Lectin‐dependent quality control of the Na,K‐ATPase maturation in the ER

Abstract

To evaluate the role of N‐glycan‐lectin interactions in maturation of the Na,K‐ATPase β2 subunit, the effects of removing all 8 or selected N‐glycans on trafficking of the β2 and its binding to calnexin were studied in MDCK cells. N‐glycan‐dependent binding of the β2 to calnexin persists for several hours after completing translation, suggesting involvement of the ER glucosyltransferase UGGT1 that is known to induce additional rounds of calnexin‐assisted folding of misfolded glycoproteins by re‐glucosylating N‐glycans next to local folding defects exposing hydrophobic amino acids. The β2 is retained in the ER when calnexin binding is prevented by castanospermine. Homology modeling of the β2 using the crystal structure of the α1‐β1 Na,K‐ATPase shows the presence of a relatively hydrophobic cluster proximal to N‐glycans 2 and 7. Combined, but not separate, removal of N‐glycosylation sites 2 and 7 dramatically impairs calnexin binding and prevents the export of the β2 from the ER. Similar effects are observed with hydrophilic substitution of two hydrophobic amino acids in the cluster, suggesting that exposure of these residues in incompletely folded conformers results in UGGT1‐catalyzed re‐glucosylation of N‐glycans 7 and 2, allowing post‐translational calnexin‐assisted re‐folding, which, in turn, is required for maturation of the Na,K‐ATPase β2 subunit.Supported by NIH grants DK077149 and DK058333.