FUT8对megalin功能及白蛋白负荷致肾小管上皮细胞单核细胞趋化蛋白1表达的影响

Effect of FUT8 on megalin and albumin over load-induced MCP expression in renal tubular epithelial cell

关键词:  核心岩藻糖基化  低密度脂蛋白相关蛋白-2  蛋白尿  HK-2细胞

Key words:  α1-6-fucosyltransferase  inflammation  megalin  proteinuria  HK-2

摘要:

［目的］探讨α1-6-岩藻糖基转移酶（α1-6-fucosyltransferase，FUT8）所催化的核心岩藻糖基化对低密度脂蛋白受体相关蛋白-2 （LRP-2，meglain）内吞白蛋白功能和对白蛋白超负荷诱导的肾小管上皮细胞炎症的影响。［方法］用10 mg/mL的牛血清白蛋白（BSA）孵育HK-2细胞4 h建立白蛋白超负荷细胞模型，分别用FUT8siRNA或FUT8过表达载体转染，用流式细胞仪测定白蛋白的绑定和内吞以及megalin的核心岩藻糖基化水平。用免疫印迹、免疫沉淀和凝集素印迹方法测定单核细胞趋化蛋白-1（MCP-1）及megalin的蛋白表达，用单因素方差检验方法进行统计学处理。［结果］BSA的绑定和内吞有时间和浓度依赖性。BSA诱导后HK-2细胞FUT8、MCP-1明显升高。白蛋白超负荷条件下，FUT8过表达组细胞MCP-1表达水平明显高于BSA组。此外，FUT8的过表达显著提高megalin的核心岩藻糖基化水平及绑定和内吞白蛋白能力。沉默FUT8基因降低megalin的核心岩藻糖基化及绑定、内吞白蛋白能力，同时抑制MCP-1的升高，使其保持在正常表达水平。 ［结论］核心岩藻糖基化是megalin发挥白蛋白绑定和内吞功能的必需过程，抑制megalin蛋白合成后的核心岩藻糖基化修饰能够减轻蛋白超负荷诱导的肾小管上皮细胞炎症介质表达。

Abstract:

［Objective］To investigate the role of core fucosylation in megalin-mediated binding and endocytosis as well as its effect on albumin overload-induced renal tubular epithelia.［Methods］An albumin overload cell model of HK-2 cell was constructed with 10 mg/mL BSA,and then FUT8siRNA or FUT8 over-expression vector was transfected into the cells.The binding and endocytosis of BSA and the core fucose on megalin were determined by flow-cytometry.The expression of MCP-1 and megalin were detected with western blot,immunoprecipitation and lectin blot.Data were assessed by ANOVA analysis. ［Results］Both of binding and endocytosis for BSA were time and dose-dependent.A significant increasing protein expressions of FUT8 and MCP-1 were found in HK-2 cells after BSA incubation.Under the condition of albumin overload,the expression of MCP-1 in cells of the group of pre-incubation with FUT8 over-expression vector was higher than that of BSA group.The core fucose and both of binding and endocytosis for megalin increased significantly in HK-2 cells with over-expression of FUT8,but following treatment with FUT8 siRNA,all of them decreased significantly and the expression of MCP-1 was down-regulated to normal level in the cells.［Conclusion］The core fucosylation of megalin is essential to its abilities in binding and endocytosis,and inhibition of catalisation of core fucosylation could suppress albumin overload-induced HK-2 cells to express MCP-1.