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: Sandra Pahr, Regina Selb, Milena Weber, Margarete Focke-Tejkl, Gerhard Hofer, Andela Dordić, Walter Keller, Nikolaos G Papadopoulos, Stavroula Giavi, Mika Mäkelä, Anna Pelkonen, Verena Niederberger, Susanne Vrtala, Rudolf Valenta
• Journal: PloS one (PLoS One) Vol. 9 Issue 11 Pg. e111483 ( 2014) ISSN: 1932-6203 [Electronic] United States
• PMID: 25368998 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Allergens
• Antimicrobial Cationic Peptides
• Epitopes
• Plant Proteins
• Immunoglobulin E
• purothionin

Topics

• Allergens (chemistry, immunology)
• Amino Acid Sequence
• Animals
• Antimicrobial Cationic Peptides (chemistry, immunology)
• Cell Line
• Epitope Mapping
• Epitopes (chemistry, immunology)
• Food Hypersensitivity (immunology)
• Humans
• Immunoglobulin E (immunology)
• Molecular Sequence Data
• Plant Proteins (chemistry, immunology)
• Rabbits
• Triticum (chemistry, immunology)