title: 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.
  
  
  RESULTS
  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
  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.
authors: UESC, Centro de Biotecnologia e Genética, Ilhéus, Bahia, Brasil.
authors: Sara Pereira
authors: SP
authors: Menezes
authors: Jane Lima
authors: JL
authors: dos Santos
authors: Thyago Hermylly Santana
authors: TH
authors: Cardoso
authors: Carlos Priminho
authors: CP
authors: Pirovani
authors: Fabienne
authors: F
authors: Micheli
authors: Fátima Soares Motta
authors: FS
authors: Noronha
authors: Andréa Catão
authors: AC
authors: Alves
authors: Ana Maria Caetano
authors: AM
authors: Faria
authors: Abelmon da Silva
authors: Ada S
authors: Gesteira
mesh_terms: D000465:Algorithms; D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D052179:Antigens, Plant / Q000737:chemistry / Q000276:immunology*; D001487:Basidiomycota / Q000166:cytology / Q000187:drug effects; D018893:Bronchoalveolar Lavage; D002099:Cacao / Q000737:chemistry / Q000276:immunology; D002452:Cell Count; D019295:Computational Biology; D030562:Databases, Protein; D005260:Female; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D007073:Immunoglobulin E / Q000276:immunology; D008168:Lung / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D012260:Ribonucleases / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein
chemical_list: D000485:Allergens; D000935:Antifungal Agents; D052179:Antigens, Plant; D050505:Mutant Proteins; D010940:Plant Proteins; D007073:Immunoglobulin E; D012260:Ribonucleases