Jump to main content
Jump to site search

Issue 30, 2015
Previous Article Next Article

O-GlcNAcylation of the human epidermal growth factor receptor

Author affiliations

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.

Graphical abstract: O-GlcNAcylation of the human epidermal growth factor receptor

Back to tab navigation

Publication details

The article was received on 05 Mar 2015, accepted on 09 Jun 2015 and first published on 09 Jun 2015


Article type: Paper
DOI: 10.1039/C5OB00443H
Author version
available:
Download author version (PDF)
Org. Biomol. Chem., 2015,13, 8196-8204

  •   Request permissions

    O-GlcNAcylation of the human epidermal growth factor receptor

    S. R. Stateva and A. Villalobo, Org. Biomol. Chem., 2015, 13, 8196
    DOI: 10.1039/C5OB00443H

Search articles by author

Spotlight

Advertisements