title: Cloning and expression analysis of interferon-γ-inducible-lysosomal thiol reductase gene in South African clawed frog (Xenopus laevis).
abstract: In this study, an interferon-γ-inducible-lysosomal thiol reductase (GILT) homologue has been cloned and identified from South African clawed frog Xenopus laevis (designated XlGILT). The open reading frame (ORF) of XlGILT consists of 771 bases encoding a protein of 256 amino acids with an estimated molecular mass of 28.76kDa and a theoretical isoelectric point of 5.12. The N-terminus of the XlGILT was found to have a putative signal peptide with a cleavage site amino acid position at 15-16. SMART analysis showed that the XlGILT contained a GILT active-site C(69)GGC(72) motif and a GILT signature motif C(114)QHGKEECIGNLIETC(129). The expression levels of XlGILT mRNA were higher in spleen and peripheral blood mononuclear cells (PBMCs), moderate in liver, intestine, heart and kidney, and lower in lung. The XlGILT mRNA expression was significantly up-regulated in spleen in vivo and PBMCs in vitro after LPS stimulation. The soluble X. laevis GILT (XlsGILT) was inserted into a pET28a vector and expressed in BL21 (DE3) cells as a His-tag fusion enzyme. After purification, further study revealed that XlsGILT was capable of catalyzing the reduction of the interchain disulfide bonds intact IgG. These results will allow for further investigation to unravel the role of this key enzyme in class II MHC-restricted antigen processing and to use X. laevis as an in vivo model for related studies.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology & Aquatic Crustacean Diseases, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
authors.forename: Xian-wei
authors.initials: XW
authors.lastname: Cui
authors.forename: Wen
authors.initials: W
authors.lastname: Xiao
authors.forename: Zhen
authors.initials: Z
authors.lastname: Ke
authors.forename: Xia
authors.initials: X
authors.lastname: Liu
authors.forename: Xing-zhou
authors.initials: XZ
authors.lastname: Xu
authors.forename: Shuang-quan
authors.initials: SQ
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D007413:Intestinal Mucosa / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013154:Spleen / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics*; D014982:Xenopus laevis / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D010088:Oxidoreductases; C571251:IFI30 protein, Xenopus; C483811:Ifi30 protein, mouse; D050862:Oxidoreductases Acting on Sulfur Group Donors