title: 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 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 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 β4Gal-T1 enzyme altered its metal-ion specificity in such a way that the M344H-β4Gal-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-β4Gal-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.
authors.affiliation: Structural Glycobiology Section, CCR-Nanobiology Program, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: Mercer
authors.forename: Boopathy
authors.initials: B
authors.lastname: Ramakrishnan
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Boeggeman
authors.forename: Luke
authors.initials: L
authors.lastname: Verdi
authors.forename: Pradman K
authors.initials: PK
authors.lastname: Qasba
mesh_terms: D000117:Acetylglucosamine / Q000032:analysis* / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002417:Cattle; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D017354:Point Mutation; D015202:Protein Engineering
chemical_list: D006001:Glycoconjugates; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I; D008274:Magnesium; D000117:Acetylglucosamine