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bioRxiv · 10.1101/2020.06.27.175075

Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling

Wu, Z.·Luo, A.·Grill, A.·Lao, T.·Zou, Y.·Chen, Y.
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Abstract

ABSTRACTO-GlcNAcylation is a reversible serine/threonine glycosylation on cytosolic and nuclear proteins that are involved in various regulatory pathways. However, the detection and quantification of O-GlcNAcylation substrates have been challenging. Here we report a highly efficient method for the identification of O-GlcNAc modification via tandem glycan labeling, in which O-GlcNAc is first galactosylated and then sialylated with a fluorophore-conjugated sialic acid residue, therefore enabling highly sensitive fluorescent detection. The method is validated on various proteins that are known to be modified by O-GlcNAcylation including CK2, NOD2, SREBP1c, AKT1, PKM and PFKFB3, and on the nuclear extract of HEK293 cells. Using this method, we then report the evidence that hypoxia-inducible factor HIF1α is a target for O-GlcNAcylation, suggesting a potential direct connection between the metabolic O-GlcNAc pathway and the hypoxia pathway.Competing Interest StatementThe authors have declared no competing interest.View Full Text

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Wu, Z., Luo, A., Grill, A., Lao, T., Zou, Y., Chen, Y.. 2020-06-28. Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling. https://doi.org/10.1101/2020.06.27.175075

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