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Nilojan, Jehanathan,Bathige, S.D.N.K.,Kugapreethan, Roopasingam,Yang, Hyerim,Kim, Myoung-Jin,Nam, Bo-Hye,Lee, Jehee Elsevier 2018 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.81 No.-
<P><B>Abstract</B></P> <P>C1r and C1s are serine proteases responsible for activating the classical complement pathway to initiate the complement cascade, which plays a crucial role in eliminating invading pathogenic microbes. In this study, cDNA sequences of C1r and C1s were identified from black rockfish and designated as <I>SsC1r</I> and <I>SsC1s</I>, respectively. In both sequences, two CUB domains, an EGF-like domain, two CCP domains, and a trypsin-like serine protease domain were identified. Multiple sequence alignments with known vertebrate homologs demonstrated that both sequences were highly conserved and, especially, the catalytic and substrate binding residues were completely conserved. In the constructed phylogenetic tree, C1r and C1s formed two separate clusters, which further branched into groups of related organisms. <I>SsC1r</I> and <I>SsC1s</I> joined with their respective teleostean clusters. Transcriptional analysis showed that the highest mRNA expression level was in the liver under normal physiological conditions. Significantly upregulated expression of both mRNAs in spleen and liver after pathologic stress, by intraperitoneal injection with different stimuli, suggested their vital role in immunity. The serine protease domains of SsC1r and SsC1s were cloned and the recombinant proteins were expressed and purified. A protease assay, conducted to confirm their functionality, indicated that both recombinant proteins had proteolytic activity. Taken together, these results indicate that SsC1r and SsC1s have significant properties to aid in the immunity of black rockfish by activating the complement system by proteolytic cleavage.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Homologs of C1r and C1s were identified from <I>Sebastes Schlegelii</I> (<I>SsC1r</I> and <I>SsC1s</I>). </LI> <LI> SsC1r and SsC1s shared similar domain architecture. </LI> <LI> Both genes were highly expressed in liver under normal physiological conditions. </LI> <LI> Highly induced transcriptional profiles observed for SsC1r and SsC1s after challenges with PAMPs and live pathogen. </LI> <LI> Both recombinant SsC1r and SsC1s possessed proteolytic activity. </LI> </UL> </P>
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>
Kwon, Hyukjae,Yang, Hyerim,Lee, Seongdo,Nilojan, Jehanathan,Bathige, S.D.N.K.,Nam, Bo-Hye,Wan, Qiang,Lee, Jehee Elsevier 2018 FISH AND SHELLFISH IMMUNOLOGY Vol.74 No.-
<P><B>Abstract</B></P> <P>Kazal-type serine protease inhibitors (KSPIs) play important roles in the regulation of endogenous proteases, cell development, blood coagulation, and immune response. In this study, we identified and characterized a KSPI homologue (<I>SsKSPI</I>) in black rockfish, <I>Sebastes schlegelii</I>. The full-length cDNA sequence of <I>SsKSPI</I> was 532 base pairs (bp), including an open reading frame (ORF) of 330 bp, which encodes a polypeptide of 110 amino acids with a signal peptide of 21 amino acids. The greatest value for identity (42.9%) and similarity (50.9%) was observed with <I>Channa striata</I> KSPI. We purified the recombinant protein of <I>SsKSPI</I> and performed protease inhibitory assays using three common serine proteases. The recombinant SsKSPI exhibited specific inhibitory activity against subtilisin A in a dose-dependent manner. Tissue distribution of <I>SsKSPI</I> mRNA has been examined amongst 10 important tissues in healthy rockfish and the liver was found to be the predominant expression organ of <I>SsKSPI.</I> The modulation of <I>SsKSPI</I> expression under immune challenges was also investigated in the liver. The <I>SsKSPI</I> mRNA expression was significantly up-regulated in response to both bacterial (<I>Streptococcus iniae</I> and lipopolysaccharide) and viral (polyinosinic:polycytidylic acid) challenges. Overall, we propose that <I>SsKSPI</I> is potentially involved in the hepatic immune response against bacterial and viral infections in black rockfish.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A KSPI homologue (<I>SsKSPI</I>) was identified in <I>Sebastes schlegelii.</I> </LI> <LI> The recombinant SsKSPI specifically inhibited subtilisin A activity. </LI> <LI> The highest constitutive expression of <I>SsKSPI</I> was detected in the liver. </LI> <LI> <I>SsKSPI</I> gene expression was significantly inducible upon bacterial and viral challenges. </LI> <LI> <I>SsKSPI</I> is potentially involved in the hepatic immune response in black rockfish. </LI> </UL> </P>