Immunoglobulin E and G4 epitopes of the major allergen of birch pollen Bet v 1 share residues critical for antibody binding
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KeywordsCircular Dichroism Food Allergy Birch Pollen IgG4 Binding Epitope Residue
Millions of patients with allergy to birch pollen develop clinically cross-reactive IgE against Bet v 1-like proteins in plant foods. Specific immunotherapy (SIT) with birch pollen extracts induces the biosynthesis of Bet v 1-specific immunoglobulin (Ig)G4. IgG4 is believed to act as a blocking antibody preventing IgE binding to Bet v 1, thus alleviating allergic symptoms. Only little information on the location and relationship of IgE and IgG4 binding sites of Bet v 1 is available. In this study we seek to identify epitopes of IgE and IgG4 antibodies on Bet v 1.
A competitive immunoscreening of phage-displayed peptides was applied to predict Bet v 1 epitopes of allergen-specific IgE and IgG4 antibodies by bioinformatic means. Predicted epitope residues potentially critical for antibody binding were substituted by site-directed mutagenesis. Recombinant Bet v 1 (rBet v 1) and rBet v 1 variants were purified from Escherichia coli. The proteins were physicochemically characterized using circular dichroism (CD) and dynamic light scattering. To test the IgE and IgG4 interactions with rBet v 1 variants, western blot analyses, ELISA, and cellular mediator release assays were performed.
Several rBet v 1 variants were expressed in E. coli. Circular dichroism and structural modeling of the variants revealed Bet v 1-like theoretical secondary structure topology. The rBet v 1 variants showed reduced IgE and IgG4 binding with sera of birch pollen allergic subjects in western blot analyses and competitive ELISAs. The rBet v 1 variants showed decreased IgE-mediated mediator release in humanized rat basophil leukemia cells sensitized with sera of birch pollen allergic subjects.
We identified critical residues in IgE and IgG4epitopes of Bet v 1. Although patient-specific variability was observed, the antibody interactions of the respective rBet v 1 variants were compromised for both IgE and IgG4, respectively. We conclude that epitopes for IgE and IgG4 share common residues critical for antibody interaction, suggesting an overlap of IgE and IgG4-binding sites on the molecular surface of Bet v 1. The knowledge of clinically relevant immunoglobulin-allergen interactions on the molecular level enables new strategies in the diagnosis, prognosis, and therapy of both birch pollen allergies and birch pollen-related food allergies.
Disclosure of interest
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