Formation of the immunogenic alpha 1,3-fucose epitope: elucidation of substrate specificity and of enzyme mechanism of core fucosyltransferase A.

摘要

Glycans of glycoproteins are often associated with IgE mediated allergic immune responses. Hymenoptera venoms, e.g., carry alpha 1,3-fucosyl residues linked to the proximal GlcNAc of glycoproteins. This epitope, formed selectively by alpha 1,3-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 K_D=37 micro M. Affinity and kinetic studies with both the core alpha 1,6-fucosylated and the unfucosylated octa- or heptasaccharides, respectively, as acceptor substrate revealed that honeybee FucTA prefers the latter structure with affinities of K_D~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 K_m=420 micro M and k_cat=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.