Mutational Analysis of Amino Acid Positions Crucial for IgE-Binding Epitopes of the Major Apple (Malus domestica) Allergen, Mal d 1

• Published in: International Archives of Allergy and Immunology 2006-01, Vol.139 (1), p.53-62
• Main Authors: Ma, Yan, Gadermaier, Gabriele, Bohle, Barbara, Bolhaar, Suzanne, Knulst, Andre, Markovic-Housley, Zora, Breiteneder, Heimo, Briza, Peter, Hoffmann-Sommergruber, Karin, Ferreira, Fatima
• Format: Article
• Language: English
• Subjects: Allergens - chemistry; Allergens - genetics; Allergens - immunology; Allergens - metabolism; Allergies; Amino Acid Sequence; Amino acids; Antibody Specificity; Antigens, Plant; Betula verrucosa; Binding sites; Binding Sites, Antibody - immunology; Biological and medical sciences; Epitopes - immunology; Escherichia coli; Food Hypersensitivity - immunology; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunoglobulin E - immunology; Immunopathology; Malus; Malus - adverse effects; Malus domestica; Medical sciences; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Original Paper; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - immunology; Plant Proteins - metabolism; Point Mutation; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Sequence Alignment
• ISSN: 1018-2438; 1423-0097; 1365-2567
• DOI: 10.1159/000089756

Background: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1. Methods: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed. Results: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88–30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays. Conclusions: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.