title: Antibodies that detect O-linked β-D-N-acetylglucosamine on the extracellular domain of cell surface glycoproteins.
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.
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Tashima
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461. Electronic address: pamela.stanley@einstein.yu.edu.
authors.forename: Pamela
authors.initials: P
authors.lastname: Stanley
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D006023:Glycoproteins; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine
keywords: Antibodies; CHO; Flow Cytometry; GlcNAc-terminating N-Glycans; Lec1; Lec8; Mutant; O-GlcNAc; O-GlcNAcylation