Formation of the immunogenic α1,3-fucose epitope: Elucidation of substrate specificity and of enzyme mechanism of core fucosyltransferase A

Glycans of glycoproteins are often associated with IgE mediated allergic immune responses. Hymenoptera venoms, e.g., carry α1,3-fucosyl residues linked to the proximal GlcNAc of glycoproteins. This epitope, formed selectively by α1,3-fucosyltransferase (FucTA), is xenobiotic and as such highly immunogenic and it also shows cross-reactivity if present on different proteins. Production of post-translationally modified proteins in insect cells is however commonly used and, thus, resulting glycoproteins can carry this highly immunogenic epitope with potentially significant side effects on mammals. To analyze mechanism, specificity and reaction kinetics of the key enzyme, we chose FucTA from Apis mellifera (honeybee) and characterized it by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR) experiments. Specifically, we show here that the donor substrate, GDP-Fucose, binds mostly via its guanine and less so via pyrophosphate and fucosyl fragments and has a KD = 37 μM. Affinity and kinetic studies with both the core α1,6-fucosylated and the unfucosylated octa- or heptasaccharides, respectively, as acceptor substrate revealed that honeybee FucTA prefers the latter structure with affinities of KD ∼ 10 mM. Establishment of progress curve analysis using an explicit solution of the integrated Michaelis–Menten equation allowed for determination of key constants of the transfer reaction of the glycosyl residue. The dominant minimum acceptor substrate is an unfucosylated heptasaccharide with Km = 420 μM and kcat = 6 min−1. Time-resolved NMR spectra as well as STD NMR allow molecular insights into specificity, activity and interaction of the enzyme with substrates and acceptors.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (105 K)Download as PowerPoint slideHighlights► A carbohydrate epitope associated with allergic reactions is found in invertebrates and on other xenobiotic material. ► The α1,3 fucosylated core epitope, is formed by transfer of a fucosyl residue from GDP-Fuc to a glycoprotein. ► FucTA synthesizes this epitope in the bee and requires an unusually large acceptor of at least seven monosaccharides. ► The enzyme has a relatively high activity that results in a fast production of the antigen.