期刊
ANALYTICAL BIOCHEMISTRY
卷 293, 期 2, 页码 169-177出版社
ACADEMIC PRESS INC
DOI: 10.1006/abio.2001.5132
关键词
O-GlcNAc; monoclonal antibody; O-glycosylation; detection; O-linked N-acetylglucosamine; CTD110.6
资金
- NCI NIH HHS [CA 42486, CA 83261] Funding Source: Medline
- NIAMS NIH HHS [AR20614] Funding Source: Medline
- NICHD NIH HHS [HD 13563] Funding Source: Medline
- NIGMS NIH HHS [GM 20528] Funding Source: Medline
beta -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-GacNAc in beta -O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha -linked Ser/Thr-O-GrlcNAc, cu-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 perm 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. (C) 2001 Academic Press.
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