title: Identification of interferon-γ-inducible-lysosomal thiol reductase (GILT) gene in goldfish (Carassius auratus) and its immune response to LPS challenge.
abstract: The interferon-γ-inducible lysosomal thiol reductase (GILT) has been demonstrated to play an important role in the processing and presentation of MHC class II restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, we cloned a GILT gene homolog from goldfish (designated gGILT), a kind of precious freshwater fish with high market value. The open reading frame of gGILT consists of 756 bases encoding a protein of 251 amino acids with an estimated molecular mass of 27.8 kDa and a theoretical isoelectric point of 5.24. The deduced protein possesses the typical structural features of known GILT proteins, including an active-site motif, a GILT signature sequence, and 10 conserved cysteines. RT-PCR results showed that gGILT and gIFN-γ (goldfish IFN-γ) mRNA were expressed in a tissue-specific manner and obviously up-regulated in splenocytes and the cells from head kidney after induction with LPS. Recombinant gGILT fused with His6 tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Further study revealed that gGILT was capable of catalyzing the reduction of the interchain disulfide bonds from intact IgG. This study shows that gGILT may be involved in the immune response to bacteria challenge and maintain first line of innate immune defense at basal level in goldfish. It also provides the basis for investigating on the role of GILT using goldfish as an animal model.
authors.affiliation: Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang Province 311121, China. Electronic address: lijianfeng609@yahoo.com.
authors.forename: Jian Feng
authors.initials: JF
authors.lastname: Li
authors.affiliation: The People's No 4 Hospital of Xiaoshan, Hangzhou, Zhejiang Province 311225, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang Province 311121, China.
authors.forename: Zhi Guo
authors.initials: ZG
authors.lastname: Wang
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu Province 210046, China.
authors.forename: Hong Zhen
authors.initials: HZ
authors.lastname: Liu
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu Province 210046, China.
authors.forename: You Long
authors.initials: YL
authors.lastname: Zhao
authors.affiliation: The People's No 4 Hospital of Xiaoshan, Hangzhou, Zhejiang Province 311225, China.
authors.forename: Jin Xi
authors.initials: JX
authors.lastname: Zhang
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu Province 210046, China. Electronic address: shuangquanz@yahoo.com.
authors.forename: Shuang Quan
authors.initials: SQ
authors.lastname: Zhang
authors.affiliation: Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang Province 311121, China. Electronic address: jun-ping.liu@monash.edu.
authors.forename: Jun Ping
authors.initials: JP
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism; D006054:Goldfish / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D029941:Fish Proteins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D050862:Oxidoreductases Acting on Sulfur Group Donors
keywords: 3D structure; Goldfish (Carassius auratus); Interferon-γ-inducible-lysosomal thiol reductase; The subcellular localization; Thiol reductase activity