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

BACKGROUND 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. RESULT 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. CONCLUSION 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