Role of glycosylation in the renal electrogenic Na+-HCO3- cotransporter (NBCe1)
Role of glycosylation in the renal electrogenic Na+-HCO3- cotransporter (NBCe1)
复制标题
DOI：
10.1152/ajprenal.00131.2002
复制
发表时间：
2003-06-01
影响因子：
4.2
通讯作者：
Boron, WF
中科院分区：
文献类型：
--
作者：
Choi, I;Hu, LH;Boron, WF
Na+-HCO3-协同转运体NBCe1对调节细胞内pH(pH(I))和许多组织(包括肾脏、胰腺和脑)上皮HCO3-转运具有重要意义。在本研究中，我们研究了NBCe1的糖基化位点。用N-糖苷酶F(PNGase F)处理大鼠肾膜提取液后，NBCe1的表观分子量(MW)从130变为116，这是根据推导的氨基酸序列预测的。内切糖苷酶F-2、H或O-糖苷酶处理不影响NBCe1的相对分子质量。凝集素结合研究以及酶数据表明，N-连接的碳水化合物是三触角或四触角类型。为了定位糖基化位点，我们通过用谷氨酰胺(Q)取代天冬酰胺(N)和评估非洲爪哇卵母细胞中突变的转运蛋白，分别突变了7个共同的N-糖基化位点。经PNGase F处理的卵母细胞膜提取物的免疫印迹表明，NBCe1通常是在N597和N617(都在第三个细胞外环)上糖基化的。然而，当其他两个位点发生突变时，N592(在同一个环上)会发生糖基化。三个突变体(N592Q/N597Q/N617Q)是完全无糖化的，但基于对卵母细胞膜电位和pH(I)的微电极测量，保留了野生型NBCe1对Na+和HCO3的依赖性和生电性。
The electrogenic Na+-HCO3- cotransporter NBCe1 is important for the regulation of intracellular pH (pH(i)) and for epithelial HCO3- transport in many tissues, including kidney, pancreas, and brain. In the present study, we investigate glycosylation sites in NBCe1. Treatment of rat kidney membrane extracts with peptide N-glycosidase F (PNGase F) shifted the apparent molecular weight (MW) of NBCe1 from 130 to 116, the MW predicted from the deduced amino acid sequence. Treatment with endoglycosidase F-2 or H or O-glycosidase did not affect the MW of NBCe1. Lectin-binding studies, together with the enzyme data, suggest that the N-linked carbohydrates are of tri- or tetra-antennary type. To localize glycosylation sites, we individually mutated the seven consensus N-glycosylation sites by replacing asparagine (N) with glutamine (Q) and assessing mutant transporters in Xenopus laevis oocytes. Immunoblotting of oocyte membrane extracts treated with PNGase F indicates that NBCe1 is normally glycosylated at N597 and N617 (both on the third extracellular loop). However, N592 (on the same loop) is glycosylated when the other two sites are mutated. The triple mutant (N592Q/N597Q/N617Q) is completely unglycosylated but, based on microelectrode measurements of membrane potential and pH(i) in oocytes, preserves the Na+ and HCO3- dependence and electrogenicity of wild-type NBCe1.