AcGlcNAcF, an OGT-tolerated but OGA-resistant regulator for O-GlcNAcylation.

O-Linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationalmonosaccaride-modification found on Ser or Thr residues of intracellular proteins in most eukaryotes. The dynamic nature of O-GlcNAc has enabled researchers to modulate the stoichiometry of O-GlcNAc on proteins in order to investigate its function. Cell permeable small moleculars have proven invaluable tools to increase O-GlcNAc levels. Herein, using in vitro substrate screening, we identified GlcNAcF as an OGT-accepted but OGA-resistant sugar mimic. Cellular experiments with cell-permeable peracetylated-GlcNAcF (AcGlcNAcF) displayed that AcGlcNAcF was a potent tool to increase O-GlcNAc levels in several cell lines. Further, NIH3T3 cells interfered with OGT (siOGT) showed significant decreasing of O-GlcNAc levels with AcGlcNAcF treatment, indicating O-GlcNAcF was an OGT-dependent modification. In addition, cellular toxic assay confirmed O-GlcNAcF production has no significant effect on cell proliferation or viability. Thus, AcGlcNAcF represents a safe and dual regulator for both OGT and OGA, which will benefit the study of O-GlcNAc.