| 20199105 |
Tokhtaeva E, Munson K, Sachs G, Vagin O: N-Glycan-Dependent Quality Control of the Na,K-ATPase beta (2) Subunit. Biochemistry. 2010 Mar 17.
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. |
86(1,1,1,6) |