Integrating GlycoSHIELD Modeling and DNA-PAINT SMLM to Map the Glycosylation-Dependent Distribution of the Na,K-ATPase

The cell surface localization of the Na,K-ATPase (sodium pump) is required for maintaining transmembrane electrochemical gradients. While glycosylation of the β1 subunit facilitates trafficking from the endoplasmic reticulum to the plasma membrane, its role in nanoscale surface organization is not characterized. This study employed GlycoSHIELD computational modeling and DNA-PAINT single-molecule localization microscopy (SMLM) to evaluate how N-glycans influence pump distribution. In-silico simulations indicated that N-glycans sequester the protein core, providing a steric shield that increases with structural complexity. To investigate this experimentally, glycosylation-deficient mutants (3NQ) were generated and confirmed via immunoblotting. Quantitative SMLM analysis of A498 cells demonstrated that wild-type pumps exhibit higher localization density and form larger (144 nm) and more frequent clusters than 3NQ mutants (109 nm). These results indicate that N-glycosylation promotes stable enzyme clustering, supporting a galectin-lattice mechanism of organization rather than steric repulsion. Generation of glycosylation-deficient Na,K-ATPase β1 mutants combined with DNA-PAINT single-molecule localization microscopy reveals that N-glycosylation promotes larger and more frequent plasma membrane nanoclusters, supporting a galectin-mediated mechanism of pump organization.