, I.,Lee, J. Academic Press 2012 Fish & shellfish immunology Vol.32 No.4
Thioredoxin (TRx) is a ubiquitous protein involved in the regulation of multiple biological processes. The TRx-2 isoform is exclusively expressed in mitochondria, where it contributes to mitochondrial redox state maintenance. In the present study, a novel thioredoxin-2 gene was identified in the Manila clam, Ruditapes philippinarum. The full-length sequence of RpTRx-2 (1561bp) consists of a 498bp coding region encoding a 166 amino acid protein. The N-terminal region of RpTRx-2 harbors a mitochondrial localization signal (56 amino acids), while the C-terminal portion contains the characteristic <SUP>89</SUP>WCGPC<SUP>93</SUP> catalytic active site. Phylogenetic analysis revealed that RpTRx-2 is closest to its ortholog from abalone. The broad distribution pattern of RpTRx-2 mRNA in healthy animal tissues implicates a generally significant function in normal clam physiology. The transcription level of RpTRx-2, however, is highest in hemocytes. Lipopolysaccharide and Vibrio tapetis bacterium caused up-regulation of the RpTrx-2 transcript levels in gill and hemocytes. Interestingly, clam manganese superoxide dismutase (MnSOD) mRNA levels in hemocytes elicited a corresponding response to these immune challenges. RpTRx-2 was recombinantly expressed in Escherichia coli BL21 (DE3) and used in insulin disulfide reduction assay as well as metal-catalyzed oxidation assay to elucidate its antioxidant property by reducing substrate and protecting super-coiled DNA from oxidative damage through free radical scavenging, respectively. Collectively, our data indicated that RpTRx-2, a mitochondrial TRx-2 family member, is an antioxidant enzyme that may be involved in antibacterial defense of clams.
Umasuthan, N.,Saranya Revathy, K.,Bathige, S.D.N.K.,Lim, B.S.,Park, M.A.,Whang, I.,Lee, J. Academic Press 2013 Fish & shellfish immunology Vol.34 No.1
In this study, we describe the identification and characterization of manganese superoxide dismutase, an important antioxidant enzyme acting as the chief reactive oxygen species (ROS) scavenger, from rock bream Oplegnathus fasciatus (Of-mMnSOD) at genomic- and transcriptional-levels as well as the biological activity of recombinant protein. The Of-mMnSOD protein portrayed distinct MnSOD family features including signature motifs, metal association sites and the typical active site topology. It was also predicted to be localized in mitochondrial matrix. The Of-mMnSOD had a quinquepartite genome organization encompassing five exons interrupted by four introns. Comparison of its sequence and gene structure with that of other lineages emphasized its strong conservation among different vertebrates. The Of-mMnSOD was ubiquitously transcribed in different rock bream tissues with higher levels in blood cells and metabolically active tissues. Transcription of Of-mMnSOD was kinetically modulated in response to investigational challenges using mitogens (lipopolysaccharide and poly I:C) and live-pathogens (Edwardsiella tarda and rock bream irido virus) in blood cells and liver tissue. The purified recombinant Of-mMnSOD possessed potential antioxidant capacity and actively survived over a range of pH (7.5-11) and temperature (15-40 <SUP>o</SUP>C) conditions. Collectively, findings of this study suggest that Of-mMnSOD combats against oxidative stress and cellular damages induced by mitogen/pathogen-mediated inflammation, by detoxifying harmful ROS (O<SUB>2</SUB><SUP>@?-</SUP>) in rock bream.
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>