1. Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2.
- Author
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Woodfolk JA, Glesner J, Wright PW, Kepley CL, Li M, Himly M, Muehling LM, Gustchina A, Wlodawer A, Chapman MD, and Pomés A
- Subjects
- Allergens immunology, Animals, Antibodies, Monoclonal immunology, Aspartic Acid Endopeptidases immunology, Asthma etiology, CD4-Positive T-Lymphocytes cytology, Crystallography, X-Ray, Epitopes, T-Lymphocyte chemistry, Humans, Immunoglobulin E immunology, Insect Proteins immunology, Mutagenesis, Mutation, Pichia, Protein Binding, Protein Conformation, Th1 Cells cytology, Th2 Cells cytology, Allergens chemistry, Aspartic Acid Endopeptidases chemistry, Cockroaches chemistry, Insect Proteins chemistry
- Abstract
Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ∼100-fold reduced capacity to bind IgE, while preserving the native molecular fold, as proven by x-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and the CD4(+) T-cell phenotype in peripheral blood mononuclear cell cultures. Although T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T-cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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