In silico Identification of IgE-Binding Epitopes of Osmotin Protein

Prerna Sharma,Naveen Arora,Shailendra Nath Gaur,Christian Schönbach

doi:10.1371/journal.pone.0054755

Prerna Sharma, Naveen Arora + Show 2 more

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https://doi.org/10.1371/journal.pone.0054755

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Abstract

The identification of B-cell epitopes is an important step to study the antigen- antibody interactions for diagnosis and therapy. The present study aimed to identify B- cell epitopes of osmotin using bioinformatic tools and further modify these regions to study the allergenic property. B-cell epitopes were predicted based on amino acid physicochemical properties. Three single point mutations M1, M2, and M3 and a multiple point mutant (M123) were selected to disrupt the IgE binding. These mutants were cloned, expressed and proteins purified to homogeneity. The IgE binding of the purified proteins was evaluated by ELISA and ELISA inhibition with patients' sera. Three regions of osmotin M1 (57–70 aa), M2 (72–85 aa) and M3 (147–165 aa) were identified as potential antibody recognition sites using in silico tools. The sequence similarity search of the predicted epitopes of osmotin using Structural Database of Allergenic proteins (SDAP) showed similarity with known allergens from tomato, kiwifruit, bell pepper, apple, mountain cedar and cypress. Mutants M1, M2 and M3 showed up to 72%, 60% and 76% reduction, respectively in IgE binding whereas M123 showed up to 90% reduction with patients' sera. The immunoblot of M123 mutant showed 40% reduction in spot density as compared to osmotin. All mutants showed decreased inhibition potency with M123 exhibiting lowest potency of 32% with osmotin positive pooled patients' sera. The three B- cell epitopes of osmotin predicted by in silico method correlated with the experimental approach. The mutant M123 showed a reduction of 90% in IgE binding. The present method may be employed for prediction of B- cell epitopes of allergenic proteins.

Highlights

The prevalence of food allergy is increasing worldwide and as many as 8% of children and 2% of adults are affected in western countries [1]
Several studies have shown the development of hypoallergenic molecules with reduced or no allergenic potential, which may be useful for specific immunotherapy [4], [5]
Mapping of IgE-binding epitopes IgE binding regions were identified by sequence based tools which fall in common regions of the sequence map

Summary

Introduction

The prevalence of food allergy is increasing worldwide and as many as 8% of children and 2% of adults are affected in western countries [1]. Several studies have shown the development of hypoallergenic molecules with reduced or no allergenic potential, which may be useful for specific immunotherapy [4], [5]. Epitopes can be identified by many methods but computational tools provide a promising and rapid alternative [8]. These epitopes may be modified to reduce allergenicity of an allergen. The key requirement is to identify IgE-binding epitopes on food allergens. The recombinant allergens have similar structure and immunologic properties as that of native counterparts, these can be modified to reduce the allergenicity while maintaining important immunologic properties [10]. Earlier studies on recombinant food allergens have demonstrated reduced allergenicity of shrimp [4], peanut allergen [11], apple [12], olive [13], etc

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