Dominating IgE-Binding Epitope of Bet v 1, the Major Allergen of Birch Pollen, Characterized by X-ray Crystallography and Site-Directed Mutagenesis

TLDR

Allergy vaccination is effective, but safer vaccines are needed, and structural determination of allergens and antibody complexes facilitates epitope mapping and rational design of hypoallergenic molecules. The study aims to identify and modify a human IgE‑binding epitope of Bet v 1 using its crystal structure with the BV16 Fab′ fragment. The authors employed the Bet v 1–BV16 Fab′ crystal structure to pinpoint and engineer the epitope. The epitope occupies ~10 % of Bet v 1’s surface, is conformational, and a Glu45‑Ser mutation abolished mAb BV16 binding and reduced human IgE binding by up to 50 %, confirming Glu45 as critical without altering the protein’s tertiary structure.

Abstract

Abstract Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab′ fragment. The epitope occupies ∼10% of the molecular surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid substitution, Glu45-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45 is a critical amino acid also in a major human IgE-binding epitope. By solving the three-dimensional crystal structure of the Bet v 1 Glu45-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu45-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.