Relevance of IgE binding to short peptides for the allergenicactivity of food allergens(1)
The food proteins and their fragments encounter the immune system during the passage through the gastrointestinal tract. Because these modified allergens are not in their native conformation, they are more likely to induce immune responses to linearized stretches of the amino acid sequence of the allergens and/or food proteins. Linear IgE-binding sequences have been identified on several food allergens, including major allergens of peanut, soybean, wheat, milk, egg, and shrimp. There is a correlation between specific epitope recognition patterns and different clinical manifestations of wheat allergy and celiac disease. Gliadins are rheomorphic proteins lacking a defined conformational structure and are therefore likely to induce antibody responses against sequential structures. The peanut allergen Ara h 2 and the shrimp tropomyosin Pen a 1, which have been described as containing sequential epitopes, as model allergens. Ara h 2 was chosen as a model for an allergen with a complex 3-dimensional structure stabilized by disulfide bonds. Pen a 1, a major shrimp allergen, served as the model for a protein containing predominantly a-helical structures and no disulfide bonds.
Comparison with folded rAra h 2 by means of CD analysis revealed that reduction and alkylation led to destruction of secondary structure. Western blot analyses of both preparations with sera of 2 individuals with peanut allergy showed that the strong IgE reactivity of the sera to the native allergen was completely abrogated or reduced by the denaturing treatment of the protein.
Subjects with shrimp allergy PI-1 and PII-3 displayed strong IgE binding to the synthetic peptides of Ara h 2 and Pen a 1, respectively.
The recognized peptides were assigned to IgE reactive regions and, within each region, amino acid sequences shared between the peptides are illustrated in bold.
Incubation of IgG-depleted sera from 2 individuals with peanut allergy with a mixture of soluble short peptides representing the identified sequences did not interfere detectably with IgE binding to rAra h 2 coated on the solid phase. No inhibition of IgE binding to rAra h 2 was detectable after incubation with the denatured protein. IgE binding to shrimp tropomyosin rPen a 1 on the solid phase was inhibited in similar experiments by incubation of IgG-depleted sera from individuals with shrimp allergy with recombinant Pen a1.
Preincubation of IgG-depleted patients’ sera with a mixture of peptides representing the sequences before sensitization of RBL cells did not affect the mediator release after stimulation with recombinant Ara h 2, and stimulation with denatured rAra h 2 did not elicit any mediator release. Preincubation of sera with peptides representing the IgE-binding Pen a 1 sequences did not reduce the capacity of the parent allergen to cause mediator release in the RBL assay.
Coincubation of patients’ sera with mouse tropomyosin caused inhibition only at the highest inhibitor concentration.
A predominance of IgE recognition of conformational epitopes on Ara h 2 and minor involvement of sequential epitopes. Moreover, the partial reduction of IgE binding in Western blot analysis, in contrast with the nearly complete abrogation of mediator release in RBL assay with unfolded rAra h 2. The identified IgE-binding peptides might represent hidden epitopes that are not accessible in the native folded allergen, preventing the inhibition of IgE binding to the native allergen by short peptides. Fluid phase binding of IgE antibodies is more relevant in relation to in vivo allergenicity, it is possible that solid phase approaches for epitope identification may generate partially misleading results.