Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine.
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-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-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.
Comer
F I
FI
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Vosseller
K
K
Wells
L
L
Accavitti
M A
MA
Hart
G W
GW
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D002457:Cell Extracts; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007075:Immunoglobulin M / Q000276:immunology; D019169:Jurkat Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D007075:Immunoglobulin M; D013912:Threonine; D012694:Serine; D000117:Acetylglucosamine