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Background-Sensitization to house dust mite allergens is strongly correlated with asthma. Der p 7 elicits strong IgE antibody and T-cell responses in mite allergic patients. However, the structure and biological function of this important allergen are unknown. Allergen function may contribute to allergenicity as shown for the protease activity of Group 1 mite allergens and the interaction with the innate immune system by Group 2 mite allergens.

Background Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein. Objective This study sought to assess the importance of Der p 23 among other Dermatophagoides allergens in a North American population, and to determine the structure for functional characterization. Methods IgE binding to Der p 23, Der p 1, Der p 2, Der p 5, Der p 7, and Der p 8 was measured by ELISA. RNA-seq data from D. pteronyssinus were compared as estimates of allergen expression levels. The structure was analyzed by X-ray crystallography and NMR. Results Despite a high prevalence of Der p 23, (75% versus 87% and 79% for Der p 1 and Der p 2, respectively), the anti-Der p 23 IgE levels were relatively low. The patient response to the 6 allergens tested was variable (n=47), but on average anti-Der p 1 and anti-Der p 2 together accounted for 85% of the specific IgE. In terms of abundance, the RNA expression level of Der p 23 is the lowest of the major allergens, 30-fold less than and 7-fold less than Der p 2. The structure of Der p 23 is a small, globular protein stabilized by two disulfide bonds, which is structurally related to allergens such as Blo t 12 that contain carbohydrate binding domains that bind chitin. Functional assays failed to confirm chitin binding by Der p 23. Conclusions and Clinical Relevance Der p 23 accounts for a small percentage of the IgE response to mite allergens, which is dominated by Der p 1 and Der p 2. The prevalence and amount of specific IgE to Der p 23 and Der p 2 are disproportionately high compared to the expressions of other Dermatophagoides allergens.

Background Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of ~100 amino acids, but the fold of the protein and the biological function are unknown. Objective To determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays. Methods Natural Bla g 1 and recombinant constructs were compared by ELISA using specific murine IgG and human IgE. The structure of Bla g 1 was determined by X-ray crystallography. Mass spectrometry and NMR were utilized to examine ligand-binding properties of the allergen. Results The structure of a recombinant Bla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by X-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic and stearic acids were associated with nBla g 1 from cockroach frass. One Unit of Bla g 1 was equivalent to 104 ng of allergen. Conclusions Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with non-specific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure.

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.

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of ϳ3000 Å 3 that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

Background It is not clear if cross-reactivity or co-sensitization to glutathione S-transferases (GST) occurs in tropical and subtropical environments. In the United States, Bla g 5 is the most important GST allergen, and lack of co-exposure to GST from certain species allows a better assessment of cross-reactivity. Objectives To examine the molecular structure of GST allergens from cockroach (Bla g 5), dust mites (Der p 8, Blo t 8) and helminth (Asc s 13) for potential cross-reactive sites, and to assess the IgE cross-reactivity of sensitized patients from a temperate climate for these allergens for molecular diagnostic purposes. Methods Four crystal structures were determined. Sera from cockroach and mite allergic patients were tested for IgE reactivity to these GST. A panel of six murine anti-Bla g 5 mAb was assessed for cross-reactivity with the other three GST using antibody binding assays. Results Comparisons of the allergen structures, formed by two-domain monomers that dimerize, revealed few contiguous regions of similar exposed residues, rendering cross-reactivity unlikely. Accordingly, anti-Bla g 5 or anti-Der p 8 IgE from North American patients did not recognize Der p 8 or Bla g 5, respectively, and neither showed binding to Blo t 8 or Asc s 13. A weaker binding of anti-Bla g 5 IgE to Der p 8 versus Bla g 5 (~100-fold) was observed by inhibition assays, similar to a weak recognition of Der p 8 by anti-Bla g 5 mAb. Patients from tropical Colombia had IgE to all four GST. Conclusions The lack of significant IgE cross-reactivity among the four GST is in agreement with the low shared amino acid identity at the molecular surface. Each GST is needed for accurate molecular diagnosis in different geographic areas.

The crystal structure of a murine monoclonal antibody, 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 to the monoclonal antibody 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 antigen-antibody contacts are mediated by water molecules and ions, most likely zinc. Antibody 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 antibody 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 amino acids Lys251, Glu233, and Ile199 are important for the recognition of Bla g 2 by the 4C3 mAb antibody. The results show the relevance of X-ray crystallographic studies of allergen-antibody complexes to identify conformational epitopes that define the antigenic surface of Bla g 2.