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Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.

Pahr S, Selb R, Weber M, Focke-Tejkl M, Hofer G, Dordić A, Keller W, Papadopoulos NG, Giavi S, Mäkelä M, Pelkonen A, Niederberger V, Vrtala S, Valenta R - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

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Related in: MedlinePlus

Effects of gastric and duodenal digestion on natural Tri a 37.Undigested wheat seed extract (undigested) or after different times of gastric (top panel) or duodenal digestion (lower panel) was subjected to SDS-PAGE and blotted to nitrocellulose. Nitrocelluloses were then tested with rabbit anti-Tri a 37 antibodies (Immune) and the corresponding pre-immune serum (Pre). Bound IgG antibodies were detected with 125I-labeled anti-rabbit IgG antibodies and visualized by autoradiography.
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pone-0111483-g005: Effects of gastric and duodenal digestion on natural Tri a 37.Undigested wheat seed extract (undigested) or after different times of gastric (top panel) or duodenal digestion (lower panel) was subjected to SDS-PAGE and blotted to nitrocellulose. Nitrocelluloses were then tested with rabbit anti-Tri a 37 antibodies (Immune) and the corresponding pre-immune serum (Pre). Bound IgG antibodies were detected with 125I-labeled anti-rabbit IgG antibodies and visualized by autoradiography.

Mentions: Figure 5 shows nitrocellulose-blotted wheat extracts which were subjected to gastric and duodenal digestion and then probed with rabbit anti-Tri a 37 antibodies. Undigested Tri a 37 was detected at 15 kDa whereas one minute of gastric digestion led to the loss of the signal. Under duodenal digestion conditions, Tri a 37 could be detected even after 45 minutes at 15 kDa and an additional smaller IgG binding band at 10 kDa became visible after 10 minutes of digestion (Figure 5).


Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.

Pahr S, Selb R, Weber M, Focke-Tejkl M, Hofer G, Dordić A, Keller W, Papadopoulos NG, Giavi S, Mäkelä M, Pelkonen A, Niederberger V, Vrtala S, Valenta R - PLoS ONE (2014)

Effects of gastric and duodenal digestion on natural Tri a 37.Undigested wheat seed extract (undigested) or after different times of gastric (top panel) or duodenal digestion (lower panel) was subjected to SDS-PAGE and blotted to nitrocellulose. Nitrocelluloses were then tested with rabbit anti-Tri a 37 antibodies (Immune) and the corresponding pre-immune serum (Pre). Bound IgG antibodies were detected with 125I-labeled anti-rabbit IgG antibodies and visualized by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219751&req=5

pone-0111483-g005: Effects of gastric and duodenal digestion on natural Tri a 37.Undigested wheat seed extract (undigested) or after different times of gastric (top panel) or duodenal digestion (lower panel) was subjected to SDS-PAGE and blotted to nitrocellulose. Nitrocelluloses were then tested with rabbit anti-Tri a 37 antibodies (Immune) and the corresponding pre-immune serum (Pre). Bound IgG antibodies were detected with 125I-labeled anti-rabbit IgG antibodies and visualized by autoradiography.
Mentions: Figure 5 shows nitrocellulose-blotted wheat extracts which were subjected to gastric and duodenal digestion and then probed with rabbit anti-Tri a 37 antibodies. Undigested Tri a 37 was detected at 15 kDa whereas one minute of gastric digestion led to the loss of the signal. Under duodenal digestion conditions, Tri a 37 could be detected even after 45 minutes at 15 kDa and an additional smaller IgG binding band at 10 kDa became visible after 10 minutes of digestion (Figure 5).

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus