Title: The NMR structure and IgE Epitopes of Ara h 1 Leader Sequence
Abstract: The vicilins from peanut, walnut, cashew and pistachio are considered major allergens and are translated with leader sequences (LS) that are cleaved before yielding the mature protein. While previous work has shown that some contain immunodominant IgE epitopes, their structure has not been solved. Linear IgE epitopes were identified using microarrays generated by printing 15-mer peptides overlapping by 10 aa on slides and incubating with peanut allergic sera. IgE binding by allergic sera was detected with a fluorescently-labeled antibody. Western blots and mass spectrometry were used to show the presence of the LS in peanut and walnut seeds. The NMR structure of the Ara h 1 LS was determined and the IgE binding sites were modeled on this structure. The epitopes with the highest degree of IgE binding were clustered within regions that were near cysteine residues. Of the patients tested, >90% showed IgE binding to those epitopes even if they recognized no other epitopes in the Ara h 1 LS. The NMR structure showed 4 of the cysteine residues are disulfide bonded and hold together two parallel alpha helices. IgE binding is shown to be located at the junction of the C-terminal region of the alpha helices and the beginning of each flexible loop. The results indicate that cysteine residues known to confer high structural stability to allergens may also coincide with areas of increased IgE binding frequency and intensity in Ara h 1 LS. The LS contains multiple immunodominant epitopes and may contribute to cross-reactivity and nut allergy.