Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling.
Zhengliang L WuAng LuoAlex GrillTaotao LaoYonglong ZouYue ChenPublished in: Bioconjugate chemistry (2020)
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.
Keyphrases
- label free
- highly efficient
- loop mediated isothermal amplification
- real time pcr
- living cells
- protein kinase
- quantum dots
- endothelial cells
- fluorescent probe
- oxidative stress
- cell proliferation
- signaling pathway
- transcription factor
- photodynamic therapy
- cell cycle arrest
- cell death
- sensitive detection
- liquid chromatography