Identification and Structure of Epitopes on Cashew Allergens Ana o 2 and Ana o 3 Using Phage Display

Abstract

Cashew (Anacardium occidentale L.) is a commercially important plant. Cashew nuts are a popular food source that belong to the tree nut family. Tree nuts are one of the eight major food allergens identified by the Food and Drug Administration in the USA. Allergies to cashew nuts cause severe and systemic immune reactions. Tree nut allergies are frequently fatal and are becoming more common. We aimed to identify the key allergenic epitopes of cashew nut proteins by correlating the phage display epitope prediction results with bioinformatics analysis. We predicted and experimentally confirmed cashew nut allergen antigenic peptides, which we named Ana o 2 (cupin superfamily) and Ana o 3 (prolamin superfamily). The Ana o 2 and Ana o 3 epitopes were predicted using DNAstar and PyMoL (incorporated in the Swiss-model package). The predicted weak and strong epitopes were synthesized as peptides. The related phage library was built. The peptides were also tested using phage display technology. The expressed antigens were tested and confirmed using microtiter plates coated with pooled human sera from patients with cashew nut allergies or healthy controls. The Ana o 2 epitopes were represented by four linear peptides, with the epitopes corresponding to amino acids 108-111, 113-119, 181-186, and 218-224. Furthermore, the identified Ana o 3 epitopes corresponding to amino acids 10-24, 13-27, 39-49, 66-70, 101-106, 107-114, and 115-122 were also screened out and chosen as the key allergenic epitopes. The Ana o 3 epitopes accounted for more than 40% of the total amino acid sequence of the protein; thus, Ana o 3 is potentially more allergenic than Ana o 2. The bioinformatic epitope prediction produced subpar results in this study. Furthermore, the phage display method was extremely effective in identifying the allergenic epitopes of cashew nut proteins. The key allergenic epitopes were chosen, providing important information for the study of cashew nut allergens.