Figure 1: G6PD is dynamically modified by O-GlcNAc at serine 84.

(a) Chemoenzymatic labelling approach for biotinylation, capture and detection of O-GlcNAcylated G6PD from cells. Endogenous O-GlcNAcylated proteins in cell lysates were chemoenzymatically tagged with an azido-galactose sugar using a mutant galactosyltransferase (GalT, Y289L) and the non-natural nucleotide sugar analogue UDP-GalNAz, and then biotinylated by reaction of the azido-galactose sugar with an alkyne-functionalized biotin molecule. The biotinylated proteins were pulled down using streptavidin beads, and eluted with SDS. Lysates before pull down (input) and the captured proteins (elution) were immunoblotted with an antibody towards G6PD. (b) Detection of O-GlcNAcylated G6PD levels from 293T cells. Lysates prior to pull down (input) and the captured proteins (elution) were immunoblotted with an antibody towards G6PD. Control experiments in the absence of GalT or UDP-GalNAz demonstrated selective labelling of the O-GlcNAcylation on G6PD. (c) Detection of O-GlcNAcylated G6PD levels from 293T cells overexpressing OGT using the chemo-enzymatic method. (d) Peptide sequence and glycosylation site identified by LTQ-Orbitrap MS/MS. Glycosylation levels of WT G6PD compared with the S84V mutant expressed in 293T cells as determined by the chemoenzymatic method. (e) Determination of O-GlcNAcylation levels of G6PD under hypoxic treatment for indicated periods of time by the chemoenzymatic method. (f) Detection of O-GlcNAcylation levels of G6PD cultured in media with different glucose concentrations by the chemo-enzymatic method. (g) Detection of O-GlcNAcylation levels of G6PD in A549 cells on serum stimulation by the chemoenzymatic method.