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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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Characterization of purified Tri a 37 expressed in E.coli cells (EcTri a 37) and in baculovirus-infected insect cells (BvTri a 37) as well as detection of natural Tri a 37 in wheat seed extract.(A) Coomassie brilliant blue-stained SDS-PAGE under reducing (lane R) and under non-reducing (lane NR) conditions. A protein molecular weight marker (kDa) is shown on the left side. (B) Detection of IgE-reactivitiy to Western-blotted EcTri a 37 and BvTri a 37, using serum from a wheat food allergic patient (P) and buffer (B) as a control. Western-blotted wheat seed extract was tested with Tri a 37-specific rabbit antibodies (Immune) or the corresponding pre-immune serum (Pre). Bound human IgE and rabbit IgG antibodies were detected with 125Iodine-labeled antibodies and visualized by autoradiography. Molecular weights are indicated in kilo Dalton (kDa). (C) Mass spectrometry of recombinant EcTri a 37 and BvTri a 37. The mass/charge ratios are shown on the x-axes, and the intensities are displayed on the y-axes as percentages of the most intensive signals obtained in the investigated mass range. (D) Circular dichroism analysis of recombinant EcTri a 37 and BvTri a 37. The spectra are expressed as mean residue ellipticities (θ) (y-axes) at given wavelengths (x-axes).
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pone-0111483-g001: Characterization of purified Tri a 37 expressed in E.coli cells (EcTri a 37) and in baculovirus-infected insect cells (BvTri a 37) as well as detection of natural Tri a 37 in wheat seed extract.(A) Coomassie brilliant blue-stained SDS-PAGE under reducing (lane R) and under non-reducing (lane NR) conditions. A protein molecular weight marker (kDa) is shown on the left side. (B) Detection of IgE-reactivitiy to Western-blotted EcTri a 37 and BvTri a 37, using serum from a wheat food allergic patient (P) and buffer (B) as a control. Western-blotted wheat seed extract was tested with Tri a 37-specific rabbit antibodies (Immune) or the corresponding pre-immune serum (Pre). Bound human IgE and rabbit IgG antibodies were detected with 125Iodine-labeled antibodies and visualized by autoradiography. Molecular weights are indicated in kilo Dalton (kDa). (C) Mass spectrometry of recombinant EcTri a 37 and BvTri a 37. The mass/charge ratios are shown on the x-axes, and the intensities are displayed on the y-axes as percentages of the most intensive signals obtained in the investigated mass range. (D) Circular dichroism analysis of recombinant EcTri a 37 and BvTri a 37. The spectra are expressed as mean residue ellipticities (θ) (y-axes) at given wavelengths (x-axes).

Mentions: rTri a 37 expressed in a prokaryotic system using E.coli cells was designated EcTri a 37, however rTri a 37 expressed in baculovirus-infected insect cells was termed BvTri a 37. Both recombinant proteins were purified by Nickel affinity chromatography and showed a similar migration behaviour in Coomassie Blue-stained SDS-PAGE under reducing conditions where they appeared as bands of approximately 18kDa which differs from their calculated molecular weights (EcTri a 37 including N-terminal methionine and C-terminal hexahistine-tag: 12.757 Da; BvTri a 37 including a D and P residue at the N-terminus and a C-terminal hexahistidine-tag: 12.838 Da) and may be explained by the high content of positively charged amino acids (Figure 1A). Under non-reducing conditions, BvTri a 37 appeared as single band of approximately 14 kDa whereas EcTri a 37 showed an additional second band of approximately 18 kDa but no high molecular weight aggregates were visible in both protein preparations (Figure 1A). Using serum IgE from a wheat food allergic patient, monomeric EcTri a 37 and BvTri a 37 were detected at approximately 18 kDa by immunoblotting. Tri a 37-specific rabbit antibodies reacted with natural, monomeric Tri a 37 at 15 kDa in a nitrocellulose-blotted aqueous wheat seed extract (Figure 1B).


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)

Characterization of purified Tri a 37 expressed in E.coli cells (EcTri a 37) and in baculovirus-infected insect cells (BvTri a 37) as well as detection of natural Tri a 37 in wheat seed extract.(A) Coomassie brilliant blue-stained SDS-PAGE under reducing (lane R) and under non-reducing (lane NR) conditions. A protein molecular weight marker (kDa) is shown on the left side. (B) Detection of IgE-reactivitiy to Western-blotted EcTri a 37 and BvTri a 37, using serum from a wheat food allergic patient (P) and buffer (B) as a control. Western-blotted wheat seed extract was tested with Tri a 37-specific rabbit antibodies (Immune) or the corresponding pre-immune serum (Pre). Bound human IgE and rabbit IgG antibodies were detected with 125Iodine-labeled antibodies and visualized by autoradiography. Molecular weights are indicated in kilo Dalton (kDa). (C) Mass spectrometry of recombinant EcTri a 37 and BvTri a 37. The mass/charge ratios are shown on the x-axes, and the intensities are displayed on the y-axes as percentages of the most intensive signals obtained in the investigated mass range. (D) Circular dichroism analysis of recombinant EcTri a 37 and BvTri a 37. The spectra are expressed as mean residue ellipticities (θ) (y-axes) at given wavelengths (x-axes).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219751&req=5

pone-0111483-g001: Characterization of purified Tri a 37 expressed in E.coli cells (EcTri a 37) and in baculovirus-infected insect cells (BvTri a 37) as well as detection of natural Tri a 37 in wheat seed extract.(A) Coomassie brilliant blue-stained SDS-PAGE under reducing (lane R) and under non-reducing (lane NR) conditions. A protein molecular weight marker (kDa) is shown on the left side. (B) Detection of IgE-reactivitiy to Western-blotted EcTri a 37 and BvTri a 37, using serum from a wheat food allergic patient (P) and buffer (B) as a control. Western-blotted wheat seed extract was tested with Tri a 37-specific rabbit antibodies (Immune) or the corresponding pre-immune serum (Pre). Bound human IgE and rabbit IgG antibodies were detected with 125Iodine-labeled antibodies and visualized by autoradiography. Molecular weights are indicated in kilo Dalton (kDa). (C) Mass spectrometry of recombinant EcTri a 37 and BvTri a 37. The mass/charge ratios are shown on the x-axes, and the intensities are displayed on the y-axes as percentages of the most intensive signals obtained in the investigated mass range. (D) Circular dichroism analysis of recombinant EcTri a 37 and BvTri a 37. The spectra are expressed as mean residue ellipticities (θ) (y-axes) at given wavelengths (x-axes).
Mentions: rTri a 37 expressed in a prokaryotic system using E.coli cells was designated EcTri a 37, however rTri a 37 expressed in baculovirus-infected insect cells was termed BvTri a 37. Both recombinant proteins were purified by Nickel affinity chromatography and showed a similar migration behaviour in Coomassie Blue-stained SDS-PAGE under reducing conditions where they appeared as bands of approximately 18kDa which differs from their calculated molecular weights (EcTri a 37 including N-terminal methionine and C-terminal hexahistine-tag: 12.757 Da; BvTri a 37 including a D and P residue at the N-terminus and a C-terminal hexahistidine-tag: 12.838 Da) and may be explained by the high content of positively charged amino acids (Figure 1A). Under non-reducing conditions, BvTri a 37 appeared as single band of approximately 14 kDa whereas EcTri a 37 showed an additional second band of approximately 18 kDa but no high molecular weight aggregates were visible in both protein preparations (Figure 1A). Using serum IgE from a wheat food allergic patient, monomeric EcTri a 37 and BvTri a 37 were detected at approximately 18 kDa by immunoblotting. Tri a 37-specific rabbit antibodies reacted with natural, monomeric Tri a 37 at 15 kDa in a nitrocellulose-blotted aqueous wheat seed extract (Figure 1B).

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