Analysis of the IgE Epitope Profile of Soybean Allergen Gly M 4

Individuals with birch pollinosis may show allergic reactions after consumption of soybean-containing food. This is caused by cross-reaction of IgE directed against the major birch pollen allergen Bet v 1 with the structurally homologous allergen Gly m 4 from soybean. Hypersensitivity reactions in birch-soy allergy range from mild reactions to severe systemic reactions. Sera of birch pollen-allergic subjects may contain IgE to Gly m 4, even though no allergy to soy is present. Thus Gly m 4-specific IgE per se is not a suitable biomarker for birch-related soy allergy. To develop novel approaches for improved diagnosis and therapy of birch-soy allergy, knowledge on epitopes and IgE epitope profile of Gly m 4 for is needed. To date, data on epitopes of Bet v 1 and its homologous allergen Gly m 4 is very limited. In this study, birch pollen-allergic patients with (27 subjects) and without (20 subjects) clinically confirmed allergy to soybean were included and analyzed regarding Gly m 4-specific serum IgE/IgG levels and epitope profiles of Gly m 4 for IgE. Specific IgE levels against Bet v 1, Gly m 4 and further soy allergens Gly m 5 and Gly m 6 were determined by ImmunoCAP™ and IgE binding to rGly m 4 and soy extract was tested in western blot. To analyze putative IgE epitopes of Gly m 4, non-allergenic Norcoclaurine synthase (NCS) from meadow rue was used as a model protein. NCS is structurally homologous to Gly m 4 but exhibits none to very little binding of Gly m 4-specific IgE antibodies enabling grafting of Gly m 4 epitopes onto the model protein. Potential candidate residues of Gly m 4 were selected by bioinformatic analysis of antibody-binding phage-displayed peptides and mapping of segments of Gly m 4 primary structure onto the molecular surface of the allergen. As a result a library of recombinant NCS variants with potential IgE binding was generated. In addition, five multiple substitutional variants of rGly m 4 with a total number of 18 amino acid substitutions crucial for IgE binding were generated and analyzed for antibody binding with patients’ sera. Furthermore a misfolded variant of each rBet v 1a and rGly m 4 was generated and defined molar ratios of folded/misfolded variants were compared in different immunological and physicochemical assays. Gly m 4-specific median IgE and IgG levels of allergic (IgE: 9.3 kUA/L, IgG: 8.1 mgA/L) and non-allergic (IgE: 4.5 kUA/L, IgG: 8.3 mgA/L) subjects were comparable. The specific IgE levels did not correlate to the (severity of) clinical phenotypes. 51 candidate residues of Gly m 4 were selected for IgE epitope analysis and 46 potential functional IgE epitopes, single residues within a structural IgE epitope which dominate the energetics of allergen-IgE binding, were identified with IgE binding to ΔNCSN42/P49 variants. The putative functional IgE epitope pattern was individual for each patient and not distinguishable between allergic and non-allergic subjects. Using five rGly m 4 variants parts of the results of the NCS-based analyses could be confirmed with 18 potential functional IgE epitopes identified. 46 potential functional IgE epitopes clustered into six distinct putative IgE-binding areas on Gly m 4 and eleven NCS variants (ΔNCSN42/P49_1-11) presenting parts of these epitope areas were purified, showing a Gly m 4-type secondary structure according to CD measurements. Densitometric analysis for binding of IgE antibodies was performed via dot blot with sera of the study population but no characteristic IgE epitope pattern could be found. In contrast ΔNCSN42/P49_9 was identified as most suitable marker to distinguish soy allergic from tolerant patients in birch-related soybean allergy with a sensitivity and specificity of 68% and 93%, respectively. Using a pool of ΔNCSN42/P49 variants a depletion of Gly m 4-specific IgE binding to about 80% and 70% in pooled sera of patients with and without soybean allergy, respectively, was possible. Therefore identified putative functional IgE epitopes are specific for interaction with IgE antibodies in study population. With ΔNCSN42/P49_9 a differentiation between sensitization to Gly m 4 and clinically confirmed soybean allergy might be possible. The usage of a Gly m 4-specific epitope library might be a more promising tool for evaluating birch-related soybean allergy compared to ImmunoCAP™ and screening of substitutional Gly m 4 variants alone. However no correlations between patient’s IgE epitope profile and specific symptoms to soy or severity of allergic reactions could be found using NCS-based epitope library. Rather patient-specific epitope pattern might be relevant for birch-related soybean allergy. Therefore a thorough diagnosis by the combination of component-resolved diagnosis and profound epitope analysis might be mandatory in the future. Using two misfolded variants of rBet v 1a and rGly m 4 the impact of unstructured allergens in rBet v 1a/rGly m 4 preparations was addressed with physico- and immunological assays. Both rBet v 1aS112P/R145P and rGly m 4S111P/L150P showed a highly disordered protein conformation and reduced IgE binding frequencies with analyzed patients’ sera. With CD spectroscopy, immunoblot, ELISA and RBL cell release assay defined combinations of native and unstructured allergen were assessed concerning secondary structure and IgE binding. Correlation of rBet v 1a content with secondary structure and IgE binding was suitable only at high rBet v 1aS112P/R145P levels in mixtures. CD spectroscopy and ELISA performed more precise compared to immunoblot and rat basophil cell mediator release assay where larger deviations between native and unstructured allergen were necessary. In addition, quantification of IgE-binding allergen was difficult for concentrations of rBet v 1a ≤10% in all assays. Overall, CD, ELISA and RBL cell release assay underestimated while immunoblot overestimated the actual level of rBet v 1a. Results of both misfolded variants rBet v 1aS112P/R145P and rGly m 4S111P/L150P might be used within screening of hypoallergenic molecules with potential use in treatment of allergies or in quality assessment of recombinant allergen preparations.