Fig. 2.

O-GlcNAcylation of AHCY on the residue T136 promotes its enzyme activity. (A) Analysis of AHCY O-GlcNAcylation in cells using a chemoenzymatic labeling method. O-GlcNAcylated proteins in E14.1 cell lysates were first labeled with an azido-N-acetylgalactosamine (GalNAz) sugar. Labeled proteins were then conjugated with biotin via Cu(I)-mediated [3 + 2] azide-alkyne cycloaddition chemistry and further captured with streptavidin-agarose beads. After stringent washing, the eluate was immunoblotted using an antibody against AHCY. The corresponding biotin blot was shown. (B) Analysis of AHCY O-GlcNAcylation upon OGT overexpression. The corresponding biotin blot was shown. (C) Analysis of glycosylation stoichiometry of AHCY by conjugation with alkyne-PEG5k. (D) Tandem mass spectrum of an O-GlcNAcylated peptide on AHCY. (E) Probing the major site of glycosylation on AHCY using various site-directed mutants. (F) Comparison of amino acid sequences containing the glycosylation site among different species. (G) Enzymatic activity of AHCY WT and T136A mutant in the presence or absence of OGT overexpression (n = 4 assays). (H) Analysis of the oligomerization state of AHCY WT or T136A mutant in the presence or absence of TMG treatment using disuccinimidyl suberate (DSS) for protein cross-linking. Error bars denote the means ± SEM. Statistical analyses were performed by unpaired Student’s t test (*P < 0.05, **P < 0.01).