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- Title
Fluorescently Labeled Substrates for Monitoring α1,3-Fucosyltransferase IX Activity.
- Authors
Lunau, Nathalie; Seelhorst, Katrin; Kahl, Stefanie; Tscherch, Kathrin; Stacke, Christina; Rohn, Sascha; Thiem, Joachim; Hahn, Ulrich; Meier, Chris
- 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.
- Subjects
FUCOSYLTRANSFERASES; GLYCANS; CELL adhesion molecules; THIN layer chromatography; MASS spectrometry; FLUORESCENCE spectroscopy
- Publication
Chemistry - A European Journal, 2013, Vol 19, Issue 51, p17379
- ISSN
0947-6539
- Publication type
Article
- DOI
10.1002/chem.201302601