Antibodies That Detect O-Linked β-d-N-Acetylglucosamine on the Extracellular Domain of Cell Surface Glycoproteins

The transfer of N -acetylglucosamine (GlcNAc) to Ser or Thr in cytoplasmic and nuclear proteins is a well known post-translational modification that is catalyzed by the O- GlcNAc transferase OGT. A more recently identified O- GlcNAc transferase, EOGT, functions in the secretory pathway and transfers O- GlcNAc to proteins with epidermal growth factor-like (EGF) repeats. A number of antibodies that detect O- GlcNAc in cytosolic and nuclear extracts have been described previously. Here we compare seven of these antibodies (CTD110.6, 10D8, RL2, HGAC85, 18B10.C7(#3), 9D1.E4(#10), and 1F5.D6 (#14) for detection of the O- GlcNAc modification on extracellular domains of membrane or secreted glycoproteins that may also carry various N- and O -glycans. We found that CTD110.6 binds not only to O- GlcNAc on proteins but also to terminal β-GlcNAc on the complex N- glycans of Lec8 Chinese hamster ovary (CHO) cells that lack UDP-Gal transporter activity and express GlcNAc-terminating, complex N- glycans. We show that CTD110.6, #3, and #10 antibodies can be used to detect cell surface glycoproteins bearing O- GlcNAc. Cell surface glycoproteins recognized by CTD110.6 antibody included NOTCH1 that possesses many EGF repeats with a consensus site for EOGT. Knockdown of CHO Eogt reduced binding of CTD110.6 to Lec1 CHO cells, and expression of a human EOGT cDNA increased the O- GlcNAc signal on Lec1 cells and the extracellular domain of NOTCH1. Thus, with careful controls, antibodies CTD110.6 (IgM), #3 (IgG), and #10 (IgG) can be used to detect membrane and secreted proteins modified by O- GlcNAc on EGF repeats. Background: Seven antibodies that detect cytosolic and nuclear O- GlcNAc were examined for recognition of O- GlcNAc on cell surface glycoproteins. Results: Three antibodies recognized cell surface O- GlcNAc and also bound terminal β-GlcNAc on N- glycans. Conclusion: With careful controls, cell surface O- GlcNAc can be detected using antibodies CTD110.6, 18B10.C7, and 9D1.E4. Significance: Antibodies to monitor cell surface O- GlcNAcylated proteins were characterized.