title: Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis.
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 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.
authors.affiliation: ALK-Abelló, Research Department, Hørsholm, Denmark. msp@dk.alk.abello.com
authors.forename: Michael D
authors.initials: MD
authors.lastname: Spangfort
authors.forename: Osman
authors.initials: O
authors.lastname: Mirza
authors.forename: Henrik
authors.initials: H
authors.lastname: Ipsen
authors.forename: R J Joost
authors.initials: RJ
authors.lastname: Van Neerven
authors.forename: Michael
authors.initials: M
authors.lastname: Gajhede
authors.forename: Jørgen N
authors.initials: JN
authors.lastname: Larsen
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D029662:Betula / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics / Q000276:immunology; D018360:Crystallography, X-Ray / Q000379:methods; D018698:Glutamic Acid / Q000235:genetics; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007073:Immunoglobulin E / Q000097:blood / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011058:Pollen / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D012694:Serine / Q000235:genetics; D013499:Surface Properties
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D010940:Plant Proteins; C061312:Bet v 1 allergen, Betula; D007073:Immunoglobulin E; D018698:Glutamic Acid; D012694:Serine