Targeting the cysteine-stabilized fold of Art v 1 for immunotherapy of Artemisia pollen allergy.
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.
Gadermaier
Gabriele
G
Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Salzburg, Austria. Gabriele.Gadermaier@sbg.ac.at
Jahn-Schmid
Beatrice
B
Vogel
Lothar
L
Egger
Matthias
M
Himly
Martin
M
Briza
Peter
P
Ebner
Christof
C
Vieths
Stefan
S
Bohle
Barbara
B
Ferreira
Fatima
F
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
D000485:Allergens; D000906:Antibodies; D052179:Antigens, Plant; C102724:Art v 1 protein, mugwort; D050505:Mutant Proteins; D010940:Plant Proteins; D007073:Immunoglobulin E; D003545:Cysteine