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Na+/K+-ATPase is a membrane glycoprotein composed of α, β, and γ subunits, generating ion gradients across plasma membranes. Ion pumping is mainly accomplished by the α subunit, while the glycosylated β subunit binds tightly to the α subunit to assemble the pump and plays an essential role in the stabilization and maturation of Na+/K+-ATPase. Accumulating evidence suggests that the N-glycans of the β subunit contribute to cell-cell interaction and the tightness of cell contacts is modulated by N-glycan branching. However, N-glycan function is not fully understood due to a lack of detailed information on the oligosaccharide structure. We, here, perform glycosylation profi ling of the N-glycans attached to pig kidney Na+/K+-ATPase in order to better understand the mechanism of Na+/K+-ATPase-mediated cell adhesion.
We purifi ed Na+/K+-ATPase from pig kidney outer medulla to homogeneity and solubilized it with the detergent C12E8. The enzyme thus obtained was identifi ed as α1β1 subtype by LC-MS/MS analysis of tryptic digests. During the course of MS analysis, we found that Lys456 of the α subunit was partially modifi ed with 4-hydroxynonenal, an aldehydric lipid peroxidation product. Three N-glycosylation sites on the β1 subunit were confi rmed to be fully occupied by time course analysis of enzymatic deglycosylation monitored by SDS-PAGE. HPLC profi ling of pyridylaminated oligosaccharides derived from Na+/K+-ATPase showed that high-mannose type oligosaccharides predominate while most of the less abundant complex-type oligosaccharides are capped with galactose residues. No glycans could be detected on the four consensus N-glycosylation sites on the α1 subunit. We briefl y discuss the possibility that oligomerization of Na+/K+- ATPase via β-β interactions assembles the N-glycans and promotes cell-cell adhesion.