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Evaluation of the allergenicity potential of TcPR-10 protein from Theobroma cacao

Menezes S.P., Dos Santos J.L., Cardoso T.H.S., Pirovani C.P., Micheli F., Noronha F.S.N., Alves A.C., Faria A.M.C., Da Silva Gesteira A.. 2012. PloS One, 7 (6) : 12 p..

DOI: 10.1371/journal.pone.0037969

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.

Mots-clés : theobroma cacao; moniliophthora; allergène; protéine; résistance aux organismes nuisibles; génie génétique; bioinformatique; moniliophthora perniciosa

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