Human IgE against the major allergen Bet v 1 – defining an epitope with limited cross‐reactivity between different PR‐10 family proteins

Abstract

BackgroundThe interaction between IgE and allergen is a key event at the initiation of an allergic response, and its characteristics have substantial effects on the clinical manifestation. Despite this, the molecular details of the interaction between human IgE and the major birch allergen Bet v 1, one of the most potent tree allergens, still remain poorly investigated.ObjectiveTo isolate Bet v 1-specific human monoclonal IgE and characterize their interaction with the allergen.MethodsRecombinant human IgE were isolated from a combinatorial antibody fragment library and their interaction with Bet v 1 assessed using various immunological assays. The structure of one such IgE in the single-chain fragment variable format was determined using X-ray crystallography.ResultsWe present four novel Bet v 1-specific IgE, for one of which we solve the structure, all with their genetic origin in the IGHV5 germline gene, and demonstrate that they target two non-overlapping epitopes on the surface of Bet v 1, thereby fulfilling the basic criteria for FcεRI cross-linkage. We further define these epitopes and for one epitope pinpoint single amino acid residues important for the interaction with human IgE. This provides a potential explanation, at the molecular level, for the differences in recognition of isoforms of Bet v 1 and other allergens in the PR-10 protein family displayed by IgE targeting this epitope. Finally, we present the first high-resolution structure of a human allergen-specific IgE fragment in the single-chain fragment variable (scFv) format.Conclusions and Clinical RelevanceWe here display the usefulness of allergen-specific human monoclonal IgE as a tool in studies of the crucial molecular interaction taking place at the initiation of an allergic response. Such studies may aid us in development of better diagnostic tools and guide us in the development of new therapeutic compounds.

Ligand Binding Modulates the Structural Dynamics and Compactness of the Major Birch Pollen Allergen

“…It is tempting to speculate that for Bet v 1, the less compact conformers that are transiently populated in the apo protein display enhanced susceptibility to proteolytic digestion by exposure of structurally buried amino acid residues. Moreover, the conformational flexibility that we observe for Bet v 1 could contribute to optimal positioning of the epitope residues on the protein surface and facilitate cross-linking of Fc ε RI receptors ( 53 ). Conformational plasticity is often a prerequisite for efficient recognition of biological targets ( 54 ).…”

Interfaces Between Allergen Structure and Diagnosis: Know Your Epitopes

“…These include an anti-Bla g 1 IgG scFv, an anti-Bet v 1 IgE scFv and three anti-Der p 1 IgG Fab (mAb 4C1: 3RVT, 3RVU; mAb 10B9: 4POZ) [90••, 92, 93]. The use of antibodies specific for Blag 1 and Bet v 1 in mutant or peptide-binding experiments, respectively, led to the molecular location of species-specific epitopes.…”

Enlarging the Toolbox for Allergen Epitope Definition with an Allergen-Type Model Protein

“…Allergen variants carrying substitutions of either individual or multiple residues to either attenuate or induce IgE antibody binding (epitope grafting) identified individual residues crucial for IgE recognition by Bet v 1 [6] – [10] and Bet v 1-type allergens from apple [11] , [12] , cherry [13] , [14] , and celeriac [15] . In addition, monoclonal antibodies have been used to localize potential IgE epitopes of Bet v 1 and related allergens [10] , [16] – [19] . The only structural information on an epitope of Bet v 1 to date was obtained from X-ray crystallography of a complex between the monoclonal Bet v 1-specific mouse IgG BV16 and the major isoform of Bet v 1, Bet v 1 a [20] .…”