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

Dennis L Y Dong, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

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-β-D-glucosaminidase (O-GlcNA-case) 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 Mr = 54,000 (α subunit) and Mr = 51,000 (β subunit). Enzyme activity sediments at Mr = 106,000 on sucrose gradients, indicating that the native O-GlcNAcase is an αβ heterodimer. The O-GlcNA-case 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.

Original languageEnglish (US)
Pages (from-to)19321-19330
Number of pages10
JournalJournal of Biological Chemistry
Volume269
Issue number30
StatePublished - Jul 29 1994

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Hexosaminidases
Cytosol
Purification
Rats
Spleen
Glycopeptides
Bearings (structural)
Enzymes
Glycosylation
Acetylglucosamine
Monosaccharides
Glycoside Hydrolases
Enzyme activity
Post Translational Protein Processing
Fractionation
Nuclear Proteins
Sugars
Sucrose
Glycoproteins
Sediments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of an O-GlcNAc selective N-acetyl-β-D-glucosaminidase from rat spleen cytosol. / Dong, Dennis L Y; Hart, Gerald Warren.

In: Journal of Biological Chemistry, Vol. 269, No. 30, 29.07.1994, p. 19321-19330.

Research output: Contribution to journalArticle

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