Abstract
BackgroundCockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2.Methodology/Principal FindingsMutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab′ fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients.Conclusions/SignificanceAnalysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates.
Highlights
Exposure and sensitization to cockroach is associated with the development of asthma, and up to 81% of children in inner-city areas of the U.S are sensitized to cockroach allergens [1,2,3]
Our aim was to map conformational antigenic determinants of Bla g 2 using the tertiary structure of the molecule as a ‘‘template’’ for mutagenesis, and to analyze the effect of amino acid substitutions on monoclonal antibodies (mAb) and IgE antibody binding in order to gain insight on the IgE antibody binding repertoire
Inhibition of IgE antibody binding by mAb 7C11 Increasing concentrations of mAb 7C11 showed dose dependent inhibition of IgE antibody binding of up to 40% (Figure 1)
Summary
Exposure and sensitization to cockroach is associated with the development of asthma, and up to 81% of children in inner-city areas of the U.S are sensitized to cockroach allergens [1,2,3]. The X-ray crystal structure of Bla g 2 shows a bilobal fold typical of pepsin-like aspartic proteases, but Bla g 2 is enzymatically inactive due to amino acid substitutions in the catalytic site [7,8]. The structures of both lobes are similar despite the low degree of the amino acid sequence homology (,15% identity) [9]. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2
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