Role of N-glycosylation in renal betaine transport.
Role of N-glycosylation in renal betaine transport.
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DOI：
10.1042/bj20131031
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发表时间：
2015-09-01
期刊：
影响因子：
--
通讯作者：
Ziegler C
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文献类型：
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作者：
Schweikhard ES;Burckhardt BC;Joos F;Fenollar-Ferrer C;Forrest LR;Kempson SA;Ziegler C
渗透压和折叠伴侣甜菜碱由肾脏钠离子偶联的GABA转运体BGT-1转运，BGT-1是SLC6家族的成员。在高渗条件下，BGT-1在肾脏细胞中的转录、翻译和质膜插入显著增加，导致甜菜碱和GABA转运增加。重建等渗性涉及BGT-1的质膜耗尽。BGT-1在渗透胁迫变化过程中调节质膜插入的分子机制尚不清楚。在这里，我们揭示了调节质膜插入和N-糖基化之间的联系。基于同源模型，我们在BGT-1的胞外环2(EL2)中确定了两个位置(N171和N183)，通过免疫金标记法、电子显微镜、诱变和双电极电压钳测量，研究了BGT-1胞外环2(EL2)的运输、插入和转运，以及放射性标记底物在MDCK和HEK细胞中的摄取。N-糖基化可明显促进BGT-1在卵母细胞和MDCK细胞中的转运和质膜插入。此外，与N171和N183位N-糖链的结合对蛋白质活性和底物亲和力的贡献是相同的。N171和N183分别被天冬氨酸取代不会导致BGT-1活性的损失，而双突变体是无效的，这表明至少一个位点的N-糖基化是功能所必需的。在任何一个位点被丙氨酸或缬氨酸取代会导致运输活动的戏剧性损失。此外，在MDCK细胞中，N183D的质膜插入不再受渗透胁迫的调节，这突显了N-糖基化在调节这种SLC6转运蛋白中的影响。
The osmolyte and folding chaperone betaine is transported by the renal Na+-coupled GABA symporter BGT-1, a member of the SLC6 family. Under hypertonic conditions, the transcription, translation and plasma membrane insertion of BGT-1 in kidney cells are significantly increased, resulting in elevated betaine and GABA transport. Re-establishing isotonicity involves plasma membrane depletion of BGT-1. The molecular mechanism of the regulated plasma membrane insertion of BGT-1 during changes in osmotic stress is unknown. Here we reveal a link between regulated plasma membrane insertion and N-glycosylation. Based on homology modelling we identified two sites (N171 and N183) in the extracellular loop 2 (EL2) of BGT-1, which were investigated with respect to trafficking, insertion, and transport by immunogold-labelling, electron microscopy, mutagenesis, and two-electrode voltage clamp measurements in Xenopus laevis oocytes, and uptake of radiolabelled substrate into MDCK and HEK cells. Trafficking and plasma membrane insertion of BGT-1 was clearly promoted by N-glycosylation in both oocytes and MDCK cells. Moreover, association with N-glycans at N171 and N183 contributed equally to protein activity and substrate affinity. Substitution of N171 and N183 by aspartate individually caused no loss of BGT-1 activity, while the double mutant was inactive, suggesting that N-glycosylation of at least one of the sites is required for function. Substitution by alanine or valine at either site caused a dramatic loss in transport activity. Furthermore, in MDCK cells plasma membrane insertion of N183D was no longer regulated by osmotic stress, highlighting the impact of N-glycosylation in regulation of this SLC6 transporter.