Sweet and Bright: Illuminating Glycoprotein-Mediated Endocytosis via Metabolic Labeling and NanoLuciferase

Glycoprotein-mediated endocytosis is a critical pathway for the cell entry of biomolecules, pathogens, and delivery vectors. Despite this, glycans are among the most analytically challenging biomolecules due to their structural complexity and dynamic behavior. Here, we report a sensitive, membrane-specific, mix-and-read bioluminescent assay to monitor cell surface glycan dynamics during endocytosis. By combining metabolic labeling and bioorthogonal chemistry with split nanoluciferase, a bright luminescent enzyme comprising two complementary peptides, HiBiT and LgBiT, we were able to differentiate between the glycan uptake of four distinct cell-penetrating peptides (CPPs) reported to have varying degrees of glycan engagement. This proof-of-concept approach provides a basis for a broader understanding of glycan dynamics during endocytosis and may support the discovery of new ligands that exploit this pathway for cellular entry.