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Characterization of endogenous betaine γ-amino-n-butyric acid cotransporter glycoform and its hyperosmotic regulation in MDCK cells

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Abstract

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 Peptide N-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.

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Acknowledgements

This work was supported by the Cystic Fibrosis Foundation grants GUGGIN00G0 and GUGGIN02P0 to SEG, a NIH fellowship grant DK07712 and a CF fellowship grant WANG-X99F0 to XT Wang, a CF student fellowship to SW Leung and a CFRDP grant to WB Guggino.

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Authors and Affiliations

  1. Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, China

    Xue-Mei Zhang

  2. Department of Medicine, School of Medicine, Johns Hopkins University, 929 Ross Bldg. 720 Rutland Ave., Baltimore, MD, 21205, USA

    Xue-Mei Zhang, Xi-Tao Wang, Hui Cai, Steve W. Leung & Sandra E. Guggino

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  1. Xue-Mei Zhang
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  2. Xi-Tao Wang
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  3. Hui Cai
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Correspondence to Xue-Mei Zhang.

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Xue-Mei Zhang and Xi-Tao Wang contributed equally to this work.

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Zhang, XM., Wang, XT., Cai, H. et al. Characterization of endogenous betaine γ-amino-n-butyric acid cotransporter glycoform and its hyperosmotic regulation in MDCK cells. Pflugers Arch - Eur J Physiol 454, 143–153 (2007). https://doi.org/10.1007/s00424-006-0179-x

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  • DOI: https://doi.org/10.1007/s00424-006-0179-x

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