title: Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2.
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.
authors.affiliation: From the Allergy Division, Department of Medicine, University of Virginia, Charlottesville, Virginia 22903.
authors.forename: Judith A
authors.initials: JA
authors.lastname: Woodfolk
authors.affiliation: INDOOR Biotechnologies, Inc., Charlottesville, Virginia 22908.
authors.forename: Jill
authors.initials: J
authors.lastname: Glesner
authors.affiliation: From the Allergy Division, Department of Medicine, University of Virginia, Charlottesville, Virginia 22903.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Wright
authors.affiliation: the Joint School of Nanoscience and Nanoengineering, University of North Carolina, Greensboro, North Carolina 27401.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Kepley
authors.affiliation: the Macromolecular Crystallography Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, Maryland 21702, and.
authors.forename: Mi
authors.initials: M
authors.lastname: Li
authors.affiliation: the Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Himly
authors.affiliation: From the Allergy Division, Department of Medicine, University of Virginia, Charlottesville, Virginia 22903.
authors.forename: Lyndsey M
authors.initials: LM
authors.lastname: Muehling
authors.affiliation: the Macromolecular Crystallography Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702.
authors.forename: Alla
authors.initials: A
authors.lastname: Gustchina
authors.affiliation: the Macromolecular Crystallography Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702.
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.affiliation: INDOOR Biotechnologies, Inc., Charlottesville, Virginia 22908.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Chapman
authors.affiliation: INDOOR Biotechnologies, Inc., Charlottesville, Virginia 22908, apomes@inbio.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Pomés
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000276:immunology; D001249:Asthma / Q000209:etiology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D003058:Cockroaches / Q000737:chemistry*; D018360:Crystallography, X-Ray; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D019476:Insect Proteins / Q000737:chemistry* / Q000276:immunology; D016296:Mutagenesis; D009154:Mutation; D010843:Pichia; D011485:Protein Binding; D011487:Protein Conformation; D018417:Th1 Cells / Q000166:cytology; D018418:Th2 Cells / Q000166:cytology
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D018984:Epitopes, T-Lymphocyte; D019476:Insect Proteins; D007073:Immunoglobulin E; D016282:Aspartic Acid Endopeptidases; C094699:allergen Bla g 2
keywords: Bla g 2; allergen; antigenic determinant; aspartic protease; asthma; cockroach allergy; epitope mapping; immunotherapy; site-directed mutagenesis; x-ray crystallography