Cloning and expression analysis of interferon-γ-inducible-lysosomal thiol reductase gene in South African clawed frog (Xenopus laevis)

• Published in: International immunopharmacology Vol. 11; no. 12; pp. 2091 - 2097
• Main Authors: Cui, Xian-wei, Xiao, Wen, Ke, Zhen, Liu, Xia, Xu, Xing-zhou, Zhang, Shuang-quan
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.12.2011; Elsevier
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Cell Line; Cells, Cultured; Cloning, Molecular; Gene Expression; Genetic Vectors; Innate immunity; Interferon-γ-inducible lysosomal thiol reductase; Intestinal Mucosa - metabolism; Kidney - metabolism; Leukocytes, Mononuclear - metabolism; Liver - metabolism; Lung - metabolism; Lysosomes - enzymology; Lysosomes - genetics; Medical sciences; Molecular Sequence Data; Myocardium - metabolism; Open Reading Frames - genetics; Oxidoreductases - biosynthesis; Oxidoreductases - genetics; Oxidoreductases Acting on Sulfur Group Donors - genetics; Pharmacology. Drug treatments; Real-time PCR; Recombinant Fusion Proteins - biosynthesis; Sequence Analysis, DNA; Spleen - metabolism; Thiol reductase activity; Xenopus laevis; Xenopus laevis - genetics; Xenopus Proteins - genetics; South Africa; Africa; Innate immunity; Interferon-γ-inducible lysosomal thiol reductase; Real-time PCR; Xenopus laevis; Thiol reductase activity; Thiol; Enzyme; Frog; Lysosome; Amphibia; Natural immunity; Salientia; Real time; In vitro; Biological activity; Polymerase chain reaction; Vertebrata; Gene; Genetics; Oxidoreductases; Molecular biology; Gamma interferon; Reductase
• ISSN: 1567-5769; 1878-1705
• DOI: 10.1016/j.intimp.2011.09.001

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 C69GGC72 motif and a GILT signature motif C114QHGKEECIGNLIETC129. 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. ► We report the gene structure and phylogenetic analysis of Xenopus laevis GILT. ► The expression pattern of GILT gene is investigated in various tissues. ► The expression was significantly up-regulated in spleen and PBMCs after stimulation. ► Results suggested that the GILT perhaps involved in the innate immune response.