page_title: O-GlcNAc
section_title: Chemoenzymatic labeling
hierarchical_section_title: O-GlcNAc / Detection and characterization / Chemoenzymatic labeling
caption_attribution_description: English: Schematic of FRET biosensor for O-GlcNAc
context_page_description: O-GlcNAc is a reversible enzymatic post-translational modification that is found on serine and threonine residues of nucleocytoplasmic proteins. The modification is characterized by a β-glycosidic bond between the hydroxyl group of serine or threonine side chains and N-acetylglucosamine. O-GlcNAc differs from other forms of protein glycosylation: O-GlcNAc is not elongated or modified to form more complex glycan structures, O-GlcNAc is almost exclusively found on nuclear and cytoplasmic proteins rather than membrane proteins and secretory proteins, and O-GlcNAc is a highly dynamic modification that turns over more rapidly than the proteins which it modifies. O-GlcNAc is conserved across metazoans.
  Due to the dynamic nature of O-GlcNAc and its presence on serine and threonine residues, O-GlcNAcylation is similar to protein phosphorylation in some respects. While there are roughly 500 kinases and 150 phosphatases that regulate protein phosphorylation in humans, there are only 2 enzymes that regulate the cycling of O-GlcNAc: O-GlcNAc transferase and O-GlcNAcase catalyze the addition and removal of O-GlcNAc, respectively. OGT utilizes UDP-GlcNAc as the donor sugar for sugar transfer.
context_section_description: Chemoenzymatic labeling provides an alternative strategy to incorporate handles for click chemistry. The Click-IT O-GlcNAc Enzymatic Labeling System, developed by the Hsieh-Wilson group and subsequently commercialized by Invitrogen, utilizes a mutant GalT Y289L enzyme that is able to transfer azidogalactose (GalNAz) onto O-GlcNAc. The presence of GalNAz (and therefore also O-GlcNAc) can be detected with various alkyne-containing probes with identifiable tags such as biotin, dye molecules, and PEG.
caption: FRET biosensor for O-GlcNAc. Under high O-GlcNAc conditions, GafD will bind the O-GlcNAc group on the CKII peptide substrate, bringing CFP and YFP into proximity for FRET. Various localization sequences can be fused to localize the sensor to various cellular compartments, e.g., nucleus, cytoplasm, and plasma membrane.
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