Journal Article

Biochemical, Biophysical and IgE-Epitope Characterization of the Wheat Food Allergen, Tri a 37

Pahr, Sandra, Hofer, Gerhard, Papadopoulos, Nikolaos G., Dordić, Andela, Weber, Milena, Focke-Tejkl, Margarete, Selb, Regina, Vrtala, Susanne, Valenta, Rudolf, Pelkonen, Anna, Giavi, Stavroula, Mäkelä, Mika, Keller, Walter, Niederberger, Verena

2014

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Allergens / Allergens - chemistry / Allergens - immunology / Allergies / Amino Acid Sequence / Anaphylaxis / Animals / Antibodies / Antimicrobial Cationic Peptides - chemistry / Antimicrobial Cationic Peptides - immunology / Baculovirus / Binding / Biology and Life Sciences / Cell Line / Chemical synthesis / Chromatography / Circular dichroism / Coding / Crosslinking / Dichroism / Digestion / E coli / Epitope Mapping / Epitopes - chemistry / Epitopes - immunology / Food / Food allergies / Food hypersensitivity / Food Hypersensitivity - immunology / Food sources / Gel electrophoresis / Hospitals / Humans / Immunization / Immunoglobulin E / Immunoglobulin E - immunology / Immunoglobulin G / Immunology / Immunotherapy / Insect cells / Insects / Laboratories / Mass spectrometry / Mass spectroscopy / Molecular Sequence Data / Patients / Pediatrics / Peptide mapping / Peptides / Plant Proteins - chemistry / Plant Proteins - immunology / Proteins / Rabbits / Radioallergosorbent test / Reactivity / Size exclusion chromatography / Sodium lauryl sulfate / Studies / Triticum - chemistry / Triticum - immunology / Wheat