Zhang Xue-Mei, Wang Xi-Tao, Yue Hongwen, Leung Steve W, Thibodeau Patrick H, Thomas Philip J, Guggino Sandra E
Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 2003 Dec 19;278(51):51232-42. doi: 10.1074/jbc.M309076200. Epub 2003 Oct 7.
The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (Delta F508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of Delta F508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of Delta F508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested Delta F508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing Delta F508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-Delta F508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-Delta F508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the Delta F508 and wild type CFTR and inhibited Delta F508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36Cl- efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of Delta F508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of Delta F508 CFTR.
囊性纤维化最常见的缺陷是囊性纤维化跨膜传导调节因子（Delta F508 CFTR）第508位的苯丙氨酸缺失，这会减少该蛋白向细胞表面膜的转运。先前的研究表明，低温以及高浓度的甘油或氧化三甲胺可以部分抵消Delta F508 CFTR的加工缺陷。本研究调查了由协同转运蛋白积累的生理相关浓度的有机溶质是否能够挽救Delta F508 CFTR的错误加工。单独的肌醇或依次给予的肌醇、甜菜碱和牛磺酸增加了核心糖基化的、在内质网滞留的Delta F508 CFTR在IB3细胞或稳定表达Delta F508 CFTR的NIH 3T3细胞中加工成完全糖基化形式的CFTR的过程。使用瞬时转染的COS7细胞进行的脉冲追踪实验表明，有机溶质也增加了绿色荧光蛋白-Delta F508 CFTR核心糖基化形式的加工。此外，GFP-Delta F508 CFTR复合糖基化形式的半衰期延长表明这种处理稳定了该蛋白的成熟形式。对纯化的NBD1稳定性和聚集的体外研究表明，肌醇稳定了Delta F508和野生型CFTR，并抑制了Delta F508的错误折叠。最显著的是，用这些可转运的有机溶质处理CF支气管气道细胞可恢复细胞表面膜中CFTR的cAMP刺激的单通道活性以及外向整流氯通道的活性，还可恢复福斯可林刺激的宏观36Cl-外流。我们得出结论，有机溶质可以通过稳定Delta F508 CFTR的复合糖基化形式来增强Delta F508 CFTR向质膜的加工，从而修复CFTR功能。
