title: Molecular structure and functional characterization of the gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in largemouth bass (Microptenus salmoides).
abstract: The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays a role in facilitating the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens and is also involved in MHC I-restricted antigens in adaptive immunity catalyzing disulfide bond reduction in mammals. In this study, we cloned a GILT gene homolog from largemouth bass (designated 'lbGILT'), a freshwater fish belonging to Perciformes and known for its nutritive value. We obtained the full-length cDNA of lbGILT by reverse transcription PCR and rapid amplification of cDNA ends. This cDNA is comprised of a 5'-untranslated region (UTR) of 87 bp, a 3'-UTR of 189 bp, and an open reading frame of 771 bp. It encodes a protein of 256 amino acids with a deduced molecular weight of 28.548 kDa and a predicted isoelectric point of 5.62. The deduced protein possesses the typical structural features of known GILTs, including an active site motif, two potential N-linked glycosylation sites, a GILT signature sequence, and six conserved cysteines. Tissue-specific expression of lbGILT was shown by real-time quantitative PCR. The expression of lbGILT mRNA was obviously up regulated in spleen and kidney after induction with lipopolysaccharide. Recombinant lbGILT was produced as an inclusion body with a His6 tag in ArcticExpress (DE3), and the protein was then washed, solubilized, and refolded. The refolded lbGILT showed reduction activity against an IgG substrate. These results suggest that lbGILT plays a role in innate immunity.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China; Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China.
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Hu
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Lu
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China; Jiangsu Province Key Laboratory for Aquatic Crustacean Diseases, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Sang
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China; Jiangsu Province Key Laboratory for Aquatic Crustacean Diseases, Life Sciences College, Nanjing Normal University, Nanjing 210046, China. Electronic address: zhangshuangquan@njnu.edu.cn.
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001492:Bass / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000662:veterinary
chemical_list: D018076:DNA, Complementary; D029941:Fish Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D050862:Oxidoreductases Acting on Sulfur Group Donors
keywords: Gamma-interferon-inducible lysosomal thiol reductase; Inclusion body; Largemouth bass; Lipopolysaccharide; Real-time quantitative PCR; cDNA cloning