N-乙酰氨基葡萄糖基转移酶V表达下调可通过GLUT1的糖基化改变和活性下降引起人肝癌SMMC-7721细胞内质网应激

Down-regulation of N-acetylglucosaminyltransferase V induces endoplasmic reticulum stress by changing glycosylation and down-regulation function of GLUT1 in SMMC-7721 cells

目的初步探讨N-乙酰氨基葡萄糖基转移酶V(GnT-V)表达下调的人肝癌细胞SMMC-7721中引起内质网应激的机制。方法利用RT-PCR检测SMMC-7721(以下简称7721)细胞中葡萄糖转运蛋白(GLUT)的表达情况,利用Western blot、免疫沉淀及凝集素印迹分析检测转染了反义GnT-V的SMMC-7721细胞(以下简称As/7721)以及转染空质粒pcDNA3的SMMC-7721细胞(以下简称Mock/7721)中GLUT1的N-糖链变化,利用葡萄糖摄取率分析来检测以上3种细胞中GLUT1葡萄糖转运活性的变化。结果在As/7721细胞中GLUT1的N-糖链糖基化水平较Mock/7721明显下降,而且其GLUT1的葡萄糖转运活性也较Mock/7721细胞明显下降。结论葡萄糖转运蛋白GLUT1N-糖基化的改变和转运活性的下降可能是GnT-V表达下调的SMMC-7721细胞中引起内质网应激的机制之一。

Purpose To study the mechanism of the endoplasmic reticulum stress （ER-stress） happened in human hepatocarcinoma cell line SMMC-7721 whose expression of N-acetylglucosaminyltrans- ferase V （GnT-V） was blocked. Methods The expression of glucose transporters in SMMC-7721 cell was detected by RT-PCR. The glycosylation change of glucose transporter 1 （GLUT1） in both antisense GnT-V transfectant （As/7721） and mock pcDNA3 transfectant （Mock/7721） cells were detected by Western blot, immunoprecipitation and lectin blot. The transport activity of GLUT1 was detected by glucose uptake measurement. Results The N- glycosylation level of GLUT1 in As/7721 cell was significantly decreased compared with that of Mock/7721 cell. The glucose transport activity of GLUT1 in As/7721 cell was also significantly declined as compared to that of Mock/7721 cell. Conclusions The glycosylation change and decrease in transport activity of GLUT1 may be one possible mechanism of ER stress induced by down-regulating GnT-V.