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Biochemistry. 2010 Apr 13;49(14):3116-28. doi: 10.1021/bi100115a.

N-glycan-dependent quality control of the Na,K-ATPase beta(2) subunit.

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Department of Physiology, School of Medicine, UCLA, and Veterans Administration Greater Los Angeles Health Care System, VAGLAHS/West LA, Building 113, Room 324, 11301 Wilshire Boulevard, Los Angeles, California 90073, USA.


Bulky hydrophilic N-glycans stabilize the proper tertiary structure of glycoproteins. In addition, N-glycans comprise the binding sites for the endoplasmic reticulum (ER)-resident lectins that assist correct folding of newly synthesized glycoproteins. To reveal the role of N-glycans in maturation of the Na,K-ATPase beta(2) subunit in the ER, the effects of preventing or modifying the beta(2) subunit N-glycosylation on trafficking of the subunit and its binding to the ER lectin chaperone, calnexin, were studied in MDCK cells. Preventing N-glycosylation abolishes binding of the beta(2) subunit to calnexin and results in the ER retention of the subunit. Furthermore, the fully N-glycosylated beta(2) subunit is retained in the ER when glycan-calnexin interactions are prevented by castanospermine, showing that N-glycan-mediated calnexin binding is required for correct subunit folding. Calnexin binding persists for several hours after translation is stopped with cycloheximide, suggesting that the beta(2) subunit undergoes repeated post-translational calnexin-assisted folding attempts. Homology modeling of the beta(2) subunit using the crystal structure of the alpha(1)-beta(1) Na,K-ATPase shows the presence of a relatively hydrophobic amino acid cluster proximal to N-glycosylation sites 2 and 7. Combined, but not separate, removal of sites 2 and 7 dramatically impairs calnexin binding and prevents the export of the beta(2) subunit from the ER. Similarly, hydrophilic substitution of two hydrophobic amino acids in this cluster disrupts both beta(2)-calnexin binding and trafficking of the subunit to the Golgi. Therefore, the hydrophobic residues in the proximity of N-glycans 2 and 7 are required for post-translational calnexin binding to these N-glycans in incompletely folded conformers, which, in turn, is necessary for maturation of the Na,K-ATPase beta(2) subunit.

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