Open AccessFluorescent Detection of O-GlcNAc via Tandem Glycan Labeling
Author(s) -
Zhengliang L. Wu,
Ang Luo,
Alex E. Grill,
Taotao Lao,
Yufei Zou,
Yue Chen
Publication year - 2020
Publication title -
bioconjugate chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.279
H-Index - 172
eISSN - 1520-4812
pISSN - 1043-1802
DOI - 10.1021/acs.bioconjchem.0c00454
Subject(s) - chemistry , glycosylation , glycan , threonine , fluorescence , biochemistry , serine , tandem , fluorophore , fluorescent labelling , residue (chemistry) , translocase , hek 293 cells , phosphorylation , gene , chromosomal translocation , glycoprotein , physics , materials science , quantum mechanics , composite material
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.