IgE epitopes of Ara h 9, Jug r 3, and Pru p 3 in peanut-allergic individuals from Spain and the US

Abstract

Non-specific lipid transfer proteins (LTPs) are well studied allergens that can lead to severe reactions, but often cause oral allergy syndrome in the Mediterranean area and other European countries. However, studies focused on LTP reactivity in allergic individuals from the United States are lacking because they are not considered major allergens. The goal of this study is to determine if differences in immunoglobulin (Ig) E binding patterns to the peanut allergen Ara h 9 and two homologous LTPs (walnut Jug r 3 and peach Pru p 3) between the US and Spain contribute to differences observed in allergic reactivity. Synthetic overlapping 15-amino acid-long peptides offset by five amino acids from Ara h 9, Jug r 3, and Pru p 3 were synthesized, and the intact proteins were attached to microarray slides. Sera from 55 peanut-allergic individuals from the US were tested for IgE binding to the linear peptides and IgE binding to intact proteins using immunofluorescence. For comparison, sera from 17 peanut-allergic individuals from Spain were also tested. Similar IgE binding profiles for Ara h 9, Jug r 3, and Pru p 3 were identified between the US and Spain, with slight differences. Certain regions of the proteins, specifically helices 1 and 2 and the C-terminal coil, were recognized by the majority of the sera more often than other regions of the proteins. While serum IgE from peanut-allergic individuals in the US binds to peptides of Ara h 9 and its homologs, only IgE from the Spanish subjects bound to the intact LTPs. This study identifies Ara h 9, Jug r 3, and Pru p 3 linear epitopes that were previously unidentified using sera from peanut-allergic individuals from the US and Spain. Certain regions of the LTPs are recognized more often in US subjects, indicating that they represent conserved and possible cross-reactive regions. The location of the epitopes in 3D structure models of the LTPs may predict the location of potential conformational epitopes bound by a majority of the Spanish patient sera. These findings are potentially important for development of peptide or protein-targeting diagnostic and therapeutic tools for food allergy.

Keywords: Ara h 9; Jug r 3; Pru p 3; allergen; allergy diagnosis section manuscript type: original research non-specific lipid transfer proteins; epitope; immunoglobulin E; peanut allergy.

Figure 1

IgE binding to Ara h 9 by peptide and region (USA vs. Spain). (A) Dot plots representing the median SNR values for each peptide. Dots are colored blue (True) if the peptide is a major IgE reactive peptide (≥50% of the subjects have positive IgE binding) and pink (False) otherwise. The horizontal line represents the definition for positive binding: SNR ≥ 3. (B) Heat maps representing the subject-specific IgE intensities, which are categorized and displayed as follows: SNR < 3 (dark blue), SNR ≥ 3 and ≤10 (medium blue), SNR > 10 (light blue). US samples depicted with (A) represent sera samples provided by Aimmune Therapeutics.

Figure 2

IgE binding to Jug r 3 and Pru p 3 by peptide and region (USA vs. Spain). (A) Dot plots representing the median SNR values for each peptide. Dots are colored blue (True) if the peptide is a major IgE reactive peptide (≥50% of the subjects have positive IgE binding) and pink (False) otherwise. The horizontal line represents the definition for positive binding: SNR ≥ 3. (B) Heat maps representing the subject-specific IgE intensities, which are categorized and displayed as follows: SNR < 3 (dark blue), SNR ≥3 and ≤10 (medium blue), SNR > 10 (light blue). US samples depicted with (A) represent sera samples provided by Aimmune Therapeutics.

Figure 3

Sequence alignment of Ara h 9, Jug r 3, and Pru p 3. ClustalW (Clustal W2, RRID:SCR_002909) sequence alignment between Ara h 9.0201, Jug r 3.0101, and Pru p 3.03. Consensus sequence is shown above the alignment. Horizontal black boxes indicate IgE binding epitopes from US sera identified in this study. Colored vertical highlights show the conserved amino acids. Amino acids are color coded based on chemistry. Vertical purple boxes indicate basic (K and R), polar (C), or aromatic (F and Y) residues within peptides 4, 14, and 15 of Ara h 9 that are conserved or semi-conserved.

Figure 4

IgE binding to intact LTPs by region. Boxplot depicting ISAC array detection of sera IgE binding to intact Ara h 9, Jug r 3, and Pru p 3 in the US and Spain. P-values indicate a significant difference between regions as determined by Wilcoxon rank-sum tests per allergen. US, N = 54; Spain, N = 17.

Figure 5

Predicted 3D structures of Ara h 9, Jug r 3, and Pru p 3. The frontal and the 180° rotated views of the predicted 3D structures of (A) Ara h 9, (B) Jug r 3, and (C) Pru p 3 using the SWISS-MODEL Protein Modeling Server (29) and PDB: 2B5S [rPru p 3.0102 (9)] as a model template. N-terminus and C-terminus are indicated in panel A with N and C, respectively. Numbers 1–4 in panel A are representative of the four major α-helices in LTPs. The small black arrow in panel A indicates the C-terminal coil. Major IgE reactive regions identified by US sera are highlighted in pink (median SNR ≥ 3 and <5) and purple (median SNR ≥ 5).

Figure 6

Sequence alignment of Pru p 3.0102 with LTPs from this study. ClustalW sequence alignment between Pru p 3.0102, Pru p 3.03, Ara h 9.0201, and Jug r 3.0101. Consensus sequence is shown above the alignment. Amino acids are color coded based on chemistry, with conserved residues being highlighted vertically. Horizontal black boxes indicate the major IgE binding epitopes identified in this study with US sera or in a previous study for Pru p 3.0102. Vertical purple boxes indicate the specific residues previously predicted to have IgE binding capabilities.