Sabina Wünschmann

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab′ fragment of a monoclonal antibody 7C11 was solved at 2.8-Å resolution. Bla g 2 binds to the antibody through four loops that include residues 60-70, 83-86, 98-100, and 129-132. Cation-π interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab′, Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an α-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less β-hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy.

Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity

To cite this article: Mueller GA, Gosavi RA, Pomés A, Wünschmann S, Moon AF, London RE, Pedersen LC. Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity. Allergy 2011; 66: 878–885.

Cockroach allergens: function, structure and allergenicity.

Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.

Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies That Inhibit IgE Antibody Binding

Background Cockroach 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 Findings Mutational 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/Significance Analysis 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.

Ara h 6 complements Ara h 2 as an important marker for IgE reactivity to peanut.

The similarities of two major peanut allergens, Ara h 2 and Ara h 6, in molecular size, amino acid sequence, and structure have made it difficult to obtain natural Ara h 6 free of Ara h 2. The objectives of this study were to purify natural Ara h 6 that is essentially free of Ara h 2 and to compare its IgE reactivity and potency in histamine release assays to Ara h 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the highly purified allergen (<0.01% Ara h 2) revealed a single 14.5 kD band, and the identity of Ara h 6 was confirmed by liquid chromatography-tandem mass spectrometry. Ara h 6 showed a higher seroprevalence in chimeric IgE enzyme-linked immunosorbent assay (n = 54) but a weaker biological activity in basophil histamine release assays than Ara h 2. Purified Ara h 6 will be useful for diagnostic IgE antibody assays as well as molecular and cellular studies to investigate the immunological mechanisms of peanut allergy.

Carbohydrates Contribute to the Interactions between Cockroach Allergen Bla g 2 and a Monoclonal Antibody

The crystal structure of a murine mAb, 4C3, that binds to the C-terminal lobe of the cockroach allergen Bla g 2 has been solved at 1.8 Å resolution. Binding of 4C3 involves different types of molecular interactions with its epitope compared with those with the mAb 7C11, which binds to the N-terminal lobe of Bla g 2. We found that the 4C3 surface epitope on Bla g 2 includes a carbohydrate moiety attached to Asn268 and that a large number of Ag–Ab contacts are mediated by water molecules and ions, most likely zinc. Ab binding experiments conducted with an enzymatically deglycosylated Bla g 2 and a N268Q mutant showed that the carbohydrate contributes, without being essential, to the Bla g 2–4C3 mAb interaction. Inhibition of IgE Ab binding by the mAb 4C3 shows a correlation of the structurally defined epitope with reactivity with human IgE. Site-directed mutagenesis of the 4C3 mAb epitope confirmed that the amino acids Lys251, Glu233, and Ile199 are important for the recognition of Bla g 2 by the 4C3 mAb. The results show the relevance of x-ray crystallographic studies of allergen–Ab complexes to identify conformational epitopes that define the antigenic surface of Bla g 2.

Indoor Allergens and Allergic Respiratory Disease

Purpose of reviewThe purpose of this review is to evaluate the most recent findings on indoor allergens and their impact on allergic diseases.Recent findingsIndoor allergens are present inside buildings (home, work environment, school), and given the chronic nature of the exposures, indoor allergies tend to be associated with the development of asthma. The most common indoor allergens are derived from dust mites, cockroaches, mammals (including wild rodents and pets), and fungi. The advent of molecular biology and proteomics has led to the identification, cloning, and expression of new indoor allergens, which have facilitated research to elucidate their role in allergic diseases. This review is an update on new allergens and their molecular features, together with the most recent reports on their avoidance for allergy prevention and their use for diagnosis and treatment.SummaryResearch progress on indoor allergens will result in the development of new diagnostic tools and design of coherent strategies for immunotherapy.

Allergen content in German cockroach extracts and sensitization profiles to a new expanded set of cockroach allergens determine in vitro extract potency for IgE reactivity

Background Cockroach allergens are an important cause of IgE‐mediated sensitization in inner‐city asthmatic patients. However, cockroach extracts used for diagnosis and immunotherapy are not standardized. Objective We sought to determine the allergen content of nonstandardized German cockroach extracts and the levels of sensitization to an expanded set of cockroach allergens as determinants of in vitro extract potency for IgE reactivity. Methods Twelve German cockroach extracts were compared for allergen content and potency of IgE reactivity. Bla g 1, Bla g 2, and Bla g 5 were measured by using immunoassays. IgE antibody levels to 8 purified recombinant allergens from groups 1, 2, 4, 5, 6, 7, 9, and 11 were measured by using ImmunoCAP. IgE antibody binding inhibition assays were performed to assess extract in vitro potencies (concentration inhibiting 30% of the total IgE antibody‐binding inhibition) relative to an arbitrarily selected reference extract in 5 patients with cockroach allergy. Results Allergen levels were highly variable. Three new major allergens (groups 6, 9, and 11), were identified among highly cockroach‐sensitized subjects (CAP class ≥ 3). Sensitization profiles were unique per subject without immunodominant allergens. The sum of IgE to 8 allergen components showed a good correlation with cockroach‐specific IgE levels (r = 0.88, P < .001). In vitro potencies varied among different extracts per subject and among subjects for each extract. Conclusions The in vitro potency of German cockroach extracts for IgE reactivity depends on allergen content and allergen‐specific IgE titers of patients with cockroach allergy. These factors are relevant for selection of potent extracts to be used for immunotherapy and for the design and interpretation of data from immunotherapy trials.

Ara h 6 complements Ara h 2 as an important marker for IgE reactivity to peanut

The similarities of two major peanut allergens, Ara h 2 and Ara h 6, in molecular size, amino acid sequence, and structure have made it difficult to obtain natural Ara h 6 free of Ara h 2. The objectives of this study were to purify natural Ara h 6 that is essentially free of Ara h 2 and to compare its IgE reactivity and potency in histamine release assays to Ara h 2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the highly purified allergen (<0.01% Ara h 2) revealed a single 14.5 kD band, and the identity of Ara h 6 was confirmed by liquid chromatography–tandem mass spectrometry. Ara h 6 showed a higher seroprevalence in chimeric IgE enzyme-linked immunosorbent assay (n = 54) but a weaker biological activity in basophil histamine release assays than Ara h 2. Purified Ara h 6 will be useful for diagnostic IgE antibody assays as well as molecular and cellular studies to investigate the immunological mechanisms of peanut allergy.