Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts

Abstract

Background: Cross-reactivity between peanuts and tree nuts implies that similar immunoglobulin E (IgE) epitopes are present in their proteins.

Objective: To determine whether walnut sequences similar to known peanut IgE-binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins, react with IgE from sera of patients with allergy to walnut and/or peanut.

Methods: Patient sera were characterized by western blotting for IgE binding to nut protein extracts and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive enzyme-linked immunosorbent assay (ELISA) was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient.

Results: Sequences from the vicilin walnut allergen Jug r 2, which had low PD values to epitopes of the peanut allergen Ara h 2, a 2S albumin, bound to IgE in sera from five patients who reacted to either walnut or peanut or both. A walnut epitope recognized by sera from six patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. This predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2.

Conclusions: Sequences with low PD value (< 8.5) to known IgE epitopes could contribute to cross-reactivity between allergens. This further validates the PD scoring method for predicting cross-reactive epitopes in allergens.

Figure 1. Immunoblots of IgE binding by peanut and or walnut allergic individuals

A) Western blots: Patients are depicted by numbers (1–6) above each blot and the clinical allergy to peanut (PNT) or walnut (WAL). The Molecular Weight Marker (MW) is shown. Ara h 1, 2, Jug r 1, 2 and 4 are indicated as A1, A2 and J1–J4, respectively. Negative control serum is Atopic Control. B) Sequence of membrane-bound, synthetic peptides in C. C) From left to right, column 1 shows IgE binding to spots of a “no serum control” (row 1) and 4 sera to the six membrane-bound synthetic peptides (row 2–5), specific allergens recognized in western blot (column 2), and peptide spots recognized by each serum (column 3).

Figure 2. Immunoblot of IgE binding to membrane-bound known and predicted synthetic peptide epitopes of peanut and walnut allergens

(A) From left to right, the first column shows the IgE binding profile of a control and 5 patient sera to the 12 membrane-bound synthetic peptides. Grey spots at the top indicate peanut peptides. The specific allergens recognized in the blots are indicated in the third column. (B), Densitometric scan of spots in panel A (indicated on x-axis) and reported in arbitrary units (a.u., y-axis).

Figure 3. 3D-characterization of peptides (Table 2, 4–12) tested in this study

a) Alignment of the Jug r 2 N-terminal region with the modeling template, 1PSY-pdb (Ric c 3). The residues are colored to show amino acid properties. Cys residues involved in disulfide bonding are highlighted in yellow, IgE binding sequences identified in this study (6, 8–10 in Table 2) are highlighted in gray. b) Mapping of peptides on a crystal structure of Ara h 2 and models, from SDAP or specifically prepared for this study. Structures are shown in ribbon format, with the potential epitopes shown space filling in red. Side chains with >30% surface exposure according to GETAREA are highlighted in red in the text below each.

Figure 4. Competitive inhibition ELISA of IgE binding to purified Ara h 2 with synthetic peptides

A and B: indicated concentrations (x-axis) of peptides: a known Ara h 2 epitope (DRRCQSQLER, ●), a walnut peptide with low PD to the Ara h 2 one (QRQCQQRECER, ■), a combination of the two peptides(▲), intact Ara h 2 (◆) and a negative control (▼) were used to compete with IgE binding to intact Ara h 2. (B) Mean of 3 peptide assays from panel A is also shown (X). The optical density at 450 nm is indicated on the y-axis.