title: Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.
abstract: Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37) and in the eukaryotic system (BvTri a 37) as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR) and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN). Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of sequential IgE epitopes indicates that sensitization to alpha-purothionin occurs via the gut. Both allergens can be used for in-vitro diagnosis of wheat food allergy. The induction of blocking IgG antibodies suggests the usefulness for immunotherapy.
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria; Christian Doppler Laboratory for the Development of Allergen Chips, Medical University of Vienna, Vienna, Austria.
authors.forename: Sandra
authors.initials: S
authors.lastname: Pahr
authors.affiliation: Department of ENT, Medical University of Vienna, Vienna, Austria.
authors.forename: Regina
authors.initials: R
authors.lastname: Selb
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Milena
authors.initials: M
authors.lastname: Weber
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Margarete
authors.initials: M
authors.lastname: Focke-Tejkl
authors.affiliation: Institute of Molecular Biosciences, Karl-Franzens University Graz, Graz, Austria.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Hofer
authors.affiliation: Institute of Molecular Biosciences, Karl-Franzens University Graz, Graz, Austria.
authors.forename: Andela
authors.initials: A
authors.lastname: Dordić
authors.affiliation: Institute of Molecular Biosciences, Karl-Franzens University Graz, Graz, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
authors.affiliation: Allergy Department, 2nd Pediatric Clinic, University of Athens, Athens, Greece.
authors.forename: Nikolaos G
authors.initials: NG
authors.lastname: Papadopoulos
authors.affiliation: Allergy Department, 2nd Pediatric Clinic, University of Athens, Athens, Greece.
authors.forename: Stavroula
authors.initials: S
authors.lastname: Giavi
authors.affiliation: Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Mika
authors.initials: M
authors.lastname: Mäkelä
authors.affiliation: Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Anna
authors.initials: A
authors.lastname: Pelkonen
authors.affiliation: Department of ENT, 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 of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria; Christian Doppler Laboratory for the Development of Allergen Chips, Medical University of Vienna, Vienna, Austria.
authors.forename: Susanne
authors.initials: S
authors.lastname: Vrtala
authors.affiliation: Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000276:immunology; D002460:Cell Line; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D005512:Food Hypersensitivity / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D014908:Triticum / Q000737:chemistry* / Q000276:immunology
chemical_list: D000485:Allergens; D023181:Antimicrobial Cationic Peptides; D000939:Epitopes; D010940:Plant Proteins; D007073:Immunoglobulin E; C016085:purothionin