Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol.

Abstract

Glycosylation of nuclear and cytoplasmic proteins by 0-linked N-acetylglucosamine (0-GlcNAc) monosaccharides is an abundant, ubiquitous, and transient posttranslational modification.To characterize enzymes involved in removal of these sugars, a neutral and cytoplasmic N-acetyl-p-D-glucosaminidase (0-GlcNAcase) with strong selectivity for 0-GlcNAc-synthetic glycopeptides has been purified over 22,000-fold from rat spleen homogenate.The purified 0-GlcNAcase has two major polypeptides of apparent M, = 54,000 (cy subunit) and M, = 51,000 ( p subunit).Enzyme activity sediments at M, = 106,000 on sucrose gradients, indicating that the native 0-GlcNAcase is an cup heterodimer.The O-GlcNAcase also shows substantially stronger relative activity against 0-GlcNAc-synthetic glycopeptides than other hexosaminidases.Unlike acidic lysosomal hexosaminidases, 0-GlcNAcase is not inhibited by GalNAc or its analogs, has no other detectable glycosidase activities, and does not cross-react with antibodies against acidic hexosaminidases.Subcellular fractionation and latency studies demonstrate the cytoplasmic and nucleoplasmic localization of the enzyme and its ubiquitous presence in tissues.These studies suggest that 0-GlcNAcase is involved in the regulated removal of 0-GlcNAc from 0- GlcNAc-bearing glycoproteins in the nucleoplasmic and cytoplasmic compartments of cells.An abundant type of intracellular protein glycosylation, in which single N-acetylglucosamine monosaccharides are O-glycosidically (0-GlcNAc)' linked to serine or threonine residues of proteins has been found exclusively in the nucleus and cytoplasm of eukaryotic cells (1, 2).0-GlcNAc has been described on many proteins in viruses, yeast, trypanosomes, frogs, ro- dents, plants, and humans, for reviews, see Refs.3-5.More than 50 0-GlcNAc-bearing proteins have thus far been identi-Health Grant R01 HD13563.The costs of publication of this article were