Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology & Aquatic Crustacean Diseases, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Fish Shellfish Immunol. 2012 Nov;33(5):1133-8. doi: 10.1016/j.fsi.2012.08.021. Epub 2012 Aug 31.
In mammals, interferon-γ-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play 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. Here, we reported the cloning of a GILT gene homologue from zebrafish (zGILT), a tropical freshwater fish. The full-length cDNA of zGILT gene is 768 nucleotides (nt) encoding a protein of 255 amino acids (aa), with a putative molecular weight of 28.33 kDa. The deduced protein is highly homologous to that of fish and mammalian GILTs and shares 57.1% sequence identity to that of Atlantic salmon and 55.7-21.6% sequence identity to that of various mammals. The deduced protein possesses all the main features characteristic of known GILT proteins including the signature sequence CQHGX2ECX2NX4C spanning residues 117-132, CXXC motif at residues 72-75, one potential sites for N-linked glycosylation at residual positions 54. The zGILT expression is obviously up-regulated in spleen and kidney after immunization with LPS although it also is constitutively expressed in heart, liver, muscle and intestine, suggesting that zGILT may be involved in the immune response to bacterial challenge. The soluble recombinant protein was successfully purified using Ni-nitrilotriacetic acid resin. Recombinant His-zsGILT appeared on SDS-PAGE in the ranges of their estimated size of 18.94-kDa. After purification, further study revealed that zsGILT 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 zebrafish as an in vivo model for related studies.
在哺乳动物中，干扰素-γ诱导的溶酶体硫醇还原酶（GILT）已被证明在 MHC Ⅱ类限制抗原（Ag）的加工和呈递中发挥关键作用，通过催化二硫键还原，从而展开天然蛋白 Ag，并促进随后的蛋白酶切割。在这里，我们报道了来自热带淡水鱼斑马鱼（zGILT）的 GILT 基因同源物的克隆。zGILT 基因的全长 cDNA 为 768 个核苷酸（nt），编码 255 个氨基酸（aa）的蛋白质，推测分子量为 28.33 kDa。推导的蛋白质与鱼类和哺乳动物 GILTs 高度同源，与大西洋鲑的序列同一性为 57.1%，与各种哺乳动物的序列同一性为 55.7-21.6%。推导的蛋白质具有已知 GILT 蛋白的所有主要特征，包括残基 117-132 之间的特征序列 CQHGX2ECX2NX4C、残基 72-75 处的CXXC 基序、残基位置 54 处一个潜在的 N-连接糖基化位点。用 LPS 免疫后，脾脏和肾脏中 zGILT 的表达明显上调，尽管它也在心脏、肝脏、肌肉和肠道中组成性表达，表明 zGILT 可能参与对细菌挑战的免疫反应。使用 Ni-亚氨基二乙酸树脂成功地纯化了可溶性重组蛋白。重组 His-zsGILT 在 SDS-PAGE 中的出现范围为其估计的 18.94-kDa。纯化后，进一步研究表明 zsGILT 能够催化完整 IgG 中链间二硫键的还原。这些结果将允许进一步研究，以揭示该关键酶在 II 类 MHC 限制抗原加工中的作用，并使用斑马鱼作为相关研究的体内模型。
