Characterization of a Mouse Monoclonal Antibody Specific for O-Linked N-Acetylglucosamine

✍ Scribed by Frank I. Comer; Keith Vosseller; Lance Wells; Mary Ann Accavitti; Gerald W. Hart

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 326 KB
• Volume: 293
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5132

✦ Synopsis

␤-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in ␤-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with ␣-linked Ser/Thr-O-GlcNAc, ␣-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.