Characterization of endogenous betaine gamma-amino-n-butyric acid cotransporter glycoform and its hyperosmotic regulation in MDCK cells.
Characterization of endogenous betaine gamma-amino-n-butyric acid cotransporter glycoform and its hyperosmotic regulation in MDCK cells.
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MDCK 细胞中内源性甜菜碱 γ-氨基-正丁酸协同转运蛋白糖型的表征及其高渗调节。
DOI：
10.1007/s00424-006-0179-x
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Guggino,SandraE
中科院分区：
文献类型：
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作者：
Zhang,Xue-Mei;Wang,Xi-Tao;Cai,Hui;Leung,SteveW;Guggino,SandraE
先前已在MDCK细胞中显示，甜菜碱γ-氨基-正丁酸协同转运蛋白（BGAT）的mRNA表达和转运活性在高渗时增加。然而，高渗透压诱导的内源性BGAT蛋白表达的反应没有详细研究。我们显示了两种形式的内源性BGAT免疫反应，在MDCK II细胞中表达。两者均对肽N-糖苷酶F（PNGase F）敏感，表明它们是N-糖基化蛋白。一条约75 kDa的条带对Endo H具有抗性，而另一条约55 kDa的条带对Endo H敏感，这表明它们分别是高尔基体后区室中的完全N-糖基化的成熟形式和内质网（ER）中的核心糖基化的未成熟形式。当用高渗透压处理时，它们显著增加。但是，BGAT加工的速率，如通过成熟与不成熟形式的比率评估的，没有增加，表明高渗透压并不促进BGAT从ER到分泌途径的输出。表面生物素化和共聚焦显微镜显示，高渗透压显着增加的量的成熟形式的BGAT的基底外侧膜上的顶膜上的一个非常小的部分。我们的结论是，BGAT是一个N-糖基化的蛋白质与两个糖型和内源性BGAT的合成，而不是加工参与了高渗胁迫的适应。
Increase in mRNA expression and transport activity of the betaine γ-amino-n-butyric acid cotransporter (BGAT) in response to hyperosmolality has been previously shown in MDCK cells. However, the hyperosmolality-induced response of endogenous BGAT protein expression was not investigated in detail. We show two forms of endogenous BGAT immunoreactivity that are expressed in MDCK II cells. Both are sensitive to PeptideN-Glycosidase F (PNGase F), suggesting that they are N-glycosylated proteins. One band, about 75 kDa, is resistant to Endo H, while the other 55 kDa band is sensitive to it, suggesting that they are fully N-glycosylated mature form in the post-Golgi compartment and core-glycosylated immature form in the endoplasmic reticulum (ER), respectively. When treated with hyperosmolality, they are significantly increased. But the rate of BGAT processing, as assessed by the ratio of mature to immature form, is not increased, suggesting that hyperosmolality does not facilitate the export of BGAT from the ER to the secretory pathway. Surface biotinylation and confocal microscopy show that hyperosmolality significantly increases the amount of the mature form of BGAT on the basolateral membrane with a very small fraction on the apical membrane. We conclude that BGAT is an N-glycosylated protein with two glycoforms and endogenous BGAT synthesis rather than processing is involved in the adaptation to the hyperosmotic stress.