Polyphenol‐oxidase‐catalyzed cross‐linking of Ara h 2: reaction sites and effect on structure and allergenicity

Abstract

Peanut is among the most common of food allergies, and one of its allergens is Ara h 2. A previous study revealed that this allergen was recognized by serum immunoglobulin E (IgE) in over 90% of a peanut-allergic patient population. Enzymatic cross-linking is a popular processing method used to tailor food functionality, such as antigenicity. The cross-linking reactions of Ara h 2 were catalyzed by polyphenol oxidase (PPO), and the relevant reaction sites were identified using mass spectrometry and StavroX software. Two pairs of intramolecular cross-linking peptides and two intermolecular cross-linking peptides were found. Intramolecular cross-linking was speculated to occur between ARG131 (amino acids 116-131) and TYR65 (amino acids 63-80) and between TYR60 (amino acids 56-62) and ARG92 (amino acids 92-102); the intermolecular cross-linking sites were ARG31 with TYR84 or TYR89 and TYR65 or TYR72 with ARG92 or ARG102 . Three out of four cross-linking peptides were found in α-helices, and destruction of this secondary structure resulted in a loose tertiary structure. Although seven linear allergen epitopes were involved in cross-linking, the IgE binding capacity of protein changed slightly, while its sensitization potential decreased in mouse model. Exploring the structural change of Ara h 2 after cross-linking is beneficial in further understanding the influence of structure on sensitization. This result indicated the future possibility of precision processing on structure of proteins to improve their properties. © 2019 Society of Chemical Industry.