Olga Vagin

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

Polarized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the trans-Golgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors.

The Gastric H,K ATPase as a Drug Target: Past, Present, and Future

The recent progress in therapy if acid disease has relied heavily on the performance of drugs targeted against the H,K ATPase of the stomach and the H2 receptor antagonists. It has become apparent in the last decade that the proton pump is the target that has the likelihood of being the most sustainable area of therapeutic application in the regulation of acid suppression. The process of activation of acid secretion requires a change in location of the ATPase from cytoplasmic tubules into the microvilli of the secretory canaliculus of the parietal cell. Stimulation of the resting parietal cell, with involvement of F-actin and ezrin does not use significant numbers of SNARE proteins, because their message is depleted in the pure parietal cell transcriptome. The cell morphology and gene expression suggest a tubule fusion-eversion event. As the active H,K ATPase requires efflux of KCl for activity we have, using the transcriptome derived from 99% pure parietal cells and immunocytochemistry, provided evidence that the KCl pathway is mediated by a KCQ1/KCNE2 complex for supplying K+ and CLIC6 for supplying the accompanying Cl−. The pump has been modeled on the basis of the structures of different conformations of the sr Ca ATPase related to the catalytic cycle. These models use the effects of site directed mutations and identification of the binding domain of the K competitive acid pump antagonists or the defined site of binding for the covalent class of proton pump inhibitors. The pump undergoes conformational changes associated with phosphorylation to allow the ion binding site to change exposure from cytoplasmic to luminal exposure. We have been able to postulate that the very low gastric pH is achieved by lysine 791 motion extruding the hydronium ion bound to carboxylates in the middle of the membrane domain. These models also allow description of the K+ entry to form the K+ liganded form of the enzyme and the reformation of the ion site inward conformation thus relating the catalytic cycle of the pump to conformational models. The mechanism of action of the proton pump inhibitor class of drug is discussed along with the cysteines covalently bound with these inhibitors. The review concludes with a discussion of the mechanism of action and binding regions of a possible new class of drug for acid control, the K+ competitive acid pump antagonists.

The Role of the β1 Subunit of the Na,K-ATPase and Its Glycosylation in Cell-Cell Adhesion

Based on recent data showing that overexpression of the Na,K-ATPase β1 subunit increased cell-cell adhesion of nonpolarized cells, we hypothesized that the β1 subunit can also be involved in the formation of cell-cell contacts in highly polarized epithelial cells. In support of this hypothesis, in Madin-Darby canine kidney (MDCK) cells, the Na,K-ATPase α1 and β1 subunits were detected as precisely co-localized with adherens junctions in all stages of the monolayer formation starting from the initiation of cell-cell contact. The Na,K-ATPase and adherens junction protein, β-catenin, stayed partially co-localized even after their internalization upon disruption of intercellular contacts by Ca2+ depletion of the medium. The Na,K-ATPase subunits remained co-localized with the adherens junctions after detergent treatment of the cells. In contrast, the heterodimer formed by expressed unglycosylated Na,K-ATPase β1 subunit and the endogenous α1 subunit was easily dissociated from the adherens junctions and cytoskeleton by the detergent extraction. The MDCK cell line in which half of the endogenous β1 subunits in the lateral membrane were substituted by unglycosylated β1 subunits displayed a decreased ability to form cell-to-cell contacts. Incubation of surface-attached MDCK cells with an antibody against the extracellular domain of the Na,K-ATPase β1 subunit specifically inhibited cell-cell contact formation. We conclude that the Na,K-ATPase β1 subunit is involved in the process of intercellular adhesion and is necessary for association of the heterodimeric Na,K-ATPase with the adherens junctions. Further, normal glycosylation of the Na,K-ATPase β1 subunit is essential for the stable association of the pump with the adherens junctions and plays an important role in cell-cell contact formation.

Characterization of a novel potassium-competitive acid blocker of the gastric H,K-ATPase, 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine monofumarate (TAK-438).

Inhibition of the gastric H,K-ATPase by the potassium-competitive acid blocker (P-CAB) 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine (TAK-438), is strictly K+-competitive with a Ki of 10 nM at pH 7. In contrast to previous P-CABs, this structure has a point positive charge (pKa 9.06) allowing for greater accumulation in parietal cells compared with previous P-CABs [e.g., (8-benzyloxy-2-methyl-imidazo(1,2-a)pyridin-3-yl)acetonitrile (SCH28080), pKa 5.6]. The dissociation rate of the compound from the isolated ATPase is slower than other P-CABs, with the t1/2 being 7.5 h in 20 mM KCl at pH 7. The stoichiometry of binding of TAK-438 to the H,K-ATPase is 2.2 nmol/mg in the presence of Mg-ATP, vanadate, or MgPi. However, TAK-438 also binds enzyme at 1.3 nmol/mg in the absence of Mg2+. Modeling of the H,K-ATPase to the homologous Na,K-ATPase predicts a close approach and hydrogen bonding between the positively charged N-methylamino group and the negatively charged Glu795 in the K+-binding site in contrast to the planar diffuse positive charge of previous P-CABs. This probably accounts for the slow dissociation and high affinity. The model also predicts hydrogen bonding between the hydroxyl of Tyr799 and the oxygens of the sulfonyl group of TAK-438. A Tyr799Phe mutation resulted in a 3-fold increase of the dissociation rate, showing that this hydrogen bonding also contributes to the slow dissociation rate. Hence, this K+-competitive inhibitor of the gastric H,K-ATPase should provide longer-lasting inhibition of gastric acid secretion compared with previous drugs of this class.

Inverse correlation between the extent of N-glycan branching and intercellular adhesion in epithelia: contribution of the Na,K- ATPase β1 subunit *,**

The majority of cell adhesion molecules are N-glycosylated, but the role of N-glycans in intercellular adhesion in epithelia remains ill-defined. Reducing N-glycan branching of cellular glycoproteins by swainsonine, the inhibitor of N-glycan processing, tightens and stabilizes cell-cell junctions as detected by a 3-fold decrease in the paracellular permeability and a 2-3-fold increase in the resistance of the adherens junction proteins to extraction by non-ionic detergent. In addition, exposure of cells to swainsonine inhibits motility of MDCK cells. Mutagenic removal of N-glycosylation sites from the Na,K-ATPase β1 subunit impairs cell-cell adhesion and decreases the effect of swainsonine on the paracellular permeability of the cell monolayer and also on detergent resistance of adherens junction proteins, indicating that the extent of N-glycan branching of this subunit is important for intercellular adhesion. The N-glycans of the Na,K-ATPase β1 subunit and E-cadherin are less complex in tight renal epithelia than in the leakier intestinal epithelium. The complexity of the N-glycans linked to these proteins gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell-cell adhesion-induced increase in expression of GnT-III (stops N-glycan branching) and a decrease in expression of GnTs IVC and V (promote N-glycan branching) as detected by real-time quantitative PCR. Consistent with these results, partial silencing of the gene encoding GnT-III increases branching of N-glycans linked to the Na,K-ATPase β1 subunit and other glycoproteins and results in a 2-fold increase in the paracellular permeability of MDCK cell monolayers. These results suggest epithelial cells can regulate tightness of cell junctions via remodeling of N-glycans, including those linked to the Na,K-ATPase β1-subunit.

The control of gastric acid and Helicobacter pylori eradication

This review focuses on the gastric acid pump as a therapeutic target for the control of acid secretion in peptic ulcer and gastro‐oesophageal reflux disease. The mechanism of the proton pump inhibitors is discussed as well as their clinical use. The biology of Helicobacter pylori as a gastric denizen is then discussed, with special regard to its mechanisms of acid resistance. Here the properties of the products of the urease gene clusters, ureA, B and ureI, E, F, G and H are explored in order to explain the unique location of this pathogen. The dominant requirement for acid resistance is the presence of a proton gated urea transporter, UreI, which increases access of gastric juice urea to the intrabacterial urease 300‐fold. This enables rapid and continuous buffering of the bacterial periplasm to ≈ pH 6.0, allowing acid resistance and growth at acidic pH in the presence of 1 m M urea. A hypothesis for the basis of combination therapy for eradication is also presented.

The Na-K-ATPase α1β1 heterodimer as a cell adhesion molecule in epithelia

The ion gradients generated by the Na-K-ATPase play a critical role in epithelia by driving transepithelial transport of various solutes. The efficiency of this Na-K-ATPase-driven vectorial transport depends on the integrity of epithelial junctions that maintain polar distribution of membrane transporters, including the basolateral sodium pump, and restrict paracellular diffusion of solutes. The review summarizes the data showing that, in addition to pumping ions, the Na-K-ATPase located at the sites of cell-cell junction acts as a cell adhesion molecule by interacting with the Na-K-ATPase of the adjacent cell in the intercellular space accompanied by anchoring to the cytoskeleton in the cytoplasm. The review also discusses the experimental evidence on the importance of a specific amino acid region in the extracellular domain of the Na-K-ATPase β(1) subunit for the Na-K-ATPase trans-dimerization and intercellular adhesion. Furthermore, a possible role of N-glycans linked to the Na-K-ATPase β(1) subunit in regulation of epithelial junctions by modulating β(1)-β(1) interactions is discussed.

Recombinant addition of N-glycosylation sites to the basolateral Na,K-ATPase β1 subunit results in its clustering in caveolae and apical sorting in HGT-1 cells

In most polarized cells, the Na,K-ATPase is localized on the basolateral plasma membrane. However, an unusual location of the Na,K-ATPase was detected in polarized HGT-1 cells (a human gastric adenocarcinoma cell line). The Na,K-ATPase α1 subunit was detected along with the β2 subunit predominantly on the apical membrane, whereas the Na,K-ATPase β1 subunit was not found in HGT-1 cells. However, when expressed in the same cell line, a yellow fluorescent protein-linked Na,K-ATPase β1 subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous α1 subunit to the basolateral membrane. The human β2 subunit has eight N-glycosylation sites, whereas the β1 isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the β2 subunit were successively introduced in the β1 subunit by site-directed mutagenesis. The mutated β1 subunits were detected on both apical and basolateral membranes. The fraction of a mutant β1 subunit present on the apical membrane increased in proportion to the number of glycosylation sites inserted and reached 80% of the total surface amount for the β1 mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous β2 subunit and the β1 mutant with additional glycosylation sites but not for the wild type β1 subunit. Hence, the N-glycans linked to the β2 subunit of the Na,K-ATPase contain apical sorting information, and the high abundance of the β2 subunit isoform, which is rich in N-glycans, along with the absence of the β1 subunit, is responsible for the unusual apical location of the Na,K-ATPase in HGT-1 cells.

Molecular mechanisms in therapy of acid-related diseases

Abstract.Inhibition of gastric acid secretion is the mainstay of the treatment of gastroesophageal reflux disease and peptic ulceration; therapies to inhibit acid are among the best-selling drugs worldwide. Highly effective agents targeting the histamine H2 receptor were first identified in the 1970s. These were followed by the development of irreversible inhibitors of the parietal cell hydrogen-potassium ATPase (the proton pump inhibitors) that inhibit acid secretion much more effectively. Reviewed here are the chemistry, biological targets and pharmacology of these drugs, with reference to their current and evolving clinical utilities. Future directions in the development of acid inhibitory drugs include modifications of current agents and the emergence of a novel class of agents, the acid pump antagonists.

Subunit isoform selectivity in assembly of Na,K-ATPase α-β heterodimers

Background: The Na,K-ATPase consists of one α (four isoforms) and one β (three isoforms) subunits. Results: The α1 preferentially assembles with β1, whereas α2 preferentially binds to β2 isoform. Conclusion: Assembly of α-β complexes is isoform-selective. Significance: This selectivity is crucial for cell- and tissue-specific functions of the Na,K-ATPase. To catalyze ion transport, the Na,K-ATPase must contain one α and one β subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three β subunit isoforms and form an active enzyme, suggesting the absence of selective α-β isoform assembly. However, it is unknown whether in vivo conditions the α-β assembly is random or isoform-specific. The α2-β2 complex was selectively immunoprecipitated by both anti-α2 and anti-β2 antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α1-β2 nor α2-β1 complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α1, β1, and β2 isoforms, a greater fraction of the β2 subunits was unassembled with α1 as compared with that of the β1 subunits, indicating preferential association of the α1 isoform with the β1 isoform. In addition, the α1-β2 complex was less resistant to various detergents than the α1-β1 complex isolated from MDCK cells or the α2-β2 complex isolated from mouse brain. Therefore, the diversity of the α-β Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and β subunit isoforms.