title: Targeting the cysteine-stabilized fold of Art v 1 for immunotherapy of Artemisia pollen allergy.
abstract: Plants of the genus Artemisia domestic in Northern and Central Europe, USA and parts of Asia are a major cause of allergic symptoms from late summer to autumn. Art v 1, the major mugwort pollen allergen appears as two-domain glycoprotein, consisting of an N-terminal defensin-like and a proline/hydroxyproline-rich C-terminal part. Patients sensitized to Art v 1 commonly display IgE antibodies against the cysteine-stabilized defensin fold. Site-directed mutagenesis of eight cysteines was used to disrupt disulfide bonds to generate molecules with altered IgE-binding capacity. Engineered constructs were expressed in E. coli and recombinant proteins were tested for their allergenic and T cell reactivity as well as for their physicochemical characteristics. Three cysteine variants (C22S, C47S, and C49S) exhibited extremely low IgE-binding activity in immunoblot and ELISA using sera from Art v 1-allergic patients. Mediator release assays using rat basophil leukemia cells showed that these variants displayed a 1x10(5)-fold reduced allergenic potency as compared to wild-type protein. All variants were able to activate allergen-specific T cells in PBMC, as well as Art v 1-specific T cell lines and clones. Variant C49S displayed an increased hydrophobic surface potential which correlated with an advanced activation of allergen-specific T cells. The low allergenicity and high immunogenic activity of Art v 1 variant C49S renders the molecule an attractive candidate for hypoallergen-based immunotherapy of Artemisia pollen allergy.
authors.affiliation: Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Salzburg, Austria. Gabriele.Gadermaier@sbg.ac.at
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gadermaier
authors.forename: Beatrice
authors.initials: B
authors.lastname: Jahn-Schmid
authors.forename: Lothar
authors.initials: L
authors.lastname: Vogel
authors.forename: Matthias
authors.initials: M
authors.lastname: Egger
authors.forename: Martin
authors.initials: M
authors.lastname: Himly
authors.forename: Peter
authors.initials: P
authors.lastname: Briza
authors.forename: Christof
authors.initials: C
authors.lastname: Ebner
authors.forename: Stefan
authors.initials: S
authors.lastname: Vieths
authors.forename: Barbara
authors.initials: B
authors.lastname: Bohle
authors.forename: Fatima
authors.initials: F
authors.lastname: Ferreira
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000276:immunology*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D052179:Antigens, Plant; D049109:Cell Proliferation; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology / Q000628:therapy*; D007073:Immunoglobulin E / Q000276:immunology; D007167:Immunotherapy; D008213:Lymphocyte Activation; D050505:Mutant Proteins / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology*; D011058:Pollen / Q000737:chemistry / Q000276:immunology*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
chemical_list: D000485:Allergens; D000906:Antibodies; D052179:Antigens, Plant; C102724:Art v 1 protein, mugwort; D050505:Mutant Proteins; D010940:Plant Proteins; D007073:Immunoglobulin E; D003545:Cysteine