Polyphenol-Oxidase-Catalyzed Cross-linking of Ara h 2: Reaction Sites and Effect on Structure and Allergenicity.

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 Ig E 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 (aa 116-131) and TYR⁶⁵ (aa 63-80) and between TYR⁶⁰ (aa 56-62) and ARG⁹² (aa 92-102); the intermolecular cross-linking sites were ARG³¹ , TYR⁸⁴ , TYR⁸⁹ and TYR⁶⁵ , TYR⁷² , ARG^92, and ARG¹⁰² . Three out of four cross-linking peptides were found in alpha-helices, and destruction of this secondary structure caused a loose tertiary structure. Although seven linear allergen epitopes were involved in cross-linking, the Ig E 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 to further understand the influence of structure on sensitization. This result indicated the future possibility of precision processing on structure of proteins to improve their properties. This article is protected by copyright. All rights reserved.