Characterization of endogenous betaine gamma-amino-n-butyric acid cotransporter glycoform and its hyperosmotic regulation in MDCK cells.
Increase in mRNA expression and transport activity of the betaine gamma-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.
Zhang
Xue-Mei
XM
Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, China. xuemzhang@shmu.edu.cn
Wang
Xi-Tao
XT
Cai
Hui
H
Leung
Steve W
SW
Guggino
Sandra E
SE
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D009997:Osmotic Pressure; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000494:pharmacology; D020033:Protein Isoforms / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D013997:Time Factors
D050485:GABA Plasma Membrane Transport Proteins; D020033:Protein Isoforms; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase