Spatiotemporal Proximity Labeling Tools to Track GlcNAc Sugar-Modified Functional Protein Hubs during Cellular Signaling.

A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked N-acetylglucosamine (O-GlcNAc) is rapidly added to and removed from diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects remain challenging for tracking functional O-GlcNAc events with chemical strategies: spatial control over subcellular locations and time control during labeling. The objective of this study was to create intracellular proximity labeling tools to identify functional changes in O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on the TurboID proximity labeling system for rapid protein biotin conjugation directed to O-GlcNAc protein modifications inside cells, a set of tools called "GlycoID." Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, labeled O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin and serum stimulation revealed functional changes in O-GlcNAc proteins as soon as 30 min following signal initiation. We demonstrated using proteomic analysis that the GlycoID strategy captured O-GlcNAcylated "activity hubs" consisting of O-GlcNAc proteins and their associated protein-protein interactions. The ability to follow changes in O-GlcNAc hubs during physiological events such as insulin signaling allows these tools to determine the mechanisms of glycobiological cell regulation. Our functional O-GlcNAc data sets in human cells will be a valuable resource for O-GlcNAc-driven mechanisms.