Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases.

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Citation

Bulter T, Schumacher T, Namdjou DJ, Gutierrez Gallego R, Clausen H, Elling L

Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases.

Chembiochem. 2001 Dec 3;2(12):884-94.

PubMed ID
11948877 [ View in PubMed
]
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-galactosamin e (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.

DrugBank Data that Cites this Article

Drug Targets
DrugTargetKindOrganismPharmacological ActionActions
N-AcetylglucosamineBeta-1,4-galactosyltransferase 4ProteinHumans
Unknown
Not AvailableDetails