Protein structure affects allergenicity, and critical structural elements, especially conformational epitopes that determine allergenicity, have attracted a great deal of interest. In this study, we aimed to identify the localized structure that affects the potential allergenicity of protein by making targeted modifications of Ara h 2 and comparing the structure and allergenicity of mutants with those of the wide-type allergen. The structures of the allergen and its mutants were characterized by circular dichroism and ultraviolet absorption spectroscopy and simulated by molecular dynamics. The allergenicity was assessed by Western blotting, an indirect competitive enzyme-linked immunosorbent assay, a cell model, and a mouse model. Then, the structures that affect allergenicity were analyzed and screened. Our results showed that mutations in amino acids changed the nearby localized structure and the overall structures. The structural changes affected the IgE binding capacity of the allergen and reduced its potential allergenicity. The solvent accessible surface area (SASA) of aromatic residues was positively correlated with the IgE binding capacity. The integrity of the disulfide bond is also critical for the binding of IgE to allergens. Interestingly, different mutations induced similar electrostatic potential and allergenicity changes, such as localized structure R62DPYSPSQDPYSPS75. In conclusion, the disulfide bond and the SASA of aromatic residues are important for the allergenicity of Ara h 2. The localized structure R62DPYSPSQDPYSPS75 is also crucial for the allergenicity of Ara h 2.

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 ARG 131 (amino acids 116-131) and TYR 65 (amino acids 63-80) and between TYR 60 (amino acids 56-62) and ARG 92 (amino acids 92-102); the intermolecular cross-linking sites were ARG 31 with TYR 84 or TYR 89 and TYR 65 or TYR 72 with ARG 92 or ARG 102 . Three out of four cross-linking peptides were found in -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.

Peanut (Arachis hypogaea) is listed among the eight major food allergens, in which Ara h 2 is the major allergen. The microbial transglutaminase (MTGase)‐catalyzed cross‐linking reaction reduces the allergenicity of Ara h 2. However, deamidation might occur and influence the cross‐linking reaction. In this work, native and reduced Ara h 2 were catalyzed by MTGase. In addition to intermolecular cross‐linking, intramolecular cross‐linking and deamidation were proven to occur simultaneously. Moreover, intramolecular cross‐linking sites were identified using mass spectrometry and the PLINK software. The reactions moved toward different directions because of different reduction conditions and different protein concentrations. Practical applications Cross‐linking is a common processing method used to improve the textural and functional properties of protein. Deamidation reaction might occur during processing and influence the cross‐linking reaction. The two types of reactions have a competitive relation, because both of them occur on glutamine residue. In Ara h 2 and MTGase system, protein concentration and reduction content influence the direction of reaction. The results can be applied to modify the reaction system of Ara h 2 catalyzed by MTGase. In addition, the relationship between structure and properties changes on Ara h 2 was discussed based on analysis of cross‐linking sites.