Abstract
BackgroundThe 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 FindingsMutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by site-directed 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/SignificanceWe 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.
Highlights
The development of genetically modified organisms (GMOs) through molecular engineering techniques is an alternative to plant genetic improvement programs for the purpose of promoting resistance against pathogens, herbicides or environmental stresses [1,2]
Conclusions/Significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of immunoglobulin E (IgE) production and cellular infiltration in the lungs
Based on the sequence of continuous amino acids, one should note that the TcPR-10 gene showed similarity to allergenic proteins especially in the region rich in glycine (P-loop motif 47GDGGVGSIK55) (Figure 1)
Summary
The development of genetically modified organisms (GMOs) through molecular engineering techniques is an alternative to plant genetic improvement programs for the purpose of promoting resistance against pathogens, herbicides or environmental stresses [1,2]. Among the allergenic proteins classified as PR-10, the Bet v 1 isolated from Betula verrucosa is the main allergen present in pollen grains [12,13] Food allergens such as Pru p 1 from pear (Prunus persica) [14], Mal d 1 from apple (Malus domestica) [15], Pru av 1 from cherry (P.avium) [16,17] and Dau c 1 in carrot (Daucus carota) [18] are reported as part of the PR-10 family. The P-loop motif present in some allergenic proteins such as Mal d 1 (Malus domestica) [22], Bet v 1 (Betula verrucosa) [12] and Api g 1 (Apium graveolens) [23] is conserved in the TcPR-10 gene identified in a cDNA library observed in the interaction between Theobroma cacao and Moniliophthora perniciosa. 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
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