Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase

An assay for the enzyme responsible for the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins, a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, is reported using the synthetic peptide YSDSPSTST as the acceptor substrate. The activity is linearly dependent on time, enz...

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Bibliographic Details
Published in:The Journal of biological chemistry 1990-02, Vol.265 (5), p.2563-2568
Main Authors: HALTIWANGER, R. S, HOLT, G. D, HART, G. W
Format: Article
Language:English
Subjects:
Acetylglucosamine - metabolism
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Cytosol - enzymology
Enzymes and enzyme inhibitors
Erythrocyte Membrane - enzymology
Fundamental and applied biological sciences. Psychology
Glucosamine - analogs & derivatives
Glucosyltransferases - metabolism
Intracellular Membranes - enzymology
Kinetics
Liver - enzymology
Male
Membrane Proteins - blood
Mice
Microsomes, Liver - enzymology
Molecular Sequence Data
N-Acetylglucosaminyltransferases
Nuclear Proteins - metabolism
Oligopeptides - metabolism
Peptides - chemical synthesis
Peptides - metabolism
Rabbits
Rats
Reticulocytes - metabolism
Substrate Specificity
Transferases
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Summary:An assay for the enzyme responsible for the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins, a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, is reported using the synthetic peptide YSDSPSTST as the acceptor substrate. The activity is linearly dependent on time, enzyme, and substrate concentration. Replacement of the proline with a glycine in the peptide renders it ineffective as a substrate, whereas changing of the aspartic acid to a glycine has no effect. Product characterization of the glycosylated peptide demonstrates that the monosaccharide covalently attached to the peptide is N-acetylglucosamine (GlcNAc) and has not been epimerized to N-acetylgalactosamine. Mild base-catalyzed beta-elimination of the in vitro glycosylated peptide quantitatively yields GlcNAcitol, indicating that the GlcNAc is attached via an O-linkage. The transferase activity is strongly inhibited by UDP but is unaffected by GlcNAc or tunicamycin. Interestingly, EDTA only slightly inhibits activity, suggesting that the enzyme may not require divalent cations. The majority of the activity is soluble, and the remainder is lost from membranes after extracting with high salt and EDTA. Consistent with the subcellular localization of most proteins bearing O-GlcNAc, the activity appears to reside in the cytosolic portion of the cell when compared to two lumenal marker enzymes, galactosyltransferase and mannose-6-phosphatase.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(19)39838-2