Mapping and Mutational Analysis of the IgE‐Binding Epitopes on Ara h 1, a Legume Vicilin Protein and a Major Allergen in Peanut Hypersensitivity

Abstract

Peanut allergy is a significant health problem because of the prevelance and potential severity of the allergic reaction. Serum IgE from patients with documented peanut hypersensitivity reactions and overlapping peptides were used to identify the IgE-binding epitopes on the major peanut allergen, Ara h 1. At least twenty-three different linear IgE-binding epitopes, located throughout the length of the Ara h 1 protein, were identified. All of the epitopes were 6-10 amino acids in length, but there was no obvious sequence motif shared by all peptides. Four of the peptides appeared to be immunodominant IgE-binding epitopes in that they were recognized by serum from more than 80% of the patients tested and bound more IgE than any of the other Ara h 1 epitopes. Mutational analysis of the immunodominant epitopes revealed that single amino acid changes within these peptides had dramatic effects on IgE-binding characteristics. The identification and determination of the IgE-binding capabilities of core amino acids in epitopes on the Ara h 1 protein will make it possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.

Reduced Allergenic Potency of VR9-1, a Mutant of the Major Shrimp Allergen Pen a 1 (Tropomyosin)

“…The individual Pen a 1 epitopes differed in two or three amino acid positions from those of the homologous vertebrate sequences. Thus, sequence identity (73%) and similarity (87-100%) of this epitope to homologous vertebrate sequences were much higher than those of Pen a 1 [43][44][45][46][47][48][49][50][51][52][53][54][55] .…”

“…The most frequently used strategy to identify amino acid positions critical for IgE Ab binding is to replace each amino acid with alanine (alanine scan) (38,39,(42)(43)(44). Instead of an alanine scan, amino acids located at homologous positions of nonallergenic tropomyosins were replaced in the allergenic peptides.…”

“…1A) were identical for all four subjects tested (Pen a 1 [43][44][45][46][47][48][49][50][51][52][53][54][55] ), VHNL QKRMQQLEN) and consisted of 13 aa residues. Pen a 1 [43][44][45][46][47][48][49][50][51][52][53][54][55] differed at nine or 10 aa positions from homologous vertebrate tropomyosin sequences. Thus, the sequence identity was very low (23-31%), and the sequence similarity ranged from 39 -54%.…”

“…To identify the IgE-binding epitopes (shortest peptide with maximal IgE Ab reactivity) 5-to 15-aa-long peptides with offsets of one or two residues were synthesized spanning the previously identified main IgE-binding regions of Pen a 1 (region 1, Pen a 1 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] ; region 2, Pen a 1 85-105 ; region 3, Pen a 1 133-153 ; region 4, Pen a 1 187-201 ; region 5, Pen a 1 247-284 ) (2). These peptide libraries were probed with patients' sera for specific IgE Abs as described previously (2).…”

“…Peptides were tested for IgE binding with sera from 18 shrimp-allergic subjects. Based on frequency and intensity of IgE binding, five major allergenic regions were identified: region 1 (Pen a 1 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] ), region 2 (Pen a 1 ), region 3 (Pen a 1 ), region 4 (Pen a 1 [187][188][189][190][191][192][193][194][195][196][197][198][199][200][201] ), and region 5 (Pen a 1 ). The IgE binding regions seemed to occur at regular intervals in the molecule spaced approximately every sixth heptad (approximately every 42 aa), suggesting some relation with the coiled-coil structure of tropomyosin.…”