title: Fluorescently labeled substrates for monitoring α1,3-fucosyltransferase IX activity.
abstract: Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP-β-L-fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3-fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3-fucosyltransferases was examined by high-performance thin-layer chromatography coupled with mass spectrometry as well as dual-color fluorescence cross-correlation spectroscopy, which revealed that both fluorescently labeled donor GDP-β-L-fucose-ATTO 550 and acceptor N-acetyllactosamine-ATTO 647N were accepted by recombinant human fucosyltransferase IX and Helicobacter pylori α1,3-fucosyltransferase, respectively. Analysis by fluorescence cross-correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.
authors.affiliation: Organic Chemistry, Department of Chemistry, Faculty of Sciences, Hamburg University, Martin-Luther-King-Platz 6, 20146 Hamburg (Germany), Fax: (+49) 40-42838-5592.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Lunau
authors.forename: Katrin
authors.initials: K
authors.lastname: Seelhorst
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kahl
authors.forename: Kathrin
authors.initials: K
authors.lastname: Tscherch
authors.forename: Christina
authors.initials: C
authors.lastname: Stacke
authors.forename: Sascha
authors.initials: S
authors.lastname: Rohn
authors.forename: Joachim
authors.initials: J
authors.lastname: Thiem
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hahn
authors.forename: Chris
authors.initials: C
authors.lastname: Meier
mesh_terms: D002855:Chromatography, Thin Layer; D005456:Fluorescent Dyes / Q000737:chemistry*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D016480:Helicobacter pylori / Q000201:enzymology; D006801:Humans; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C519500:FUT9 protein, human; D005647:Fucosyltransferases
keywords: carbohydrates; chromatographic methods; enzymes; fluorescent probes; glycosylation