title: N-glycan-dependent quality control of the Na,K-ATPase beta(2) 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 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.
authors.affiliation: 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.
authors.forename: Elmira
authors.initials: E
authors.lastname: Tokhtaeva
authors.forename: Keith
authors.initials: K
authors.lastname: Munson
authors.forename: George
authors.initials: G
authors.lastname: Sachs
authors.forename: Olga
authors.initials: O
authors.lastname: Vagin
mesh_terms: D000818:Animals; D037281:Calnexin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D008164:Luminescent Proteins / Q000235:genetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D011134:Polysaccharides / Q000502:physiology*; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D018832:Molecular Chaperones; D011134:Polysaccharides; D021122:Protein Subunits; D037281:Calnexin; D000254:Sodium-Potassium-Exchanging ATPase