Fine resolution of the N-terminal IgE-binding epitope of Ara h 2: Discovery of variants with enhanced IgE binding

Background: IgE binding to linear peptides from the N-terminal region of Ara h 2 (epitope 1) is associated with the achievement of sustained unresponsiveness in young children receiving oral immunotherapy and may be important for cross-reactivity between peanuts and tree nuts. This region is also part of the binding site for neutralizing IgG monoclonal antibodies associated with sustained unresponsiveness following oral immunotherapy. Objective: We sought to perform alanine scanning of this epitope to determine the importance of individual amino acids and then amino acid scanning to screen for sequences with enhanced binding of IgE. Methods: A streptavidin IgE ELISA with biotinylated peptides was used to measure the binding of IgE to full-length and truncated peptides to identify a core sequence (DRRCQSQLERAN, amino acids 30-41 in the Ara h 2 sequence). Peptide microarrays were used to screen multiple peptides and quantitate binding of IgE. Statistical analysis included one-way ANOVA followed by the Dunnett multiple comparison test. Results: IgE binding was greatly reduced when alanine was substituted for arginine at positions 31, 32, and 39 (R31, P < .001; R32, P < .01; R39, P < .001); glutamine at positions 34 and 36 (Q34, P < .01; Q36, P < .001); and glutamate at position 38 (E38, P < .01). Substitution of aspartate with asparagine at position D30 in conjunction with substitution of asparagine at position N41 with either leucine or lysine gave enhanced binding (P < .0001). Molecular modeling of these data suggests a conformational basis for recognition by polyclonal IgE. Conclusions: IgE binding assays using pooled and individual sera demonstrated the importance of amino acids throughout the sequence of epitope 1 for immune recognition. The results of alanine scanning indicated residues that could be changed as part of a larger strategy to generate hypoallergenic forms of Ara h 2, whereas sequence variants with enhanced binding were identified that may be useful for improving diagnostics.