title: Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases.
abstract: The enzymatic oxidation of uridine 5'-diphospho-alpha-D-galactose (UDP-Gal) and uridine 5'-diphospho-N-acetyl-alpha-D-galactosamine (UDP-GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin-epsilon-amidocaproylhydrazide in a one-pot synthesis. The novel nucleotide sugar derivatives uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-alpha-D-galactose (UDP-6-biotinyl-Gal) and uridine 5'-diphospho-6-biotin-epsilon-amidocaproylhydrazino-N-acetyl-alpha-D-galactosamine (UDP-6-biotinyl-GalNAc) were synthesized on a 100-mg scale and characterized by mass spectrometry (fast atom bombardment and matrix-assisted laser desorption/ionization time of flight) and one/two dimensional NMR spectroscopy. It could be demonstrated for the first time, by use of UDP-6-biotinyl-Gal as a donor substrate, that the human recombinant galactosyltransferases beta3Gal-T5, beta4Gal-T1, and beta4Gal-T4 mediate biotinylation of the neoglycoconjugate bovine serum albumin-p-aminophenyl N-acetyl-beta-D-glucosaminide (BSA-(GlcNAc)17) and ovalbumin. The detection of the biotin tag transferred by beta3Gal-T5 onto BSA-(GlcNAc)17 with streptavidin-enzyme conjugates gave detection limits of 150 pmol of tagged GlcNAc in a Western blot analysis and 1 pmol of tagged GlcNAc in a microtiter plate assay. The degree of Gal-biotin tag transfer onto agalactosylated hybrid N-glycans present at the single glycosylation site of ovalbumin was dependent on the Gal-T used (either beta3Gal-T5, beta4Gal-T4, or beta4Gal-T1), which indicates that the acceptor specificity may direct the transfer of the Gal-biotin tag. The potential of this biotinylated UDP-Gal as a novel donor substrate for human galactosyltransferases lies in the targeting of distinct acceptor structures, for example, under-galactosylated glycoconjugates, which are related to diseases, or in the quality control of glycosylation of recombinant and native glycoproteins.
authors.affiliation: Institute of Enzyme Technology, Heinrich-Heine University of Düsseldorf, Research Center Jülich, 52426 Jülich, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Bülter
authors.forename: T
authors.initials: T
authors.lastname: Schumacher
authors.forename: D J
authors.initials: DJ
authors.lastname: Namdjou
authors.forename: R
authors.initials: R
authors.lastname: Gutiérrez Gallego
authors.forename: H
authors.initials: H
authors.lastname: Clausen
authors.forename: L
authors.initials: L
authors.lastname: Elling
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D005692:Galactose Oxidase / Q000737:chemistry*; D005700:Galactosyltransferases / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010047:Ovalbumin / Q000737:chemistry; D019809:Streptavidin / Q000737:chemistry; D014531:Uridine Diphosphate Galactose / Q000737:chemistry*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry*
chemical_list: D014531:Uridine Diphosphate Galactose; D014537:Uridine Diphosphate N-Acetylglucosamine; D001710:Biotin; D010047:Ovalbumin; D019809:Streptavidin; D005692:Galactose Oxidase; D016695:Glycosyltransferases; D005700:Galactosyltransferases