Cross-reactive carbohydrate determinants (CCD) from plants and insects are N-glycans recognized by IgE, with minimal or no clinical significance [35]C[37]. 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. Introduction 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]C[3]. Among such allergens, Bla g 2 is of particular importance, eliciting IgE responses in 58C70% of cockroach allergic patients [4]C[6]. 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 presence of a zinc binding site and five disulfide bridges in Bla g 2 adds stability to the protein, thus favoring persistence of the allergen in the environment [7]. YIL 781 Chronic exposure to low doses (<1 g/g dust) of this stable cockroach allergen may explain the association between sensitization to Bla g 2 and asthma [3], [6]. Most reports on epitope mapping of allergens focus on the identification of linear epitopes by using libraries of overlapping synthetic peptides, recombinant fragments of the allergen, epitope expression cDNA libraries, or digested YIL 781 allergens. These approaches are especially useful for food allergens where linear epitopes are common due to allergen digestion (in addition to conformational epitopes) [10], [11]. However, conformational IgE antibody binding epitopes are important for inhaled allergens which reach the respiratory system mostly in their original globular structure. Little is known about the IgE antibody binding repertoire, the clonality and affinity of the interactions, the location and structure of epitopes and the kind of interactions involved in antibody recognition of the allergen [12]C[15]. 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 mAb and IgE antibody binding in order to gain insight on the IgE antibody binding repertoire. The epitope mapping approach presented here was based on the determination of the molecular structure of two allergen-antibody complexes by X-ray crystallography, which provided an accurate molecular structure of the conformational epitopes. Subsequently, site-directed mutagenesis was performed to confirm that the amino acids found at the interfaces are essential for antibody binding. For mapping B cell epitopes, it is not feasible to co-crystallize IgE antibodies with the allergen, due to the polyclonal nature of IgE and its paucity in sera (<1 g/ml compared with approximately 10 mg/ml for IgG). Therefore, mAb 7C11 and 4C3 which interfere with the binding of IgE antibodies, were selected to facilitate the identification of residues and kinds of interactions involved in such binding. Fragments of the non-overlapping mAb 7C11 and 4C3, raised MPL against natural Bla g 2, were individually co-crystallized with recombinant Bla g 2 (rBla YIL 781 g 2) and the constructions were identified [16], [17]. Subsequent site-directed mutagenesis of the residues involved in the mAb epitopes led to the recognition of amino acids that are important for the allergen-mAb connection. Additionally, amino acids involved in.
