Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao

Background: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. Methodology/Principal Findings: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by sitedirected mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. Conclusions/Significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties. (Résumé d'auteur

Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1

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-type 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. Copyright (C) 2006 S. Karger AG, Basel

Identification of European allergy patterns to the allergen families PR-10, LTP, and profilin from rosaceae fruits

High fruit intakes are associated with significant health benefits but fruit allergy sufferers may be discouraged from eating fruit due to the symptoms they experience. Knowledge about allergens involved in fruit allergy and the frequent cross-reactions to other allergens is essential to (a) design the best strategy for fruit allergy testing (b) prescribe optimal avoidance diets, and (c) design technological solutions for development of hypoallergenic fruits. The objective of this review was to investigate whether some characteristic disease entities could be identified in Europe for allergy to Rosaceae fruits. Five allergy patterns were found involving the allergen families PR-10, LTP, and profilin. In the Western Mediterranean area allergies to Rosaceae fruits are caused by monosensitization to LTP, monosensitization to profilin, or co-sensitization to both these allergens. On the contrary, monosensitization to PR-10 and, to a lesser degree, co-sensitization to profilin and PR-10 is dominant in Northern and Central Europe. LTP sensitization is present both in pollinosis and non-pollinosis patients and is associated with peach allergy in particular. The disease pattern for patients sensitized to profilin is characterized by several concomitant allergies including grass and other pollens, Rosaceae and non-Rosaceae fruits. Finally, PR-10 sensitization is primarily associated to concomitant birch pollen and apple allergy