Dynamic O-glycosylation of nuclear and cytosolic proteins

Cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain

Yuan Gao, Lance Wells, Frank I. Comer, Glendon J. Parker, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

Dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) on Ser/Thr residues is ubiquitous in higher eukaryotes and is analogous to protein phosphorylation. The enzyme for the addition of this modification, O-GlcNAc transferase, has been cloned from several species. Here, we have cloned a human brain O-GlcNAcase that cleaves O-GlcNAc off proteins. The cloned cDNA encodes a polypeptide of 916 amino acids with a predicted molecular mass of 103 kDa and a pI value of 4.63, but the protein migrates as a 130-kDa band on SDS-polyacrylamide gel electrophoresis. The cloned O-GlcNAcase has a pH optimum of 5.5-7.0 and is inhibited by GlcNAc but not by GalNAc. p-Nitrophenyl (pNP)-β-GlcNAc, but not pNP-β-GalNAc or pNP-α-GlcNAc, is a substrate. The cloned enzyme cleaves GlcNAc, but not GalNAc, from glycopeptides. Cell fractionation suggests that the overexpressed protein is mostly localized in the cytoplasm. It therefore has all the expected characteristics of O-GlcNAcase and is distinct from lysosomal hexosaminidases. Northern blots show that the transcript is expressed in every human tissue examined but is the highest in the brain, placenta, and pancreas. An understanding of O-GlcNAc dynamics and O-GlcNAcase may be key to elucidating the relationships between O-phosphate and O-GlcNAc and to the understanding of the molecular mechanisms of diseases such as diabetes, cancer, and neurodegeneration.

Original languageEnglish (US)
Pages (from-to)9838-9845
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number13
DOIs
StatePublished - Mar 30 2001

Fingerprint

Glycosylation
Acetylglucosaminidase
Cloning
Nuclear Proteins
Organism Cloning
Brain
Proteins
Cell Fractionation
Hexosaminidases
Phosphorylation
Acetylglucosamine
Glycopeptides
Molecular mass
Enzymes
Fractionation
Medical problems
Electrophoresis
Eukaryota
Northern Blotting
Placenta

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dynamic O-glycosylation of nuclear and cytosolic proteins : Cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain. / Gao, Yuan; Wells, Lance; Comer, Frank I.; Parker, Glendon J.; Hart, Gerald Warren.

In: Journal of Biological Chemistry, Vol. 276, No. 13, 30.03.2001, p. 9838-9845.

Research output: Contribution to journalArticle

Gao, Yuan ; Wells, Lance ; Comer, Frank I. ; Parker, Glendon J. ; Hart, Gerald Warren. / Dynamic O-glycosylation of nuclear and cytosolic proteins : Cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 13. pp. 9838-9845.
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