Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155–159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.
- allergen structure
- hypoallergenic mutants
- IgE-epitope analysis
↵2 Present address: JASCO, Robert-Bosch-Strasse 11, 64823 Gross-Umstadt, Germany.
↵3 Present address: Forschungszentrum Juelich, IBI-2, 52425 Juelich, Germany, and Institut fuer Physikalische Biologie, Heinrich-Heine-Universitaet, 40225 Duesseldorf, Germany.
Abbreviations used: 1D/2D/3D, one-/two-/three-dimensional; EAST, enzyme allergosorbent test; HMQC, heteronuclear multiple quantum correlation spectroscopy; HSQC, heteronuclear single-quantum coherence; mAb, monoclonal antibody; OAS, oral allergy syndrome; RMSD, root-mean-square deviation; SIT, specific immunotherapy.
- The Biochemical Society, London ©2003