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

Intermediate Tyrosyl Radical and Amyloid Structure in Peroxide-Activated Cytoglobin.

We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity

Changes in the EA spectrum of Cygb during the reaction with hydrogen peroxide.

<p>A) Bleaching of Soret and Q bands of EA spectra of Cygb in the course of the reaction with hydrogen peroxide. The black line represents the EA spectrum of resting Cygb, red, green and blue lines corresponds to the spectra obtained at 30, 60 and 200 s after addition of hydrogen peroxide and indicated by the arrows. B) Normalized spectra of Cygb resting form and 200 s after hydrogen peroxide addition. C) Differential spectra of Cygb obtained 30 and 200 s after the addition of hydrogen peroxide The experiments of EA spectroscopy were performed using 65 μmol.L^-1 Cygb and 0.1 cm optical length. When present, the concentration of peroxide was 650 μmol.L^-1. These results are representative of three independent replicates.</p

Formation of Cygb amyloid structure after challenge by peroxides.

<p>A), B) and C) show, respectively the epifluorescence images of Cygb control, control plus GSH and challenged by hydrogen peroxide obtained immediately (left panels) 24 h (right panels) after incubation and staining by thioflavine-T. For the low-vacuum SEM experiments, it was used 7 μmol.L^-1 cygb solution with 70 μmol.L^-1 peroxide solutions. For the epifluorescence experiments 70 μmol.L^-1 protein solution was incubated for 1 h with 700 μmol.L^-1 peroxide solution in the presence of thioflavin-T. For FTIR measurements, 7 μmol.L^-1 protein solution was incubated with 70 μmol.L^-1 peroxide solutions for 1 h. The results are representative of three independent experiments.</p

Changes in the EA spectrum of Cygb during the reaction with <i>t</i>-BuOOH.

<p>A) Bleaching of Soret and Q bands of EA spectra of Cygb in the course of the reaction with <i>t</i>-BuOOH. The black line represents the EA spectrum of resting Cygb, red, green and blue lines corresponds to the spectra obtained at 30, 60 and 200 s after addition of <i>t</i>-BuOOH and indicated by the arrows. B) Normalized spectra of Cygb resting form and 200 s after <i>t</i>-BuOOH addition. C) Differential spectra of Cygb obtained 30 and 200 s after the addition of <i>t</i>-BuOOH. The experiments of EA spectroscopy were performed using 65 μmol.L^-1 Cygb and 0.1 cm optical length. When present, the concentration of peroxides was 650 μmol.L^-1. These results are representative of three independent replicates.</p

Interatoma of rat Cygb with hydrogen peroxide.

<p>The network shows, in each node, a protein predicted to have functional links with Cygb and hydrogen peroxide. Inside the figure the abbreviations are SOD1 (superoxide dismutase [Cu-Zn]), Hmox2 (heme oxygenase 2 [HO-2]), Mb (myoglobin), Mpo (myeloperoxidase), cat (catalase), Cygb (cytoglobin), Prdx1 (peroxyredoxin-1), Prdx5 (peroxyredoxin-5) and Srxn1 (Ab2-390). In the figure light green, cyan and magenta lines correspond, respectively, to textmining, databases and experiments supporting the relationship among the proteins and hydrogen peroxide.</p

Spectroscopic characteristics of Cygb.

<p>The upper panel shows the EA spectrum of Cygb and the respective inset the corresponding far-UV CD spectrum. The lower panel shows the CD (light gray line) and the MCD spectra of Cygb obtained by addition and subtraction of the original spectra obtained at positive and negative magnetic fields. MCD is shown at increasing magnetic fields and the respective inset shows the linear increase of Soret band intensity promoted by increasing the magnetic field. The experiments of EA spectroscopy were performed with 65 μmol.L^-1 Cygb using 0.1 cm optical length. The experiments of CD and MCD were performed using 20 μmol.L^-1 protein solution in 20 mmol.L^-1 phosphate buffer, pH 7.4. These results are representative of three independent replicates.</p

Changes in the EPR spectrum of resting Cygb during the reaction with hydrogen peroxide.

<p>The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of hydrogen peroxide. The inset shows a zoom in the spectra of the free radical produced concomitantly with the formation of high valence species. For EPR experiments, the protein concentration was of 1.2 mmol.L^-1and when present, the peroxide concentration was of 12 mmol.L^-1. These results are representative of three independent replicates.</p

EPR spectrum of resting Cygb and the spectral components obtained by simulation.

<p>The red line corresponds to simulation of the composite spectrum and the blue and green lines correspond to the high and low spin components, respectively. The g values of the low spin state component are: g1 = 3.228, g2 = 2.033 and g3 = 1.385 with rhombic distortion and for the high spin state the g values are: g1 = 6.062, g2 = 5.785 and g3 = 2.0409. The simulation was done by the software Symphonia. For EPR experiments, the protein concentration was of 1.2 mmol.L^-1. These results are representative of three independent replicates.</p