Identification and Key Amino Acid Analysis of Epitopes of Pin y 2, a Novel Allergen from Pinus yunnanensis

Pine nut allergy is a global food safety concern, yet the epitope of the major allergen from Pinus yunnanensis Pin y 2 remains unknown. We identified linear B-cell epitopes of Pin y 2 using five bioinformatics tools, followed by indirect ELISA with rabbit anti-Pin y 2 IgG and sera from pine nut-allergic patients. Four IgE epitopes and three IgG epitopes were validated. All epitopes are surface-located and predominantly in negatively charged electrostatic regions. Alanine scanning revealed that leucine (L97, L98), glutamic acid (E419, E434, E436), and glutamine (Q442) are key residues, with their alanine substitution causing a reduction of more than 50% in IgE/IgG binding OD values. Comparative epitope mapping with other tree nut vicilin allergens showed the positional conservation but sequence divergence of the Pin y 2 epitope. These findings reveal the epitope map and epitope key residues of Pin y 2, which provides rational targets for hypoallergenic design and diagnostics.