title: Engineered hypoallergenic variants of osmotin demonstrate hypoallergenicity with in vitro and in vivo methods.
abstract: The molecular structure of protein and epitope mapping strategies are required to engineer the epitopes of a protein. In the present study, IgE binding regions of osmotin were identified and mutated to obtain hypoallergenic variant. The three dimensional (3-D) model of osmotin obtained by homology modeling comprised of a characteristic thaumatin-like fold. This model was used to predict IgE binding regions of osmotin. These regions were mutated and three mutant proteins with four mutations (Ma, Mb and Mc) and one with six mutations (Mabc) were expressed and purified to homogeneity. IgE binding of the mutant proteins was evaluated by in vitro studies using patients' sera. Ma, Mb and Mc demonstrated reduction in IgE binding of 73%, 83% and 77%, respectively, whereas Mabc showed complete abrogation of IgE binding. Ma, Mb and Mc showed inhibition of 48%, 44% and 38%, respectively to osmotin, while Mabc showed 24% inhibition at 10 μg with pooled patients' sera. Osmotin reached effective concentration at 50% inhibition (EC50) at 3 ng and none of the mutant proteins reach the EC50 value. The immunological response to mutant proteins was examined in mice. Blood, bronchoalveolar lavage fluid spleen and lung tissue were excised from mice for analysis. The mice treated with mutant proteins showed significant reduction in IgE and IgG1 levels as compared to mice given osmotin (p<0.001). Th2 cytokines level in splenocyte supernatant and BALF of mice given mutant proteins were significantly lower (p<0.001), accompanied with significant reduction in cellular infiltration in lungs (p<0.001). In conclusion, osmotin structure was predicted by homology modeling and IgE binding regions predicted were mutated to obtain a hypoallergenic protein.
authors.affiliation: Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, Delhi, India; Department of Biotechnology, University of Pune, Ganeshkhind, Pune, India.
authors.forename: Prerna
authors.initials: P
authors.lastname: Sharma
authors.affiliation: Department of Respiratory Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India.
authors.forename: Shailendra Nath
authors.initials: SN
authors.lastname: Gaur
authors.affiliation: Department of Respiratory Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India.
authors.forename: Nitin
authors.initials: N
authors.lastname: Goel
authors.affiliation: Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, Delhi, India. Electronic address: navdelhi@hotmail.com.
authors.forename: Naveen
authors.initials: N
authors.lastname: Arora
mesh_terms: D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D003198:Computer Simulation; D016207:Cytokines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008168:Lung / Q000276:immunology / Q000473:pathology; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D011485:Protein Binding; D015202:Protein Engineering; D040681:Structural Homology, Protein; D055815:Young Adult
chemical_list: D000485:Allergens; D016207:Cytokines; D050505:Mutant Proteins; D010455:Peptides; D010940:Plant Proteins; C073139:osmotin protein, Nicotiana tabacum; D007073:Immunoglobulin E
keywords: Allergenicity; B-cell epitopes; Genetically modified; Hypoallergenic variant; Mutants; Osmotin