Investigating cross-reactivity: Alanine scanning mutations of key epitopes in Ara h 1 and Jug r 2 vicilin-buried peptides

Authors: SWIENTONIEWSKI, LAUREN - Oak Ridge Institute For Science And Education (ORISE); RAMBO, IAN - Oak Ridge Institute For Science And Education (ORISE); NESBIT, JACQUELINE - Oak Ridge Institute For Science And Education (ORISE); Cheng, Hsiaopo; GIPSON, STEPHEN - Oak Ridge Institute For Science And Education (ORISE); JONES, STACIE - University Of Arkansas; DRESKIN, STEPHEN - University Of Colorado; MUSTAFA, SHAHAZAD - University Of Rochester; MUELLER, GEOFFREY - National Institutes Of Health (NIH); Maleki, Soheila

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 11/20/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Peptides called vicilin-buried peptides (VBPs) in the leader sequences of proteins from peanuts and walnuts are important because they can cause allergic reactions to both types of nuts. This study looks into how certain antibodies (IgE and IgG4) in people with nut allergies react to these VBPs found in peanuts and walnuts. Researchers took blood samples from 30 people who have allergies to peanuts, walnuts, or both. They tested how well their antibodies could recognize small segments (15-amino-acid pieces) of the VBPs from peanut (Ara h 1) and walnut (Jug r 2). They also altered these segments to see how changes in their makeup affected the antibodies’ reactions. The results showed that by replacing specific amino acids in the peptides with another amino acid called alanine, the binding of IgE (the antibody related to allergies) and IgG4 (another type of antibody) decreased significantly at certain positions in the peptide structure. This suggests that those specific amino acids are crucial for how well the antibodies interact with the peptides. Overall, the study highlights the importance of these specific amino acids in understanding nut allergies. The knowledge gained could lead to better allergy tests and potentially the development of safer nut products for allergic individuals.

Technical Abstract: Rationale: Vicilin-buried peptides (VBPs), short segments within N-terminal vicilin leader sequences (LS), contribute to clinically relevant cross-reactivity between peanuts and tree nuts due to their highly conserved a-hairpinin structural motif. We investigate and compare IgE and IgG4 binding patterns to linear peptides of VBPs from peanut and walnut. Methods: Sera from OFC+ (n=30) subjects with peanut (PN), walnut (WN), and PN+WN (PW) allergy were assessed for IgE and IgG4 binding to linear peptides and alanine scanning mutations thereof with microarrays containing overlapping 15-mers offset by 5 amino acids of VBPs in peanut Ara h 1 (AH1.1) and walnut Jug r 2 (JR2.1, JR2.2, JR2.3). These analyses were also performed using a monoclonal IgE antibody specific to major epitopes within walnut Jug r 2 LS. Results: IgE and IgG4 binding to previously identified clinically relevant linear epitopes was significantly reduced (p = 0.01) when alanine was substituted for amino acids in various locations within the a-helices and the loop of the a-hairpinin motifs, indicating their potential role in peptide-antibody contact. Increased IgE and IgG4 binding was also observed for different residues within the same epitopes due to alanine substitutions. Conclusions: IgE and IgG4 binding to peptide microarrays using individual sera and monoclonal IgE antibodies demonstrated the importance of certain amino acids in the sequences of major linear epitopes in the VBPs of Ara h 1 and Jug r 2. These findings can contribute to improving diagnostics involved with cross-reactive allergens and developing tools such as hypoallergenic forms of VBPs.