title: O-GlcNAcylation of the human epidermal growth factor receptor.
abstract: The reversible O-linked attachment of single β-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence suggesting that the epidermal growth factor receptor (EGFR) is subjected to O-GlcNAcylation in human carcinoma epidermoid A431 cells and human lung carcinoma A549 cells. However, no signal was detected in human cervix adenocarcinoma HeLa cells or in mouse EGFR-T17 fibroblasts ectopically expressing the human EGFR. We detected a positive O-GlcNAcylation signal in the immunoprecipitated EGFR by Western blotting using two distinct specific anti-O-GlcNAc antibodies even after N-deglycosylation of the receptor using peptide-N-glycosidase F (PNGase F). Conversely, the presence of EGFR was detected by Western blotting using an anti-EGFR antibody in the immunocomplex of O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when the O-linked β-N-acetylglucosaminidase (OGA) inhibitor Thiamet G was added to prevent the deglycosylation of the GlcNAc moiety(ies). Moreover, we also detected a positive signal in the immunoprecipitated and N-deglycosylated EGFR using PNGase F, and tunicamycin when the cells were metabolically labeled with azido-GlcNAc (GlcNAz), biotinylated and probed with a streptavidin-labeled peroxidase. Finally, EGFR and O-linked β-N-acetylglucosamine transferase (OGT) co-immunoprecipitate, and incubation of the immunoprecipitated EGFR with the immunoprecipitated OGT in the presence of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) resulted in a significant enhancement of the EGFR O-GlcNAcylation signal as detected by Western blotting using an anti-O-GlcNAc antibody. We conclude that the human EGFR is subjected to O-GlcNAcylation in the A431 and A549 tumor cell lines.
authors.affiliation: Department of Cancer Biology, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/Arturo Duperier 4, E-28029 Madrid, Spain. antonio.villalobo@iib.uam.es.
authors.forename: Silviya R
authors.initials: SR
authors.lastname: Stateva
authors.forename: Antonio
authors.initials: A
authors.lastname: Villalobo
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D013194:Staining and Labeling
chemical_list: D004791:Enzyme Inhibitors; C512478:EGFR protein, human; D066246:ErbB Receptors; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine