N-Glycan-Dependent Quality Control of the Na,K-ATPase β2 Subunit

Abstract

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 β₂ subunit in the ER, the effects of preventing or modifying the β₂ 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 β₂ subunit to calnexin and results in the ER retention of the subunit. Furthermore, the fully N-glycosylated β₂ 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 β₂ subunit undergoes repeated post-translational calnexin-assisted folding attempts. Homology modeling of the β₂ subunit using the crystal structure of the α₁-β₁ 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 β₂ subunit from the ER. Similarly, hydrophilic substitution of two hydrophobic amino acids in this cluster disrupts both β₂−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 β₂ subunit.