Inhibition of endocytosis by glycans arises from steric rather than electrostatic repulsion

Clathrin-mediated endocytosis is an essential cellular process that facilitates nutrient uptake, cell signaling, and recycling of membrane proteins. Many membrane proteins that are internalized by endocytosis are glycosylated. Interestingly, glycosylation of transmembrane receptors has been shown to work against receptor uptake by clathrin-mediated endocytosis. This observation suggests that cells may use glycosylation to tune the residence time of individual transmembrane proteins at the cell surface. However, it is unclear which characteristics of glycans confer the ability to avoid endocytosis. Physically, glycans contribute both steric bulk, owing to glycans extending from the protein, and negative charge, mainly due to the sialic acid groups that frequently terminate these glycans. Here, we sought to determine the relative impact of electrostatic versus steric repulsion during endocytosis of glycoproteins. We employed an artificial ligand-receptor system that allowed us to recruit ligands with a known extent of glycosylation to the cell surface and assess their impact on endocytosis of a model receptor. In this system, the ligand was composed of 10 tandem repeats from the ectodomain of Muc1, a protein known for its dense O-glycosylation, and was prepared with and without sialic acid residues. We found that sialic acid moieties had little effect on either receptor uptake or the hydrodynamic radii of glycosylated ligands. These results suggest that the resistance of O-glycans to endocytosis arises primarily from their steric bulk rather than electrostatic repulsion. This finding has implications for glycoprotein recycling and signaling in both healthy and diseased cells.