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Enlarging the toolbox for allergen epitope definition with an allergen-type model protein.

Berkner H, Seutter von Loetzen C, Hartl M, Randow S, Gubesch M, Vogel L, Husslik F, Reuter A, Lidholm J, Ballmer-Weber B, Vieths S, Rösch P, Schiller D - PLoS ONE (2014)

Bottom Line: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE.BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding.Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biopolymers, University of Bayreuth, Bayreuth, Bavaria, Germany.

ABSTRACT

Background: Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.

Method: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.

Results: We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.

Conclusion: Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

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Structural alignment of the Bet v 1 epitope forBV16 and amino acids critical for Ig cross-reactivity in PR-10 allergens.Upper panel: model structures of Δ29NCS_5x and PR-10 allergens showing IgE cross-reactivity with Δ29NCS_5x. The amino acids comprising the BV16-binding epitope and lysine 55 of Bet v 1 and the corresponding residues of Δ29NCS_5x, Gly m 4, and Cor a 1 are listed. Residues of the epitope and of position 55 of Bet v 1 that are critical for Ig cross-reaction are highlighted in green. Lower panel: model structures of Δ29NCS and allergens that do not show IgE cross-reactivity with Δ29NCS_5x. The amino acids corresponding to the epitope in the cross-reactive proteins are listed and highlighted in green.
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pone-0111691-g004: Structural alignment of the Bet v 1 epitope forBV16 and amino acids critical for Ig cross-reactivity in PR-10 allergens.Upper panel: model structures of Δ29NCS_5x and PR-10 allergens showing IgE cross-reactivity with Δ29NCS_5x. The amino acids comprising the BV16-binding epitope and lysine 55 of Bet v 1 and the corresponding residues of Δ29NCS_5x, Gly m 4, and Cor a 1 are listed. Residues of the epitope and of position 55 of Bet v 1 that are critical for Ig cross-reaction are highlighted in green. Lower panel: model structures of Δ29NCS and allergens that do not show IgE cross-reactivity with Δ29NCS_5x. The amino acids corresponding to the epitope in the cross-reactive proteins are listed and highlighted in green.

Mentions: The differential interaction of Δ29NCS_5x and Pr-10 allergens as above with both serum IgE and the Bet v 1-specific mouse monoclonal BV16 let us to hypothesize that Δ29NCS_5x displays an epitope shared only with Bet v 1, Cor a 1.04, and Gly m 4, but not with Dau c 1.01, Api g 1.01, and Pru av 1. Thus, we structurally aligned the epitope grafted onto Δ29NCS_5x and the corresponding surface areas of cross-reactive and non-cross-reactive Bet v 1-related proteins (Figure 4). Residue by residue comparison of the epitope revealed that N43, E45, N47, and K55 of Bet v 1 are shared with Δ29NCS_5x, Gly m 4, and Cor a 1.04, but not with Pru av 1, Api g 1.01, and Dau c 1.01, thus mirroring the IgE/IgG inhibition results (cf. Figure 3) and suggesting that they are critical determinants of IgE cross-reactivity and BV16 binding. To challenge this hypothesis we investigated the substitutional variant Bet v 1_4x (Bet v 1N43I/E45S/N47D/K55A). As for Δ29NCS_5x and Δ29NCS_4x, the amino acid substitutions did not alter the protein conformation according to CD and 1H15N-HSQC NMR spectroscopy (Figure S2). Serum IgE binding to Bet v 1_4x was virtually identical to that of wild type Bet v 1 (Figure 5A). However, inhibition of IgE binding to Δ29NCS_5x was strongly reduced by Bet v 1_4x as compared to Bet v 1 (Figure 5B), and interaction of monoclonal BV16 with Bet v 1_4x was virtually abolished (Figure 5C). The capacity of Bet v 1_4x to induce mediator release in humanized rat basophil leukemia cells via allergen mediated cross-linking of receptor bound human IgE was only slightly reduced as compared to wild type Bet v 1, suggesting the presence of further IgE epitopes compensating for the loss of one IgE epitope in Bet v 1_4x (Figure 5D). As expected, the Δ29NCS variants did not induce mediator release due to the lack of a second IgE epitope needed for allergen-mediated IgE cross-linking. Since Bet v 1_4x could not compete with Δ29NCS_5x for binding to serum IgE, we conclude that we have identified an epitope in a subset of PR-10 allergens that binds cross-reactive IgE and IgG.


Enlarging the toolbox for allergen epitope definition with an allergen-type model protein.

Berkner H, Seutter von Loetzen C, Hartl M, Randow S, Gubesch M, Vogel L, Husslik F, Reuter A, Lidholm J, Ballmer-Weber B, Vieths S, Rösch P, Schiller D - PLoS ONE (2014)

Structural alignment of the Bet v 1 epitope forBV16 and amino acids critical for Ig cross-reactivity in PR-10 allergens.Upper panel: model structures of Δ29NCS_5x and PR-10 allergens showing IgE cross-reactivity with Δ29NCS_5x. The amino acids comprising the BV16-binding epitope and lysine 55 of Bet v 1 and the corresponding residues of Δ29NCS_5x, Gly m 4, and Cor a 1 are listed. Residues of the epitope and of position 55 of Bet v 1 that are critical for Ig cross-reaction are highlighted in green. Lower panel: model structures of Δ29NCS and allergens that do not show IgE cross-reactivity with Δ29NCS_5x. The amino acids corresponding to the epitope in the cross-reactive proteins are listed and highlighted in green.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214763&req=5

pone-0111691-g004: Structural alignment of the Bet v 1 epitope forBV16 and amino acids critical for Ig cross-reactivity in PR-10 allergens.Upper panel: model structures of Δ29NCS_5x and PR-10 allergens showing IgE cross-reactivity with Δ29NCS_5x. The amino acids comprising the BV16-binding epitope and lysine 55 of Bet v 1 and the corresponding residues of Δ29NCS_5x, Gly m 4, and Cor a 1 are listed. Residues of the epitope and of position 55 of Bet v 1 that are critical for Ig cross-reaction are highlighted in green. Lower panel: model structures of Δ29NCS and allergens that do not show IgE cross-reactivity with Δ29NCS_5x. The amino acids corresponding to the epitope in the cross-reactive proteins are listed and highlighted in green.
Mentions: The differential interaction of Δ29NCS_5x and Pr-10 allergens as above with both serum IgE and the Bet v 1-specific mouse monoclonal BV16 let us to hypothesize that Δ29NCS_5x displays an epitope shared only with Bet v 1, Cor a 1.04, and Gly m 4, but not with Dau c 1.01, Api g 1.01, and Pru av 1. Thus, we structurally aligned the epitope grafted onto Δ29NCS_5x and the corresponding surface areas of cross-reactive and non-cross-reactive Bet v 1-related proteins (Figure 4). Residue by residue comparison of the epitope revealed that N43, E45, N47, and K55 of Bet v 1 are shared with Δ29NCS_5x, Gly m 4, and Cor a 1.04, but not with Pru av 1, Api g 1.01, and Dau c 1.01, thus mirroring the IgE/IgG inhibition results (cf. Figure 3) and suggesting that they are critical determinants of IgE cross-reactivity and BV16 binding. To challenge this hypothesis we investigated the substitutional variant Bet v 1_4x (Bet v 1N43I/E45S/N47D/K55A). As for Δ29NCS_5x and Δ29NCS_4x, the amino acid substitutions did not alter the protein conformation according to CD and 1H15N-HSQC NMR spectroscopy (Figure S2). Serum IgE binding to Bet v 1_4x was virtually identical to that of wild type Bet v 1 (Figure 5A). However, inhibition of IgE binding to Δ29NCS_5x was strongly reduced by Bet v 1_4x as compared to Bet v 1 (Figure 5B), and interaction of monoclonal BV16 with Bet v 1_4x was virtually abolished (Figure 5C). The capacity of Bet v 1_4x to induce mediator release in humanized rat basophil leukemia cells via allergen mediated cross-linking of receptor bound human IgE was only slightly reduced as compared to wild type Bet v 1, suggesting the presence of further IgE epitopes compensating for the loss of one IgE epitope in Bet v 1_4x (Figure 5D). As expected, the Δ29NCS variants did not induce mediator release due to the lack of a second IgE epitope needed for allergen-mediated IgE cross-linking. Since Bet v 1_4x could not compete with Δ29NCS_5x for binding to serum IgE, we conclude that we have identified an epitope in a subset of PR-10 allergens that binds cross-reactive IgE and IgG.

Bottom Line: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE.BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding.Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biopolymers, University of Bayreuth, Bayreuth, Bavaria, Germany.

ABSTRACT

Background: Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.

Method: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.

Results: We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.

Conclusion: Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

Show MeSH
Related in: MedlinePlus