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

beta -O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslatio nal modification of resident nuclear and cytoplasmic proteins in eukaryotes . Increasing evidence suggests that O-GlcNAc plays a regulatory role in num erous cellular processes. Here we report on the production and characteriza tion of a highly specific mouse monoclonal antibody, MAb CTD110.6, that spe cifically 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-a cetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin an d immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes im munoreactivity, further demonstrating specificity toward O-GlcNAc, Furtherm ore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreact ivity. 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 nuclea r extracts and readily detects in vivo changes in O-GlcNAc modification. Th ese studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, w ith no cross-reactivity toward similar carbohydrate antigens or toward pept ide determinants. (C) 2001 Academic Press.