title: The alpha and beta subchain of Amb a 1, the major ragweed-pollen allergen show divergent reactivity at the IgE and T-cell level.
abstract: Ragweed is one of the most important pollen allergens in North America and parts of Europe. Although the major allergen Amb a 1 was isolated and cloned in 1991, recombinant Amb a 1 was not explored further to improve diagnosis and specific immunotherapy of ragweed-pollen allergy. In the present study the immunological properties of natural Amb a 1 and its proteolytical cleavage products was investigated in detail and compared with recombinant produced Amb a 1 variants. Characterization of natural Amb a 1 and the identification of its proteolytic fragments, designated Amb a 1 alpha and Amb a 1 beta, was performed by N-terminal sequencing and mass spectroscopy. Amb a 1 and fragments were further produced in Escherichia coli, purified, and immunologically characterized. Amb a 1-specific T-cell cultures were used to compare the T-cell response to the different Amb a 1 variants. Divergent immunological properties of Amb a 1 alpha (aa 181-396) and Amb a 1 beta (aa 26-180) were revealed. Amb a 1 beta contained important IgE epitopes, whereas Amb a 1 alpha showed low IgE binding. When compared to natural Amb a 1, all recombinant variants possessed >100-fold reduced IgE-mediated mediator release activity. At the T-cell level recombinant and natural Amb a 1 stimulated comparable T-cell responses and the T-cell reactivity was largely directed to the C-terminal part. The results demonstrated that recombinant Amb a 1 alpha behaves as hypoallergen with reduced IgE binding but preservation of the major T-cell reactivity. In addition, recombinant Amb a 1 alpha can be easily purified to homogeneity in large quantity and therefore represents an ideal candidate for specific immunotherapy.
authors.affiliation: Department of Molecular Biology, University of Salzburg, Austria. nicole.wopfner@sbg.ac.at
authors.forename: Nicole
authors.initials: N
authors.lastname: Wopfner
authors.forename: Beatrice
authors.initials: B
authors.lastname: Jahn-Schmid
authors.forename: Georg
authors.initials: G
authors.lastname: Schmidt
authors.forename: Tanja
authors.initials: T
authors.lastname: Christ
authors.forename: Gudrun
authors.initials: G
authors.lastname: Hubinger
authors.forename: Peter
authors.initials: P
authors.lastname: Briza
authors.forename: Christian
authors.initials: C
authors.lastname: Radauer
authors.forename: Barbara
authors.initials: B
authors.lastname: Bohle
authors.forename: Lothar
authors.initials: L
authors.lastname: Vogel
authors.forename: Christof
authors.initials: C
authors.lastname: Ebner
authors.forename: Riccardo
authors.initials: R
authors.lastname: Asero
authors.forename: Fatima
authors.initials: F
authors.lastname: Ferreira
authors.forename: Robert
authors.initials: R
authors.lastname: Schwarzenbacher
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D031164:Ambrosia / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D052179:Antigens, Plant; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology*; D011058:Pollen / Q000276:immunology*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000276:immunology*; D051381:Rats; D020539:Sequence Analysis, Protein; D040681:Structural Homology, Protein; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000485:Allergens; C026967:Amb a I protein, Ambrosia artemisiifolia; D052179:Antigens, Plant; D010940:Plant Proteins; D021122:Protein Subunits; D007073:Immunoglobulin E