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: Nathalie Lunau, Katrin Seelhorst, Stefanie Kahl, Kathrin Tscherch, Christina Stacke, Sascha Rohn, Joachim Thiem, Ulrich Hahn, Chris Meier
• Journal: Chemistry (Weinheim an der Bergstrasse, Germany) (Chemistry) Vol. 19 Issue 51 Pg. 17379-90 (Dec 16 2013) ISSN: 1521-3765 [Electronic] Germany
• PMID: 24258785 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical References

• Fluorescent Dyes
• Recombinant Proteins
• FUT9 protein, human
• Fucosyltransferases

Topics

• Chromatography, Thin Layer
• Fluorescent Dyes (chemistry)
• Fucosyltransferases (genetics, metabolism)
• Glycosylation
• Helicobacter pylori (enzymology)
• Humans
• Mass Spectrometry
• Recombinant Proteins (genetics, metabolism)
• Substrate Specificity