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 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.
Highlights
Na+/K+-ATPase is an integral membrane protein that is responsible for the ATP-dependent transport of Na+ and K+ across the cell membrane [1]
CID-MS/MS analysis of a peptide from 444 to 456 revealed that Lys456 was partially modified with 4-hydroxynonenal (HNE) (Figure 3B), an aldehyde produced from the peroxidation of ω-6 polyunsaturated fatty acids [52]
HNE is known to be one of the major end products of lipid peroxidation in cells and recent studies suggest that HNE is a key signaling molecule to regulate stress-mediated signaling [55]
Summary
Na+/K+-ATPase is an integral membrane protein that is responsible for the ATP-dependent transport of Na+ and K+ across the cell membrane [1]. This transport generates the electrochemical gradients that are required for electrical excitability, cellular uptake of ions, nutrients and neurotransmitters, and regulation of cell volume and intracellular pH. Na+/K+-ATPase is expressed in several isozymes, and in mammalian cells, four α isoforms (α1, α2, α3 and α4) and three β isoforms (β1, β2 and β3) have been identified [12,13,14]. The α1 isoform in association with the β1 subunit is found in almost every tissue and α1β1 is the principal isozyme of the kidney, a tissue often used as a source of Na+/K+-ATPase [12]
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