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.