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Use of novel mutant galactosyltransferase for the bioconjugation of terminal N-acetylglucosamine (GlcNAc) residues on live cell surface.


ABSTRACT: On the basis of the crystal structure of bovine ?4Gal-T1 enzyme, mutation of a single amino acid Y289 to L289 (Y289L) changed its donor specificity from Gal to N-acetyl-galactosamine (GalNAc). A chemoenzymatic method that uses GalNAc analogues like GalNAz or 2-keto-Gal as sugar donors with the enzyme Y289L-?4Gal-T1 has identified hundreds of cytosolic and nuclear proteins that have O-GlcNAc modifications. To avoid potential cytotoxicity at Mn(2+) concentrations required to selectively modify GlcNAc residues on the surface of live cells, we have engineered a Mg(2+)-dependent enzyme. Previously, we found that the mutation of the metal-binding residue Met-344 to His-344 in bovine ?4Gal-T1 enzyme altered its metal-ion specificity in such a way that the M344H-?4Gal-T1 enzyme exhibits better catalytic activity with Mg(2+) than with Mn(2+). Here, we find that, when these two mutations are combined, the double mutant, Y289L-M344H-?4Gal-T1, transfers GalNAc and its analogue sugars to the acceptor GlcNAc in the presence of Mg(2+). Using this mutant enzyme, we have detected free GlcNAc residues on the surface glycans of live HeLa cells and platelets. The specific transfer of a synthetic sugar with a chemical handle to the terminal GlcNAc residues on the surface of live cells provides a novel tool for selective modification, detection, and isolation of GlcNAc-ending glycans present on the cellular surface.

SUBMITTER: Mercer N 

PROVIDER: S-EPMC3547369 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Use of novel mutant galactosyltransferase for the bioconjugation of terminal N-acetylglucosamine (GlcNAc) residues on live cell surface.

Mercer Natalia N   Ramakrishnan Boopathy B   Boeggeman Elizabeth E   Verdi Luke L   Qasba Pradman K PK  

Bioconjugate chemistry 20130103 1


On the basis of the crystal structure of bovine β4Gal-T1 enzyme, mutation of a single amino acid Y289 to L289 (Y289L) changed its donor specificity from Gal to N-acetyl-galactosamine (GalNAc). A chemoenzymatic method that uses GalNAc analogues like GalNAz or 2-keto-Gal as sugar donors with the enzyme Y289L-β4Gal-T1 has identified hundreds of cytosolic and nuclear proteins that have O-GlcNAc modifications. To avoid potential cytotoxicity at Mn(2+) concentrations required to selectively modify Glc  ...[more]

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