Yang Lin, Cao Xiang, Ji Xuemei, Liu Hongzhen, Wu Haitao, Gu Wei, Zhang Shuangquan
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Vet Immunol Immunopathol. 2013 May 15;153(1-2):140-5. doi: 10.1016/j.vetimm.2013.01.011. Epub 2013 Feb 19.
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, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. In this study, we reported the cloning of a GILT gene homologue from chicken (designated cGILT). The open reading frame (ORF) of cGILT consists of 762 bases, encoding a protein of 253 amino acids, with a putative molecular weight of 28kDa. The deduced protein possesses the typical structural feature of known GILT proteins, including an active-site motif, a GILT signature sequence, and 6 conserved cysteines. Genomic analysis revealed that cGILT gene, spanning a 1868bp fragment, contained seven exons interrupted by six introns. The result of real-time PCR showed that cGILT mRNA was expressed in a tissue-specific manner, while the cGILT mRNA expression was obviously up-regulated in spleen and PBMCs after stimulation with lipopolysaccharide (LPS). After expression as a soluble protein in Escherichia coli and purification by Ni-NTA affinity chromatography, cGILT was demonstrated to exhibit thiol reductase activity on IgG substrate.
γ-干扰素诱导的溶酶体巯基还原酶（GILT）通过催化二硫键还原，在MHC II类限制性抗原（Ag）的加工和呈递中起关键作用，从而使天然蛋白Ag解折叠，并促进随后蛋白酶的切割。在本研究中，我们报道了从鸡中克隆的一个GILT基因同源物（命名为cGILT）。cGILT的开放阅读框（ORF）由762个碱基组成，编码一个253个氨基酸的蛋白质，推测分子量为28kDa。推导的蛋白质具有已知GILT蛋白的典型结构特征，包括一个活性位点基序、一个GILT特征序列和6个保守的半胱氨酸。基因组分析表明，cGILT基因跨越一个1868bp的片段，包含7个外显子，被6个内含子打断。实时PCR结果显示，cGILT mRNA以组织特异性方式表达，而在用脂多糖（LPS）刺激后，脾脏和外周血单核细胞（PBMC）中的cGILT mRNA表达明显上调。在大肠杆菌中表达为可溶性蛋白并通过Ni-NTA亲和层析纯化后，cGILT被证明对IgG底物具有巯基还原酶活性。
