Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Original Paper Int Arch Allergy Immunol 2006;139:53–62 DOI: 10.1159/000089756 Mutational Analysis of Amino Acid Positions Crucial for IgE-Binding Epitopes of the Major Apple (Malus domestica) Allergen, Mal d 1 Yan Ma a Gabriele Gadermaier b Barbara Bohle a Suzanne Bolhaar c Andre Knulst c Zora Markovic-Housley d Heimo Breiteneder a Peter Briza b Karin Hoffmann-Sommergruber a Fatima Ferreira b a Department of Pathophysiology, Medical University of Vienna, Vienna, b Department of Molecular Biology, University of Salzburg, Salzburg, Austria; c Department of Dermatology/Allergology, University Medical Center Utrecht, Utrecht, The Netherlands; d Department of Structural Biology, Biozentrum, University of Basel, Basel, Switzerland tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to in- duce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule ac- cording to peripheral blood mononuclear cell prolifera- tion assays. Conclusions: Our findings suggest that it is possible to modulate the IgE-binding properties of aller- gens by single amino acid substitutions at crucial posi- tions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy. Copyright © 2006 S. Karger AG, Basel Introduction Patients suffering from allergy to birch pollen often develop allergic reactions after ingestion of various fruits (e.g. apple, pear, peach, cherry), nuts (e.g. hazelnut, wal- nut), and vegetables (e.g. celery, carrot, potato) [1–3]. The major birch pollen allergen, Bet v 1, and its correspond- ing homologues in these foods could be identified as the cross-reactive molecules mainly responsible for this form of food allergy [4–8] . The deduced amino acid sequence of Mal d 1, the major apple allergen, shows 55% identity to Bet v 1a (EMBL, Genbank Database access No. Key Words Mal d 1  Bet v 1  Apple allergy  Hypoallergenic  Recombinant allergen  Point mutation Abstract 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 appli- cable 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 aller- genic 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 com- pared to wild-ype Mal d 1 in in vitro assays in the major- ity of the sera tested. In ELISA, 10 out of 14 serum sam- ples displayed an 88–30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick Received: June 8, 2005 Accepted after revision: September 14, 2005 Published online: November 15, 2005 Correspondence to: Dr. Karin Hoffmann-Sommergruber Department of Pathophysiology, Medical University Vienna AKH-EBO 3Q Währinger Gürtel 18–20, AT–1090 Vienna (Austria) Tel. +43 1 40400 5132, Fax +43 1 40400 5130 E-Mail Karin.Hoffmann-Sommergruber@meduniwien.ac.at © 2006 S. Karger AG, Basel 1018–2438/06/1391–0053$23.50/0 Accessible online at: www.karger.com/iaa Y.M. and G.G. contributed equally. Downloaded by: Universitätsbibliothek Medizin Basel 131.152.211.61 - 10/11/2017 3:12:14 PM