Identification of IgE-binding epitopes of the major peach allergen Pru p 3
| Autor: | F. Polo, Gabriel Salcedo, Domingo Barber, Rosa Sánchez-Monge, Luis F. Pacios, Gloria Garcia-Casado, Manuel Lombardero, F.J. García-Sellés, Araceli Díaz-Perales |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular DNA Plant Protein Conformation Molecular Sequence Data Static Electricity Immunology In Vitro Techniques medicine.disease_cause Immunoglobulin E Protein Structure Secondary Epitope law.invention Epitopes Allergen law medicine Humans Immunology and Allergy Amino Acid Sequence Peptide sequence Plant Proteins Base Sequence biology Chemistry Allergens Antigens Plant medicine.disease Peptide Fragments Biochemistry Mutagenesis Site-Directed biology.protein Recombinant DNA Prunus Antibody Carrier Proteins Plant lipid transfer proteins Food Hypersensitivity Fruit allergy |
| Zdroj: | Journal of Allergy and Clinical Immunology. 112:599-605 |
| ISSN: | 0091-6749 |
| DOI: | 10.1016/s0091-6749(03)01605-1 |
| Popis: | Background Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro. Objective We sought to locate major IgE-binding epitopes of Pru p 3. Methods A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immu-nodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by 125 I–anti-IgE immunodetection. Results Pru p 3 showed a 3-dimensional structure comprising 4 α-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immu-nodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes. Conclusions Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an α-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms. |
| Databáze: | OpenAIRE |
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