Tagging Glycoproteins with Fluorescently Labeled GDP-Fucoses by Using α1,3-Fucosyltransferases.

ABSTRACT

Fucose-containing glycans mediate a variety of biological processes, but there is little information on reaction processes and mechanisms mediated by fucosyltransferases. We recently reported on fluorescently labeled GDP-ß-L-fucose-ATTO 550, which enabled monitoring of α1,3-fucosyltransferase activity. Here we present an extension to the previously described results, based on the synthesis of a fluorescein-isothiocyanate (FITC)-labeled and two carboxyfluorescein-labeled (FAM-labeled) NDP-ß-L-fucose derivatives, and applied all four compounds in labeling of different glycoproteins with the aid of four different fucosyltransferases. The labeling processes were analyzed by in-gel fluorescence and fluorescence polarization measurements. Comparison with the ATTO-labeled sugar revealed that the FITC-labeled fucose was the best of these substrates, and that the bacterial enzyme HP-FucT tolerated the fluorescent substrates better than human fucosyltransferases.