Hypoallergenic Mutants of the Timothy Grass Pollen Allergen Phl p 5 Generated by Proline Mutations
| Autor: | Martin Wald, Helmut Fiebig, Helga Kahlert, Andreas Nandy, Gerald Reese, Walter Keller, Oliver Cromwell, Nina Krontal, Domen Zafred |
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| Rok vydání: | 2012 |
| Předmět: |
Adult
Male Models Molecular Allergy Proline T-Lymphocytes Immunology Mutant In Vitro Techniques medicine.disease_cause Protein Structure Secondary Microbiology law.invention Phleum Allergen law otorhinolaryngologic diseases medicine Humans Immunology and Allergy Aged Plant Proteins Sequence Deletion Timothy-grass biology Rhinitis Allergic Seasonal Hypoallergenic General Medicine Allergens Immunoglobulin E Middle Aged biology.organism_classification medicine.disease Recombinant Proteins Basophils Amino Acid Substitution Solubility Desensitization Immunologic Mutagenesis Site-Directed Recombinant DNA Pollen Female Mutant Proteins Protein Multimerization |
| Zdroj: | International Archives of Allergy and Immunology. 159:130-142 |
| ISSN: | 1423-0097 1018-2438 |
| Popis: | Background: Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions. Methods: All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays. Results: Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen. Conclusions: The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens. |
| Databáze: | OpenAIRE |
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