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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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Localization of synthetic peptides in the Tri a 37 sequence and surface accessibility prediction.(A) Amino acid sequence of Tri a 37 showing the N- and C-terminal domain and the originally isolated IgE-reactive fragment (underlined). Cysteins are boxed, basic and acidic amino acids are coloured in red and blue, respectively. Synthetic Tri a 37 peptides P1–P5 are indicated. (B) Surface accessibility plot of the Tri a 37 amino acid sequence. The numbers of the amino acids, the thionin domain (green), the C-terminal acidic extension domain (pink), the synthetic peptides (yellow) are indicated on the x-axis. The calculated accessibility of the residues is indicated on the y-axis. Scores above 5.5 (bold horizontal line) represent regions of high surface accessibility.
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pone-0111483-g003: Localization of synthetic peptides in the Tri a 37 sequence and surface accessibility prediction.(A) Amino acid sequence of Tri a 37 showing the N- and C-terminal domain and the originally isolated IgE-reactive fragment (underlined). Cysteins are boxed, basic and acidic amino acids are coloured in red and blue, respectively. Synthetic Tri a 37 peptides P1–P5 are indicated. (B) Surface accessibility plot of the Tri a 37 amino acid sequence. The numbers of the amino acids, the thionin domain (green), the C-terminal acidic extension domain (pink), the synthetic peptides (yellow) are indicated on the x-axis. The calculated accessibility of the residues is indicated on the y-axis. Scores above 5.5 (bold horizontal line) represent regions of high surface accessibility.

Mentions: In order to study the IgE epitopes of Tri a 37 in more detail, we tested the sera from the wheat allergic patients also for IgE reactivity with synthetic Tri a 37 peptides (Figure 2B). Five overlapping peptides spanning the Tri a 37 sequence were synthesized and purified (Figure 3, Table 2). With the exception of peptide 4 which was 29 amino acids long, the synthesized peptides were 30 amino acids long with overlaps of ten amino acids (Figure 3A). The molecular weights of the peptides range from 3095 to 3291 Dalton. Three of the synthesized peptides were from the original IgE-reactive recombinant fragment whereas peptides 1 and 2 also included portions of the N-terminal thionin domain which is rich in basic amino acids and has a pI of 9.75 whereas the C-terminal acidic extension domain is acidic (pI 3.53) (Figure 3A; Table 2). Hence, the isoelectric points of the individual peptides ranged from 3.71 to 9.69 depending on the domain from which they originated (Table 2). Peptides 1, 3 and 4 from both domains were recognized by IgE antibodies from Tri a 37-sensitized patients and also by Tri a 37-specific rabbit antibodies. Peptides 2 and 5 did not react with IgE from the tested patients and also not with rabbit IgG antibodies raised by immunization with rTri a 37 (Figure 2B). According to the prediction of surface-exposed portions of Tri a 37, the IgE-reactive peptides were derived from surface-exposed regions (e.g., peptides 3 and 4) but included also less surface-exposed areas (i.e., peptide 1) (Figure 3B).


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)

Localization of synthetic peptides in the Tri a 37 sequence and surface accessibility prediction.(A) Amino acid sequence of Tri a 37 showing the N- and C-terminal domain and the originally isolated IgE-reactive fragment (underlined). Cysteins are boxed, basic and acidic amino acids are coloured in red and blue, respectively. Synthetic Tri a 37 peptides P1–P5 are indicated. (B) Surface accessibility plot of the Tri a 37 amino acid sequence. The numbers of the amino acids, the thionin domain (green), the C-terminal acidic extension domain (pink), the synthetic peptides (yellow) are indicated on the x-axis. The calculated accessibility of the residues is indicated on the y-axis. Scores above 5.5 (bold horizontal line) represent regions of high surface accessibility.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111483-g003: Localization of synthetic peptides in the Tri a 37 sequence and surface accessibility prediction.(A) Amino acid sequence of Tri a 37 showing the N- and C-terminal domain and the originally isolated IgE-reactive fragment (underlined). Cysteins are boxed, basic and acidic amino acids are coloured in red and blue, respectively. Synthetic Tri a 37 peptides P1–P5 are indicated. (B) Surface accessibility plot of the Tri a 37 amino acid sequence. The numbers of the amino acids, the thionin domain (green), the C-terminal acidic extension domain (pink), the synthetic peptides (yellow) are indicated on the x-axis. The calculated accessibility of the residues is indicated on the y-axis. Scores above 5.5 (bold horizontal line) represent regions of high surface accessibility.
Mentions: In order to study the IgE epitopes of Tri a 37 in more detail, we tested the sera from the wheat allergic patients also for IgE reactivity with synthetic Tri a 37 peptides (Figure 2B). Five overlapping peptides spanning the Tri a 37 sequence were synthesized and purified (Figure 3, Table 2). With the exception of peptide 4 which was 29 amino acids long, the synthesized peptides were 30 amino acids long with overlaps of ten amino acids (Figure 3A). The molecular weights of the peptides range from 3095 to 3291 Dalton. Three of the synthesized peptides were from the original IgE-reactive recombinant fragment whereas peptides 1 and 2 also included portions of the N-terminal thionin domain which is rich in basic amino acids and has a pI of 9.75 whereas the C-terminal acidic extension domain is acidic (pI 3.53) (Figure 3A; Table 2). Hence, the isoelectric points of the individual peptides ranged from 3.71 to 9.69 depending on the domain from which they originated (Table 2). Peptides 1, 3 and 4 from both domains were recognized by IgE antibodies from Tri a 37-sensitized patients and also by Tri a 37-specific rabbit antibodies. Peptides 2 and 5 did not react with IgE from the tested patients and also not with rabbit IgG antibodies raised by immunization with rTri a 37 (Figure 2B). According to the prediction of surface-exposed portions of Tri a 37, the IgE-reactive peptides were derived from surface-exposed regions (e.g., peptides 3 and 4) but included also less surface-exposed areas (i.e., peptide 1) (Figure 3B).

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