title: Formation of the immunogenic α1,3-fucose epitope: elucidation of substrate specificity and of enzyme mechanism of core fucosyltransferase A.
abstract: 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 K(D) = 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 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 μM 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.
authors.affiliation: Institute of Organic Chemistry, University of Hamburg, Germany.
authors.forename: Miriam P
authors.initials: MP
authors.lastname: Kötzler
authors.forename: Simon
authors.initials: S
authors.lastname: Blank
authors.forename: Henning N
authors.initials: HN
authors.lastname: Behnken
authors.forename: Dirk
authors.initials: D
authors.lastname: Alpers
authors.forename: Frank I
authors.initials: FI
authors.lastname: Bantleon
authors.forename: Edzard
authors.initials: E
authors.lastname: Spillner
authors.forename: Bernd
authors.initials: B
authors.lastname: Meyer
mesh_terms: D000818:Animals; D001516:Bees / Q000201:enzymology*; D000939:Epitopes; D005647:Fucosyltransferases / Q000378:metabolism*; D007150:Immunohistochemistry; D019476:Insect Proteins / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000939:Epitopes; D019476:Insect Proteins; D011994:Recombinant Proteins; D005647:Fucosyltransferases