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Sarithaa Sellaththurai,K.A.S.N. Shanaka,D.S. Liyanage,Hyerim Yang,Thanthrige Thiunuwan Priyathilaka,Jehee Lee 제주대학교 해양과학연구소 2020 해양과환경연구소 연구논문집 Vol.44 No.-
Malectin is a carbohydrate-binding lectin protein found in the endoplasmic reticulum (ER). It selectivity binds to Glc2-N-glycan and is involved in a glycoprotein quality control mechanism. Even though malectin may play a role in immunity, its role in innate immunity is not fully known. In the present study, we identified and characterized the malectin gene from Hippocampus abdominalis (HaMLEC). We analyzed sequence features, spatial expression levels, temporal expression profiles upon immune responses, bacterial and carbohydrate binding abilities and anti-viral properties to investigate the potential role of HaMLEC in innate immunity. The molecular weight and isoelectric point (pI) were estimated to be 31.99 kDa and 5.17, respectively. The N-terminal signal peptide, malectin superfamily domain and C-terminal transmembrane region were identified from the amino acid sequence of HaMLEC. The close evolutionary relationship of HaMLEC with other teleosts was identified by phylogenetic analysis. According to quantitative PCR (qPCR) results, HaMLEC expression was observed in all the examined tissues and high expression was observed in the ovary and brain, compared to other tested tissues. Temporal expression of HaMLEC in liver and blood tissues were significant modulated upon exposure to immunogens Edwardasiella tarda, Streptococcus iniae, polyinosinic:polycytidylic and lipopolysaccharide. The presence of carbohydrate binding modules (CBMs) of bacterial glycosyl hydrolases were functionally confirmed by a bacterial binding assay. Anti-viral activity significantly reduced viral hemorrhagic septicemia virus (VHSV) replication in cells overexpressing HaMLEC. The observed results suggested that HaMLEC may have a significant role in innate immunity in Hippocampus abdominalis.
K.P. Madushani,K.A.S.N. Shanaka,Rajamanthrilage Kasun Madusanka,Jehee Lee 제주대학교 해양과학연구소 2021 해양과환경연구소 연구논문집 Vol.45 No.-
Remedies toward sustainable aquaculture rely upon research that unveils the molecular mechanisms behind host immunity and their interactions with pathogens. Antiviral defense is a major innate immune response in fish. The antiviral protein GCHV-induced gene-2 (Gig2), a member of the interferon-stimulated gene (ISG), was identified and characterized from rockfish (Sebastes schlegelii). Gig2 exists in two isoforms, namely, SsGig2-I1 and SsGig2-I2, in rockfish with lengths of 163 and 223 bp, respectively. Bioinformatic analysis indicated the availability of poly (ADP-ribose) polymerase domain in both proteins, and 51.3% identity and 71.3% similarity between both isoforms were observed. The basal expression pattern revealed the highest tissue-specific expression in rockfish gills for both isoforms. The immune challenge experiment disclosed a distinctive and strong expression of each transcript in the presence of poly I:C. Both isoforms are localized in the endoplasmic reticulum. Interferon (IFN) pathway gene analysis revealed no significant upregulation of IFN related genes. Viral hemorrhagic septicemia virus (VHSV) gene expression analysis revealed strong downregulation of viral transcripts after 48 h of infection in the presence of Gig2 isoforms. Collectively, these results indicate the protective role of Gig2 in rockfish against VHSV infection and help broaden our understanding of the innate immunity of fish.
K. A. S. N. Shanaka,Sumi Jung,N. D. Janson,J. R. P. Jayasingha,K. P. Madushani,Myoung-Jin Kim,Jehee Lee 제주대학교 해양과학연구소 2021 해양과환경연구소 연구논문집 Vol.45 No.-
Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.