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

2005; Karger Publishers; Volume: 139; Issue: 1 Linguagem: Inglês

10.1159/000089756

1423-0097

Yan Ma, Gabriele Gadermaier, Barbara Bohle, S. T. H. P. Bolhaar, André C. Knulst, Z. Marković-Housley, Heimo Breiteneder, Peter Briza, Karin Hoffmann‐Sommergruber, Fátima Ferreira,

Contact Dermatitis and Allergies

<i>Background:</i> 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. <i>Methods:</i> A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in <i>Escherichia coli</i> 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. <i>Results:</i> 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. <i>Conclusions:</i> 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.