T细胞活化接头蛋白棕榈酰化位点突变抑制CD59 GPI介导的T淋巴细胞活化信号转导

Mutation of palmitoylation site of linker for activation of T cells inhibits signal transduction mediated by glycosyl phosphatidyl inositol-anchored CD59 in T cells

目的构建T细胞活化接头蛋白(LAT)棕榈酰化位点突变的慢病毒,感染Jurkat细胞,建立稳定转染的细胞株,观察LAT棕榈酰化位点突变对CD59介导的T淋巴细胞活化信号转导的影响。方法构建阴性对照(neg-EGFP)、LAT-M-EGFP融合蛋白基因载体,包装成慢病毒,分别感染Jurkat细胞,建立稳定感染的细胞株。激光共聚焦显微镜观察病毒感染效率及融合蛋白在细胞上的定位;CCK-8法检测CD59单克隆抗体交联前后各组细胞的增殖活性,流式细胞术检测细胞凋亡情况,Western blot法检测磷脂酶Cγ1(PLC-γ1)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)蛋白的表达。结果共聚焦显微镜观察发现LAT-M组细胞的LAT分子在细胞膜上散在分布,CD59抗体交联刺激后,未出现明显的点簇状聚集区。与阴性对照细胞相比,LAT-M细胞增殖活性明显降低,中晚期凋亡细胞明显增加;Western blot结果显示,LAT-M组的PLC-γ1、LCK蛋白表达水平和阴性对照组大致相同,经抗体活化后无明显变化,而阴性对照细胞的PLC-γ1、LCK蛋白表达量下降。结论 LAT棕榈酰化位点突变的Jurkat细胞脂筏定位功能缺失,细胞处于抑制状态,对CD59糖基磷脂酰肌醇(GPI)介导的T淋巴细胞活化信号转导有抑制作用。

Objective To construct the lentivirus carrying the mutated palmitoylation site of the linker for activation of T cells（ LAT） and infect Jurkat cells with it to establish stable cel line,and to investigate the effect of LAT palmitoylation mutation on T cell signaling induced by CD59. Methods Negative control（ neg-EGFP） and LAT-M-EGFP fusion protein gene vectors were respectively constructed and then packaged using lentivirus. Subsequently,Jurkat cells were infected with them to establish stable cell lines. Confocal laser scanning microscopy was used to observe the infection efficiency and the distribution of fusion proteins in Jurkat cells. CCK-8 assay was used to detect the change of cell proliferation activity after CD59 m Ab supplementation. Flow cytometry was used to determine the apoptosis rate. Western blotting was used to examine the levels of phospholipase C-γ1（ PLC-γ1） and lymphocyte-specific protein tyrosine kinase（ LCK）. Results Confocal laser scanning microscopy revealed that LAT molecules of LAT-M group scattered on cell membrane,and there was no obvious clustered region after cross linkage with CD59 m Ab. Compared with the negative control group,the cell proliferation activity of LAT-M group significantly decreased,and the quantity of middle-late apoptotic cells significantly increased; Western blotting showed that the expression levels of PLC-γ1 and LCK in LAT-M group was roughly the same with those in negative control group,and after CD59 m Ab stimulation,there was no obvious change in LAT-M group,while the levels in negative control group were reduced. Conclusion LAT-M-EGFP fusion protein could not locate on lipid rafts of Jurkat cells infected with LAT palmitoylation mutation. In addition,the growth of the cells carrying the LAT-M-EGFP was inhibited. The palmitoylation mutation of LAT attenuated the signal transduction induced by glycosylphosphatidylinositol-anchored CD59 in T cells.