Ma Lei, Cao Fang, Tang Runze, Zhang Jiaxin, Zhang Shuangquan
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China; Qilu Institute of Pharmaceutical Research, Qilu Pharmaceutical Co. Ltd, Jinan 250100, China.
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Res Vet Sci. 2017 Apr;111:81-84. doi: 10.1016/j.rvsc.2016.12.006. Epub 2016 Dec 28.
Gamma-interferon-inducible lysosomal thiol reductase (GILT) is a key enzyme in the antigen processing and presentation pathway whereby it reduces disulfide bonds at an acidic pH. In this study, a homolog of GILT from guinea pigs (designated gpGILT) was identified and characterized using bioinformatic methods and bioactivity assays. The open reading frame of gpGILT is 705bp in length and encodes 234 amino acids, with a putative molecular weight of about 25.85kDa. The structure of gpGILT is similar to those of humans and zebrafish, containing six introns and seven exons. The deduced primary structure of the gpGILT protein includes all of the typical features of other known GILT proteins, including an active-site motif, CXXC, a GILT signature sequence, CQHGXECXNXC, three potential Asn-linked glycosylation sites, and six other conserved cysteines. The predicted tertiary structures of gpGILT, human GILT, and mouse GILT are quite similar in shape and positional arrangement of the key motifs modeled on the same template. Amino acid sequence-based alignment and phylogenetic analysis showed that gpGILT is most closely related to that from the rat, with an identity of 68.40%. Additionally, the constitutive expression and immune response to lipopolysaccharide (LPS) challenge of gpGILT were tested using real-time quantitative polymerase chain reaction. A tissue-specific expression pattern in selected tissues and remarkable up-regulation of gpGILT mRNA in spleen and blood within 12h of LPS stimulation were observed, suggesting that GILT functions as an immunological surveillance-related factor in both innate and adaptive immunity. Soluble recombinant gpGILT produced in E. coli could reduce the interchain disulfide bonds of IgG in an acidic reaction system in vitro, suggesting thiol reductase activity in antigen processing. The results of this study provide a better understanding of the molecular characteristics of gpGILT and are a useful reference for further investigation of its involvement in antigen processing and immunological surveillance using the laboratory guinea pig.
γ-干扰素诱导的溶酶体硫醇还原酶（GILT）是抗原加工和呈递途径中的关键酶，它可在酸性pH条件下还原二硫键。在本研究中，利用生物信息学方法和生物活性测定对豚鼠GILT的同源物（命名为gpGILT）进行了鉴定和表征。gpGILT的开放阅读框长度为705bp，编码234个氨基酸，推测分子量约为25.85kDa。gpGILT的结构与人类和斑马鱼的相似，包含6个内含子和7个外显子。推导的gpGILT蛋白一级结构包括其他已知GILT蛋白的所有典型特征，包括活性位点基序CXXC、GILT特征序列CQHGXECXNXC、3个潜在的天冬酰胺连接的糖基化位点以及其他6个保守的半胱氨酸。基于相同模板建模的关键基序，gpGILT、人GILT和小鼠GILT预测的三级结构在形状和位置排列上非常相似。基于氨基酸序列的比对和系统发育分析表明，gpGILT与大鼠的GILT关系最为密切，同一性为68.40%。此外，使用实时定量聚合酶链反应检测了gpGILT的组成性表达以及对脂多糖（LPS）刺激的免疫反应。观察到在选定组织中的组织特异性表达模式以及LPS刺激后12小时内脾脏和血液中gpGILT mRNA的显著上调，表明GILT在先天免疫和适应性免疫中均作为免疫监视相关因子发挥作用。在大肠杆菌中产生的可溶性重组gpGILT可在体外酸性反应体系中还原IgG的链间二硫键，表明其在抗原加工中具有硫醇还原酶活性。本研究结果有助于更好地了解gpGILT的分子特征，并为进一步利用实验豚鼠研究其在抗原加工和免疫监视中的作用提供有用参考。
