title: Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.
abstract: The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.|Christian Doppler Laboratory for Allergy Research, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Tetiana
authors.initials: T
authors.lastname: Garmatiuk
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.|Christian Doppler Laboratory for Allergy Research, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ines
authors.initials: I
authors.lastname: Swoboda
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Anna
authors.initials: A
authors.lastname: Twardosz-Kropfmüller
authors.affiliation: Division of Structural Biology, Department of Molecular Biosciences, Karl-Franzens-University Graz, Graz, Austria.
authors.forename: Fabio
authors.initials: F
authors.lastname: Dall'Antonia
authors.affiliation: Division of Structural Biology, Department of Molecular Biosciences, Karl-Franzens-University Graz, Graz, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
authors.affiliation: Plant Molecular Biology and Biotechnology Laboratory, Australian Research Council Centre of Excellence for Integrative Legume Research, Faculty of Land and Food Resources, The University of Melbourne, Parkville, VIC, Australia.
authors.forename: Mohan B
authors.initials: MB
authors.lastname: Singh
authors.affiliation: Plant Molecular Biology and Biotechnology Laboratory, Australian Research Council Centre of Excellence for Integrative Legume Research, Faculty of Land and Food Resources, The University of Melbourne, Parkville, VIC, Australia.
authors.forename: Prem L
authors.initials: PL
authors.lastname: Bhalla
authors.affiliation: Plant Molecular Biology and Biotechnology Laboratory, Australian Research Council Centre of Excellence for Integrative Legume Research, Faculty of Land and Food Resources, The University of Melbourne, Parkville, VIC, Australia.
authors.forename: Takashi
authors.initials: T
authors.lastname: Okada
authors.affiliation: Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
authors.forename: Kinya
authors.initials: K
authors.lastname: Toriyama
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Milena
authors.initials: M
authors.lastname: Weber
authors.affiliation: Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.
authors.forename: Minoo
authors.initials: M
authors.lastname: Ghannadan
authors.affiliation: Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Sperr
authors.affiliation: Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Blatt
authors.affiliation: Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Valent
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Klein
authors.affiliation: Department of Otorhinolaryngology, Medical University of Vienna, Vienna, Austria.
authors.forename: Verena
authors.initials: V
authors.lastname: Niederberger
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.|Christian Doppler Laboratory for Allergy Research, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Mirela
authors.initials: M
authors.lastname: Curin
authors.affiliation: Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria.
authors.forename: Nadja
authors.initials: N
authors.lastname: Balic
authors.affiliation: Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria.
authors.forename: Susanne
authors.initials: S
authors.lastname: Spitzauer
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.|Christian Doppler Laboratory for Allergy Research, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000328:Adult; D000485:Allergens / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation / Q000187:drug effects; D052179:Antigens, Plant / Q000235:genetics / Q000627:therapeutic use; D001491:Basophils / Q000187:drug effects* / Q000276:immunology; D029741:Brassica rapa / Q000276:immunology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use*; D015550:Cell Degranulation / Q000187:drug effects; D002478:Cells, Cultured; D003429:Cross Reactions; D003888:Desensitization, Immunologic / Q000379:methods*; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use*; D011058:Pollen / Q000009:adverse effects / Q000276:immunology; D011487:Protein Conformation; D015202:Protein Engineering; D011817:Rabbits; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000628:therapy*; D055815:Young Adult
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C416543:Bra r 1 protein, Brassica rapa; D002135:Calcium-Binding Proteins; D010940:Plant Proteins; D007073:Immunoglobulin E
keywords: Allergen; Allergy; Brassica; Calcium-binding allergen; Cross-reactivity; Hypoallergenic mutants; Immunotherapy