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J Biol Chem. 2014 Apr 18;289(16):11132-42. doi: 10.1074/jbc.M113.492512. Epub 2014 Feb 26.

Antibodies that detect O-linked β-D-N-acetylglucosamine on the extracellular domain of cell surface glycoproteins.

Author information

1
From the Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461.

Abstract

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.

KEYWORDS:

Antibodies; CHO; Flow Cytometry; GlcNAc-terminating N-Glycans; Lec1; Lec8; Mutant; O-GlcNAc; O-GlcNAcylation

PMID:
24573683
PMCID:
PMC4036252
DOI:
10.1074/jbc.M113.492512
[Indexed for MEDLINE]
Free PMC Article

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