TY - JOUR

T1 - Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis

AU - Spangfort, Michael D

AU - Mirza, Osman

AU - Ipsen, Henrik

AU - Van Neerven, R J Joost

AU - Gajhede, Michael

AU - Larsen, Jørgen

PY - 2003

Y1 - 2003

N2 - 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 approximately 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, Glu(45)-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 Glu(45)-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu(45)-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.

AB - 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 approximately 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, Glu(45)-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 Glu(45)-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu(45)-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.

KW - Allergens

KW - Amino Acid Sequence

KW - Animals

KW - Antigens, Plant

KW - Betula

KW - Binding Sites, Antibody

KW - Binding, Competitive

KW - Crystallography, X-Ray

KW - Glutamic Acid

KW - Immunodominant Epitopes

KW - Immunoglobulin E

KW - Mice

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Peptide Fragments

KW - Plant Proteins

KW - Pollen

KW - Serine

KW - Surface Properties

M3 - Journal article

C2 - 12960334

VL - 171

SP - 3084

EP - 3090

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 6

ER -