title: Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1.
abstract: Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1-6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.
authors.affiliation: Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Azra
authors.initials: A
authors.lastname: Dedic
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gadermaier
authors.forename: Lothar
authors.initials: L
authors.lastname: Vogel
authors.forename: Christof
authors.initials: C
authors.lastname: Ebner
authors.forename: Stefan
authors.initials: S
authors.lastname: Vieths
authors.forename: Fátima
authors.initials: F
authors.lastname: Ferreira
authors.forename: Matthias
authors.initials: M
authors.lastname: Egger
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D019306:Artemisia / Q000737:chemistry*; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D011058:Pollen / Q000276:immunology*; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C102724:Art v 1 protein, mugwort; D010940:Plant Proteins; D020033:Protein Isoforms