Alanine scanning of IgE-binding to the N-terminal epitope of Ara h 2 reveal critical amino acids

IgE binding to linear peptides of the N-terminal epitope (epitope 1: DRRCQSQLERAN) of Ara h 2 correlates with sensitivity to challenge in infants with peanut allergy and may be important for tree nut and peanut cross reactivity. ELISA was used to measure IgE binding to peptides of full length and truncated regions of epitope 1. We defined a core sequence (above) for further analysis. Peptide microarray technology was used to perform alanine scanning to determine the importance of individual amino acids. IgE binding for a pool of 10 sera and 4 individual sera was measured in duplicate on three separate occasions. Signals from alanine substituted peptides were normalized to the value obtained from the native sequence. Statistical analysis included one way ANOVA followed by Dunnett’s multiple comparison test. IgE binding using the pool of 10 sera was greatly reduced when alanine was substituted for arginine at position 2 (R2, P<0.001), arginine at position 3 (R3, P<0.01), glutamine at position 5 (Q5, P<0.01), glutamine at position 7 (Q7, P<0.001), glutamate at position 9 (E9, P<0.01), and arginine at position 10 (R10, P<0.001). IgE binding using individual sera was similar, except the importance of Q7 varied amongst individual sera. Molecular modeling of these data demonstrates a conformational basis for the recognition of these sequences by polyclonal IgE. IgE binding assays using pooled sera and individual sera implicated amino acids throughout the sequences of Epitope 1. These results are likely important for designing hypoallergenic forms of Ara h 2.