O-GlcNAcylation: New Tools to Investigate this Important Post-Translational Modification

O-GlcNAcylation: New Tools to Investigate this Important Post-Translational Modification

O-GlcNAcylation is a post-translational modification consisting of the addition of a single N-acetyl-glucosamine residue (GlcNAc) to specific serine/threonine residues of proteins. The addition of GlcNAc has been compared to phosphorylation, and there may be significant interplay between the two processes (1). Like phosphorylation, O-GlcNAcylation has putative involvement in a range of cellular activities, including the stress response, transcription, translation, cell signaling, and cell cycle regulation (2). Changes in O-GlcNAc modification have also been implicated in a host of diseases such as diabetes, Alzheimer’s disease, and cancer (3, 4). Despite potential roles in these important areas of biology, a lack of tools has hampered the understanding of O-GlcNAcylation and the advancement of therapies that might target this process.

O-GlcNAcylation is regulated by a discrete set of enzymes. It was originally thought to exist solely on nuclear and cytosolic proteins catalyzed by a single O-GlcNAc transferase (OGT) (5). However, O-GlcNAcylation has recently been discovered on extracellular proteins, where O-GlcNAc addition is catalyzed by extracellular OGT (EOGT) (6). O-GlcNAc is removed by a single O-GlcNAcase (OGA) (7).

Recombinant OGT (New), Recombinant EOGT (New), and Recombinant OGA are part of our extensive portfolio of glycobiology reagents. Our offering also includes a line of ancillary reagents that can be utilized for labeling and detection of O-GlcNAc modification (Table 1). Figure 1 demonstrates the results of an assay used for the detection of OGT-specific targets.

Figure 1. Assessing O-GlcNAc labeling in Cell Extracts.
Nuclear (NE) and cytoplasmic extracts (CE) of HEK-293 human embryonic kidney cells were labeled with 35S using the GlcNAC-specific sulfotransferase, Recombinant Human CHST2, and resolved on 12% SDS-PAGE under reducing conditions. PNGase F treatment removes N-Glycans, and when combined with Recombinant Human O-GlcNAcase (OGA), most labeling is eliminated. In contrast, treatment with Recombinant Human O-GlcNAc Transferase (OGT) augments the labeling.
More details and additional related experiments can be found in our recent publication in Glycobiology:
Wu, Z.L. et al. (2014) Detecting O-GlcNAc Using In Vitro Sulfation. Glycobiology 24:740.
Table 1.
Reagents for O-GlcNAc detection
Product Description
O-GlcNAc Transferase (OGT) Catalyzes O-GlcNAc addition
Extracellular OGT (EOGT) Catalyzes O-GlcNAc addition
O-GlcNAcase (OGA) Catalyzes O-GlcNAc removal
Carbohydrate Sulfotransferase 2 Transfers sulfate to GlcNAc
Carbohydrate Sulfotransferase 4 Transfers sulfate to GlcNAc
PAPS Synthesis Kit Substrate for enzymatic labeling of O-GlcNAc
Notch EGF20 EOGT substrate
PNGase F Enzyme for removing N-glycans
Thiamet G       Tocris Product Potent O-GlcNAcase inhibitor
(Z)-Pugnac       Tocris Product O-GlcNAcase and O-GlcNAcase/beta-hexosaminidase inhibitor


  1. Hart, G.W. et al. (2011) Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. Ann. Rev. Biochem. 80:825.
  2. Hart, G.W. et al. (2007) Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446:1017.
  3. Ma, J. and G.W. Hart (2013) Protein O-GlcNAcylation in diabetes and diabetic complications. Expert Rev. Proteom. 10:365.
  4. Pearson, J.S. et al. (2013) A type III effector antagonizes death receptor signalling during bacterial gut infection. Nature 501:247.
  5. Comer, F.I. and G.W. Hart (2001) Reciprocity between O-GlcNAc and O-phosphate on the carboxyl terminal domain of RNA polymerase II. Biochemistry 40:7845.
  6. Love, D.C. and J.A. Hanover (2005) The hexosamine signaling pathway: deciphering the "O-GlcNAc code". Science's STKE: signal transduction knowledge environment. 2005:re13.
  7. Gao, Y. et al. (2001) Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain. J. Biol. Chem. 276:9838.