Zheng Wenbiao, Chen Xinhua
School of Life Sciences, Xiamen University, Xiamen 361005, PR China.
Mol Immunol. 2006 May;43(13):2135-41. doi: 10.1016/j.molimm.2006.01.001. Epub 2006 Feb 14.
In mammals, interferon-gamma-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 the spleen of large yellow croaker, a marine fish (LycGILT). The full-length cDNA of LycGILT gene is 1033 nucleotides (nt) encoding a protein of 256 amino acids (aa), with a putative molecular weight of 28.9 kDa. The deduced protein is highly homologous to that of mammalian and zebrafish GILTs and shares 54.1% sequence identity to that of zebrafish and 43.2-39.2% sequence identity to that of various mammals. The deduced LycGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX2ECX2NX4C, and other six cysteines responsible for the formation of disulfide bonds in the C-terminus. Genomic analysis revealed that LycGILT gene, spanning a 3159nt fragment, contained seven exons interrupted by six introns and exhibited a similar exon-intron organization to human and mouse GILT genes except for a slightly more compact intron arrangement. The LycGILT expression is obviously up-regulated in spleen and kidney after immunization with inactivated trivalent bacterial vaccine consisting of Vibrio alginolyticus, V. paraphaemolyticus, and Aeromonas hydrophila although it also is constitutively expressed in liver, gills, brain, and heart, suggesting that LycGILT may be involved in the immune response to bacterial challenge in large yellow croaker. A search of NCBI sequence data with LycGILT cDNA identified a pufferfish (fugu rubrides) GILT homologue cDNA and its genomic DNA sequence, where two putative interferon-gamma activation sites (GAS) were found within the promoter region. This provided evidence that a fish GILT homologue like mammalian GILT, may also be regulated by interferon-gamma (IFN-gamma) through the JAK-STAT signal pathway. These results indicate that the bony fish GILT is a functional homologue of mammalian GILT.
在哺乳动物中，γ-干扰素诱导溶酶体巯基还原酶（GILT）已被证明通过催化二硫键还原，在MHC II类限制性抗原（Ag）的加工和呈递中发挥关键作用，从而使天然蛋白质Ag解折叠，并促进蛋白酶随后的切割。在此，我们报道了从海洋鱼类大黄鱼脾脏中克隆出GILT基因同源物（LycGILT）。LycGILT基因的全长cDNA为1033个核苷酸（nt），编码一个由256个氨基酸（aa）组成的蛋白质，推测分子量为28.9 kDa。推导的蛋白质与哺乳动物和斑马鱼的GILT高度同源，与斑马鱼的序列同一性为54.1%，与各种哺乳动物的序列同一性为43.2 - 39.2%。推导的LycGILT具有哺乳动物GILT的典型结构特征，包括活性位点CXXC基序、GILT特征序列CQHGX2ECX2NX4C，以及在C末端负责形成二硫键的其他六个半胱氨酸。基因组分析表明，LycGILT基因跨越一个3159 nt的片段，包含七个外显子，被六个内含子打断，并且除了内含子排列稍紧凑外，其外显子 - 内含子组织与人和小鼠的GILT基因相似。在用由溶藻弧菌、副溶血性弧菌和嗜水气单胞菌组成的灭活三价细菌疫苗免疫后，LycGILT在脾脏和肾脏中的表达明显上调，尽管它也在肝脏、鳃、脑和心脏中组成性表达，这表明LycGILT可能参与大黄鱼对细菌攻击的免疫反应。用LycGILT cDNA搜索NCBI序列数据，鉴定出河豚（红鳍东方鲀）GILT同源物cDNA及其基因组DNA序列，在启动子区域发现了两个推测的γ-干扰素激活位点（GAS）。这提供了证据，表明鱼类GILT同源物与哺乳动物GILT一样，可能也通过JAK - STAT信号通路受γ-干扰素（IFN-γ）调节。这些结果表明硬骨鱼GILT是哺乳动物GILT的功能同源物。
