title: Epitope mapping of the house-dust-mite allergen Der p 2 by means of site-directed mutagenesis.
abstract: Recombinant Der p 2, expressed in the baker's yeast Saccharomyces cerevisiae, was used as a tool to determine IgE- and monoclonal antibody (mAb)-binding sites on this allergen. For this purpose, mutant molecules were produced by application of site-directed mutagenesis. The amino-acid residues spanning cys21-cys27 and cys73-cys78 were deleted, thus preventing loop formation through disulfide bonds. Charged residues in three predicted antigenic sites (residues 45-48, 67 + 69, and 88-90) were replaced by alanine residues, IgE- and mAb reactivity to these mutants was compared to that to "wild type" Der p 2. Residues spanning cys73-cys78 were involved in the antigenic binding site for mAb alpha DpX. Mutations in the areas adjacent to this loop (i.e., 67 + 69 and 88-90) had similar effects on this mAb (10- to 20-fold decreases in reactivity were observed), supporting the suggestion that these areas are involved in this antigenic structure. The area of residues 45-48 was shown to be involved in an epitope for mAb 2B12. The reactivity of mAb 7A1 was influenced by substitutions of residues 45-48 as well as 88-90. Deletion of the residues spanning cys21-cys27 resulted in decreased reactivity to three mAbs (10E11, alpha DpX, and 7A1). From these observations, it may be concluded that binding of different mAbs is influenced by the same mutations and that the binding of single mAbs is influenced by two or more mutations scattered over the allergen molecule. These findings can point in two directions: minor amino-acid changes result in disruption of the overall conformation of the allergen, or distant sites are close together in the three-dimensional structure of the allergen. Decreased IgE reactivity was observed with all mutant molecules, varying between patients. The observed effects ranged from 5- to 1000-fold. Deletion of the amino-acid residues spanning cys21-cys27 and cys73-cys78 had the strongest effect on IgE reactivity, where decreases up to 1000-fold were observed. Such mutants might be useful tools to improve the safety of allergen-specific immunotherapy.
authors.affiliation: Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, University of Amsterdam, The Netherlands.
authors.forename: G A
authors.initials: GA
authors.lastname: Hakkaart
authors.forename: R C
authors.initials: RC
authors.lastname: Aalberse
authors.forename: R
authors.initials: R
authors.lastname: van Ree
mesh_terms: D000485:Allergens / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D039741:Antigens, Dermatophagoides; D018076:DNA, Complementary / Q000235:genetics; D018604:Epitope Mapping / Q000379:methods*; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D018076:DNA, Complementary; D006023:Glycoproteins; D007073:Immunoglobulin E