Epithelial Junctions Depend on Intercellular Trans-interactions between the Na,K-ATPase β1 Subunits

Elmira Tokhtaeva,George Sachs,Puneet Souda,Sara Bassilian,Julian P Whitelegge,Liora Shoshani,Olga Vagin

doi:10.1074/jbc.m111.252247

Abstract

N-Glycans of the Na,K-ATPase β₁ subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the β₁ subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous β₁ subunit with various YFP-linked β₁ subunits expressed in Madin-Darby canine kidney cells was used to assess β₁-β₁ interactions. The amount of co-precipitated endogenous dog β₁ was greater with dog YFP-β₁ than with rat YFP-β₁, showing that amino acid-mediated interactions are important for β₁-β₁ binding. Co-precipitation of β₁ was also less with the unglycosylated YFP-β₁ than with glycosylated YFP-β₁, indicating a role for N-glycans. Mixing cells expressing dog YFP-β₁ with non-transfected cells increased the amount of co-precipitated β₁, confirming the presence of intercellular (YFP-β₁)-β₁ complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated β₁ subunits. The decrease in β₁ co-precipitation both with rat YFP-β₁ and unglycosylated YFP-β₁ was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. Reducing N-glycan branching by specific inhibitors increased (YFP-β₁)-β₁ co-precipitation and strengthened intercellular junctions. Therefore, interactions between the β₁ subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the β₁ subunit N-glycan structure can regulate stability and tightness of intercellular junctions.

Highlights

N-Glycans of the Na,K-ATPase ␤1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the ␤1 subunits of neighboring cells
To determine whether ␤1-␤1 interaction is dependent on the presence of N-glycans or particular amino acid residues and whether this interaction is required for normal cell-cell adhesion, we investigated the effects of removing or modifying N-glycans of rat or dog YFP-linked ␤1 subunits overexpressed in MDCK cells on 1) co-immunoprecipitation of these subunits with the endogenous ␤1 subunits, 2) detergent resistance of adhesion proteins, and 3) permeability of intercellular junctions
The results indicate that N-glycans of the ␤1 subunit and their structure are critical for integrity of intercellular junctions in MDCK cell monolayers due to their stabilizing effect on direct amino acid-mediated interactions between the extracellular domains of the ␤1 subunits of neighboring cells

Summary

Introduction

N-Glycans of the Na,K-ATPase ␤1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the ␤1 subunits of neighboring cells. To determine whether ␤1-␤1 interaction is dependent on the presence of N-glycans or particular amino acid residues and whether this interaction is required for normal cell-cell adhesion, we investigated the effects of removing or modifying N-glycans of rat or dog YFP-linked ␤1 subunits overexpressed in MDCK cells on 1) co-immunoprecipitation of these subunits with the endogenous ␤1 subunits, 2) detergent resistance of adhesion proteins, and 3) permeability of intercellular junctions.

Results

Conclusion