The alpha and beta subchain of Amb a 1, the major ragweed-pollen allergen show divergent reactivity at the IgE and T-cell level.
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.
Wopfner
Nicole
N
Department of Molecular Biology, University of Salzburg, Austria. nicole.wopfner@sbg.ac.at
Jahn-Schmid
Beatrice
B
Schmidt
Georg
G
Christ
Tanja
T
Hubinger
Gudrun
G
Briza
Peter
P
Radauer
Christian
C
Bohle
Barbara
B
Vogel
Lothar
L
Ebner
Christof
C
Asero
Riccardo
R
Ferreira
Fatima
F
Schwarzenbacher
Robert
R
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*
D000485:Allergens; C026967:Amb a I protein, Ambrosia artemisiifolia; D052179:Antigens, Plant; D010940:Plant Proteins; D021122:Protein Subunits; D007073:Immunoglobulin E