RESUMO
Although many allergens have been detected in eggplant (Solanum melongena L.), their identity have not been elucidated. The aim of this study was to investigate whether polyphenol oxidase (PPO), an important eggplant enzyme, acts as an allergen. The proteins of eggplant peel extract were separated on phenyl-Sepharose (PS), and analyzed by skin prick test (SPT), ELISA and IgE-immunoblotting; the components were analyzed for PPO activity, presence of protein-bound copper, and recognition by rabbit polyclonal anti-sweet potato PPO antiserum. LC-MS/MS and in silico analysis were employed to identify the separated allergens and prediction of IgE epitopes. Eggplant allergens were separated into 5 components (PS1-PS5), of which component PS2 exhibited high specific PPO activity. SPT and ELISA with PPO-rich pool (PS2) were positive in all 6 eggplant-allergic subjects; the 43, 64 and 71kDa proteins displayed strong IgE-binding ability. The 64 and 71kDa IgE-binding proteins show PPO activity, presence of copper, and recognition by anti-sweet potato PPO antiserum, clearly identifying them as PPOs; the 43kDa protein appears to be a degradation product of the 64 or 71kDa proteins based on enzymic activity and recognition by PPO antiserum. The 64kDa protein upon further resolution by SDS-PAGE displayed two components (identified as eggplant PPO1 and PPO4 by LC-MS/MS). Based on bioinformatics approaches, PPO4 has been identified as an allergen since it harbors an IgE epitope. This study clearly demonstrates that the 64 and 71kDa allergens in eggplant peel are PPOs based on enzymic activity and recognition by PPO antiserum; the 64kDa copper-containing protein is identified as one of the several eggplant allergens (Sola m PPO4). This is the first instance of polyphenol oxidase being identified as a new food allergen.
