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

Abstract

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 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/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.

Figure 1. Amino acid sequence alignment of TcPR-10 with the allergens from the SDAP database (

http://align.genome.jp/sit-bin/clustalw ). The three point mutations for TcPR-10 (T10P, I30V, H45S) are marked in sequence alignment and P-loop was underlined. The identical, highly conserved, and conserved amino acids among the sequences are denoted with (*), (:), and (.), respectively. Matching regions of contiguous amino acids are highlighted in black.

Figure 2. Three-dimensional structure of TcPR-10 obtained by homology modeling with Pru av1 (Protein Data Bank, 1e09_A) as template using SWISS-MODEL. A.

The secondary structure elements are colored: alpha-helices in red, anti-parallel beta-sheets in yellow and P-loops in green. B. Molecular surface of TcPR-10 wild with matching regions of contiguous amino acids: 47GDGGVG52 in blue; 59FPEGSHFKY67 in brown; 116TSHYHT121 in gray; 129EEEIKAGK136 in peach. C e E Molecular surface of TcPR-10 wild type with amino acids for mutations highlighted in orangen (Thr10, Ile30, His45). D e F. TcPR-10 mutant type with point mutations in blue (Pro10, Val30, Ser45).

Figure 3. SDS-PAGE (15%) analysis of recombinants TcPR-10 Wild Types (wt) and Mutant (mut) proteins expressed in E. coli BL21(DE3).

1– Soluble fraction TcPR-10 mut; 2– Insoluble fraction TcPR10 mut; 3– Soluble fraction TcPR-10 wt; 4– Insoluble fraction TcPR10 wt; 5 - pET28a-TcPR-10 mut without induction; 6 - pET28a-TcPR-10 mut after induction; 7 - pET28a-TcPR-10 wt without induction; 8 - pET28a-TcPR-10 mut 3 h after induction; M- Protein molecular weight markers.

Figure 4. Ribonuclease activity of recombinant TcPR-10 Wild Types (wt) and Mutant visualized in 1% agarose gel. 1 µg RNA from M. perniciosa was incubated with 1 µg of recombinats proteins at 25°C at different times.

Lane 1. RNA without protein; Lane 2. RNA with boiled TcPR10 mut 2 h incubation; Lane 3. RNA with boiled TcPR10 wt 2 h incubation; Lane 4, 6, 8, 10 and 12 RNA incubated with TcPR10 mut by 10 min, 20 min, 1 h, 2 h and 3 h, respectively; Lane 5, 7, 9, 11 and 13 RNA incubated with TcPR10 wt by 10 min 20 min, 1 h, 2 h and 3 h, respectively. Arrows indicate RNA bands without degradation.

Figure 5. Survival of M. perniciosa dikaryotic broken hyphae incubated with different TcPR-10 wild and mutant type protein concentrations (4, 8 and 10 µg/ml).

Figure 6. Quantification of polyclonal IgE, BAL total cell count and histological illustration of the lung of BALB/c mice.

A. Quantification of polyclonal IgE antibody levels in serum of BALB/c mice. B. Cell counting in BAL fluid. The set average values per se quantification of antibodies and showed normal (p<0,05; Shapiro Wilk Test) using the comparison test of means the parametric Tukey Test (α = 0,05). *Significantly high values compared to control. **Significantly reduced values compared to TcPR-10 wt. Horizontal bars represent the mean value of each group. C. Lung were removed twenty-four hours after the last challenge. Lung tissue was fixed, embedded, cut into slices and stained with hematoxylin e eosin (H&E) solution. C: Sections from control; D: wild TcPR-10; E: mutant TcPR-10.

Figure 7. Experimental design of sensitization and challenge with TcPR-10 wild types and mutant.