Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine

Frank I. Comer, Keith Vosseller, Lance Wells, Mary Ann Accavitti, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

β-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 β-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with α-linked Ser/Thr-O-GlcNAc, α-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.

Original languageEnglish (US)
Pages (from-to)169-177
Number of pages9
JournalAnalytical Biochemistry
Volume293
Issue number2
DOIs
StatePublished - Jun 15 2001

Keywords

  • CTD110.6
  • Detection
  • Monoclonal antibody
  • O-GlcNAc
  • O-glycosylation
  • O-linked N-acetylglucosamine

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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  • Cite this

    Comer, F. I., Vosseller, K., Wells, L., Accavitti, M. A., & Hart, G. W. (2001). Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Analytical Biochemistry, 293(2), 169-177. https://doi.org/10.1006/abio.2001.5132