Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol.
Glycosylation of nuclear and cytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is an abundant, ubiquitous, and transient post-translational modification. To characterize enzymes involved in removal of these sugars, a neutral and cytoplasmic N-acetyl-beta-D-glucosaminidase (O-GlcNAcase) with strong selectivity for O-GlcNAc-synthetic glycopeptides has been purified over 22,000-fold from rat spleen homogenate. The purified O-GlcNAcase has two major polypeptides of apparent M(r) = 54,000 (alpha subunit) and M(r) = 51,000 (beta subunit). Enzyme activity sediments at M(r) = 106,000 on sucrose gradients, indicating that the native O-GlcNAcase is an alpha beta heterodimer. The O-GlcNAcase also shows substantially stronger relative activity against O-GlcNAc-synthetic glycopeptides than other hexosaminidases. Unlike acidic lysosomal hexosaminidases, O-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 O-GlcNAcase is involved in the regulated removal of O-GlcNAc from O-GlcNAc-bearing glycoproteins in the nucleoplasmic and cytoplasmic compartments of cells.
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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D000118:Acetylglucosaminidase / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D013154:Spleen / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases