O-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 potential target for O-GlcNAcylation, suggesting a possibly direct connection between the metabolic O-GlcNAc pathway and the hypoxia pathway.
Bibliographical noteFunding Information:
We thank Hai-Bin Ruan for the critical reading of the manuscript. This work is supported by R&D Systems, a Bio-Techne brand, and by National Institute of Health (R35GM124896 to Y.C.).
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