Structural Analysis of N-glycans attached to Pig Kidney Na+/K+-ATPase

Abstract

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 profiling 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 purified Na + /K +-ATPase from pig kidney outer medulla to homogeneity and solubilized it with the detergent C 12 E 8. The enzyme thus obtained was identified 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 modified with 4-hydroxynonenal, an aldehydric lipid peroxidation product. Three N-glycosylation sites on the β1 subunit were confirmed to be fully occupied by time course analysis of enzymatic deglycosylation monitored by SDS-PAGE. HPLC profiling 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 briefly discuss the possibility that oligomerization of Na + /K +-ATPase via β-β interactions assembles the N-glycans and promotes cell-cell adhesion.

Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation

“…The β subunit consists of an extracellular domain and one transmembrane helix, which interacts with transmembrane helices M 7 and M 10 of the α subunit. The extracellular domain of the β subunit contains three glycosylation sites [ 27 , 28 ], in which the asparagine residues are glycosylated by oligosaccharides containing N -acetylglucosamine, mannose, and partially also galactose [ 29 ]. The glycosylation level of the β subunit affects its folding and subsequent translocation into the cell plasma membrane [ 30 ].…”

A Data Set of Ion Mobility Collision Cross Sections and Liquid Chromatography Retention Times from 71 Pyridylaminated N-Linked Oligosaccharides

“…Separation of isomeric glycans is expedited by liquid chromatography (LC), and the LC method has been used to identify N -glycan structures often in combination with NMR analysis . Recently, ion mobility mass spectrometry (IM-MS) has emerged as a complementary technique for discriminating between isomers .…”