Organelle-directed metabolic glycan labeling

Metabolic Glycan Labeling (MGL) is a powerful tool to introduce diverse functional entities on cell surface by bioorthogonal labeling of abiotic sugars expressed on the glycocalyx. Akin to the plasma membrane, the luminal face of the lysosomal membrane is covered with a dense layer of glycans. To date, MGL has been mostly executed to modify cell surfaces. Herein we detail organelle-specific MGL (OMGL) by selective labeling of 9-azidosialic acid (^AzSia) residues on the lysosomal inner membrane while sparing those on the cell surface. OMGL entails: (1) metabolic incorporation of ^AzSia into the global cell glycome; (2) staining of the ^AzSia-expressing cells with dibenzocyclooctyne (DBCO)-bearing lyso-probes, which can promptly accumulate in lysosomes driven by the acidotropic effect; (3) bioorthogonal ligation of lyso-probes enriched in lysosomes with ^AzSia on the inner membrane of lysosomes. Overcoming the liability of conventional chemical probes to dissipation from stressed lysosomes, OMGL enables optical tracking of stressed lysosomes in exocytosis and cell death.