title: Molecular structure, phylogenetic analysis, tissue distribution, and function characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene in sheep (Ovis aries).
abstract: Interferon-γ-inducible-lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, a sheep cDNA, sGILT, encoding GILT protein was isolated from the spleen cDNA library of Ovis aries. It codes for a deduced protein of 244 amino acids with a putative molecular weight of 27.6 kDa, which has all the typical structural features of GILT protein including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 5 conserved cysteines. Sequence comparison indicated the amino acid sequence of sGILT showed high identity to cow GILT (93.03%). Phylogenetic analysis showed that sGILT and cow GILT shared the greatest homology. The result of real-time PCR suggested that sGILT mRNA was expressed in a tissue-specific manner and obviously up-regulated in splenocytes and PBMCs after induction with lipopolysaccharide (LPS). Recombinant sGILT fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Its expression was confirmed by SDS-PAGE and Western blotting analysis. Thiol reductase activity was assessed using antibody as the substrate. These results suggest that sGILT has the activity of disulfide bond reduction and indicate that sheep also express a protein that has been found to maintain first line of innate immune defense at basal level.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Hong-xin
authors.initials: HX
authors.lastname: Ai
authors.forename: Zhen-zhen
authors.initials: ZZ
authors.lastname: Zhang
authors.forename: Yue-fen
authors.initials: YF
authors.lastname: Shen
authors.forename: Jia-xin
authors.initials: JX
authors.lastname: Zhang
authors.forename: Xu-ming
authors.initials: XM
authors.lastname: Zhou
authors.forename: Cui
authors.initials: C
authors.lastname: Min
authors.forename: Shan-yuan
authors.initials: SY
authors.lastname: Zhu
authors.forename: Shuang-quan
authors.initials: SQ
authors.lastname: Zhang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D005260:Female; D007113:Immunity, Innate / Q000235:genetics; D007371:Interferon-gamma / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016366:Open Reading Frames; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012756:Sheep / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007371:Interferon-gamma; D050862:Oxidoreductases Acting on Sulfur Group Donors