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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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Related in: MedlinePlus

Δ29NCS_5x presents a cross-reactive Ig epitope.(A) Inhibition of IgE binding to Δ29NCS_5x. Binding of serum IgE to Δ29NCS (lane 3) and to Δ29NCS_5x (lane 4) in the presence of increasing amounts of inhibitors Δ29NCS_5x (lanes 5–8) or Bet v 1 (lanes 9–12). No serum (lane 1) and negative serum pool (lane 2) served as control. (B) Dose-dependent inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of increasing concentrations of PR-10 allergens in ELISA. (C) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of both Bet v 1 and serial dilutions of Bet v 1-binding monoclonal mouse IgG antibody BV16 (–: no BV16; 10−5 to 10−3: dilutions of BV16) in ELISA. (D) Binding of mouse monoclonal IgG antibody BV16 to surface-coated Bet v 1-related proteins. 250 ng of protein (50 ng of Api g 1) was coated and incubated with dilution series of the monoclonal BV16 in ELISA.
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pone-0111691-g003: Δ29NCS_5x presents a cross-reactive Ig epitope.(A) Inhibition of IgE binding to Δ29NCS_5x. Binding of serum IgE to Δ29NCS (lane 3) and to Δ29NCS_5x (lane 4) in the presence of increasing amounts of inhibitors Δ29NCS_5x (lanes 5–8) or Bet v 1 (lanes 9–12). No serum (lane 1) and negative serum pool (lane 2) served as control. (B) Dose-dependent inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of increasing concentrations of PR-10 allergens in ELISA. (C) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of both Bet v 1 and serial dilutions of Bet v 1-binding monoclonal mouse IgG antibody BV16 (–: no BV16; 10−5 to 10−3: dilutions of BV16) in ELISA. (D) Binding of mouse monoclonal IgG antibody BV16 to surface-coated Bet v 1-related proteins. 250 ng of protein (50 ng of Api g 1) was coated and incubated with dilution series of the monoclonal BV16 in ELISA.

Mentions: Since the majority of the patients' sera IgE bound Δ29NCS_5x, we asked whether this interaction was due to a Bet v 1-specific IgE epitope presented by the Δ29NCS variant. Therefore we used serum with sIgE to both Bet v 1 and Δ29NCS_5x and tested its IgE interaction with the Δ29NCS_5x variant in the presence of Bet v 1 (Figure 3). As expected, no IgE interaction of the serum pool with Δ29NCS was observed (Figure 3A). In contrast, IgE binding to Δ29NCS_5x was inhibited by Δ29NCS_5x itself and Bet v 1 in a dose-dependent manner, suggesting the presence of an IgE epitope shared by the two proteins.


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)

Δ29NCS_5x presents a cross-reactive Ig epitope.(A) Inhibition of IgE binding to Δ29NCS_5x. Binding of serum IgE to Δ29NCS (lane 3) and to Δ29NCS_5x (lane 4) in the presence of increasing amounts of inhibitors Δ29NCS_5x (lanes 5–8) or Bet v 1 (lanes 9–12). No serum (lane 1) and negative serum pool (lane 2) served as control. (B) Dose-dependent inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of increasing concentrations of PR-10 allergens in ELISA. (C) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of both Bet v 1 and serial dilutions of Bet v 1-binding monoclonal mouse IgG antibody BV16 (–: no BV16; 10−5 to 10−3: dilutions of BV16) in ELISA. (D) Binding of mouse monoclonal IgG antibody BV16 to surface-coated Bet v 1-related proteins. 250 ng of protein (50 ng of Api g 1) was coated and incubated with dilution series of the monoclonal BV16 in ELISA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111691-g003: Δ29NCS_5x presents a cross-reactive Ig epitope.(A) Inhibition of IgE binding to Δ29NCS_5x. Binding of serum IgE to Δ29NCS (lane 3) and to Δ29NCS_5x (lane 4) in the presence of increasing amounts of inhibitors Δ29NCS_5x (lanes 5–8) or Bet v 1 (lanes 9–12). No serum (lane 1) and negative serum pool (lane 2) served as control. (B) Dose-dependent inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of increasing concentrations of PR-10 allergens in ELISA. (C) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of both Bet v 1 and serial dilutions of Bet v 1-binding monoclonal mouse IgG antibody BV16 (–: no BV16; 10−5 to 10−3: dilutions of BV16) in ELISA. (D) Binding of mouse monoclonal IgG antibody BV16 to surface-coated Bet v 1-related proteins. 250 ng of protein (50 ng of Api g 1) was coated and incubated with dilution series of the monoclonal BV16 in ELISA.
Mentions: Since the majority of the patients' sera IgE bound Δ29NCS_5x, we asked whether this interaction was due to a Bet v 1-specific IgE epitope presented by the Δ29NCS variant. Therefore we used serum with sIgE to both Bet v 1 and Δ29NCS_5x and tested its IgE interaction with the Δ29NCS_5x variant in the presence of Bet v 1 (Figure 3). As expected, no IgE interaction of the serum pool with Δ29NCS was observed (Figure 3A). In contrast, IgE binding to Δ29NCS_5x was inhibited by Δ29NCS_5x itself and Bet v 1 in a dose-dependent manner, suggesting the presence of an IgE epitope shared by the two proteins.

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