【摘要】 目的:探究N-糖基化在KCNE1功能中的作用。方法:根据N-糖基化序列特征"Asn-X-Ser/Thr"分析和N-糖基化特异消化酶实验检测KCNE1是否存在N-糖基化,通过点突变构建N-糖基化位点突变体并通过蛋白质印迹法检测其蛋白表达,细胞免疫荧光染色检测突变体亚细胞定位;免疫共沉淀检测突变体与KCNQ1的相互作用。结果:KCNE1存在N-糖基化,N5和N26是其可能位点;点突变获得KCNE1-N5Q、KCNE1-N26Q和KCNE1-N5,26Q 3个突变体。蛋白质印迹法确定N5和N26为KCNE1的两个糖基化位点;与野生型相比,突变体在细胞内呈现不同程度的蛋白囤积现象,KCNE1-N26Q表现尤为显著,KCNE1-N5,26Q和KCNE1-N5Q则表现略轻;KCNE1 N-糖基化突变体仍能与KCNQ1结合。结论:N-糖基化在KCNE1转运中发挥重要作用,其中N26较为关键;N-糖基化在KCNE1与KCNQ1之间的相互作用中不起重要作用。更多还原

【Abstract】 Objective: To investigate the role of N-glycosylation in KCNE1 function.Methods: "Asn-X-Ser/Thr" sequence analysis and specific de-N-glycosylation treatment were used to define and confirm N-glycosylation in KCNE1.Three de-N-glycosylation mutants of KCNE1 were created with site-directed mutagenesis.The expression and suncellular localization of different KCNE1 sunubits were carried out in HEK293B cells with Western blot and immunocytochemistry.The interaction between KCNQ1 and KCNE1 was investigated with co-immunoprecipitation.Results: Two N-glycosylation sites in wild type KCNE1 were confirmed at N5 and N26 in vivo.Comparing to KCNE1 wide type,all three KCNE1 mutants showed a similar pattern of subcellular localization with various degrees of intracellular aggression in which KCNE1-N26Q was the most severe one,while KCNE1-N5Q and KCNE1-N5,26Q were less.All three KCNE1 mutants showed the ability to physically interact with wild type KCNQ1.Conclusion: N-glycosylation is important to KCNE1 trafficking,especially at N26.De-N-glycosylation of KCNE1 does not affect its interaction with wild type KCNQ1 in vivo.更多还原