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The Cockroach Allergen Bla g 1 Forms Alpha Helical Capsules with an Internal Lipid Binding Cavity: Implications for Allergenicity
AB16 Abstracts
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The Anionic Surfactant Lipid, Palmitoyl-Oleoyl-Phosphatidylglycerol (POPG), Suppresses Der p 2-Stimulated Inflammatory Responses in Lung Epithelial Cells and Macrophages Sasipa Tanyaratsrisakul, PhD1, Surapon Piboonpocanun, PhD2, Dennis R. Voelker, PhD1; 1National Jewish Health, Denver, 2Institute of Molecular Biosciences, Mahidol University, Thailand. RATIONALE: Der p 2 is the major allergen from house dust mite (HDM). More than 80% of HDM allergic patients have high Der p 2-specific IgE reactivity. Previous studies showed that the lung surfactant lipid, POPG, inhibits LPS-induced inflammatory responses by mouse and human macrophages. This study reports the protective effects of POPG on Der p 2-induced allergic responses in human bronchial epithelial cells (BEAS2B) and mouse alveolar macrophages (RAW264.7). METHODS: Recombinant Der p 2 (rDer p 2) was produced in Pichia pastoris. The structure and IgE reactivity were confirmed by Circular Dichroism (CD) and rDer p 2 inhibition of serum-IgE binding to natural Der p 2. Pro-inflammatory responses were determined using BEAS2B and RAW 264.7 incubated with rDer p 2, Pam3cys, or LPS in the presence, or absence of surfactant phospholipids. RESULTS: rDer p 2-stimulated IL-8 secretion from BEAS2B cells and TNFa secretion from RAW 264.7 cells. POPG suppressed TNFa secretion with IC50 of 13.5 mg/ml and suppressed IL-8 secretion from BEAS 2 B cell with IC50 of 11 mg/ml. A negatively charged phospholipid, palmitoyloleoyl-phosphatidic acid, which has a structure similar to POPG, was ineffective as an inhibitor of IL-8 and TNFa secretion. The site of action of POPG is upstream of JNK and p38 MAP Kinase and appears to be at the level of cell surface receptors for Der p 2. CONCLUSIONS: The Der p 2-TLR2 activated proinflammatory responses of macrophages and epithelial cells occur via JNK and p38 MAPK signaling pathways which can be inhibited by the lung surfactant lipid POPG.
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The Cockroach Allergen Bla g 1 Forms Alpha Helical Capsules with an Internal Lipid Binding Cavity: Implications for Allergenicity Geoffrey Mueller, PhD1, Lars Pedersen1, Fred Lih1, Jill Glesner2, Martin D. Chapman, PhD, FAAAAI2, Ken Tomer1, Robert London1, Anna Pomes, PhD, FAAAAI2; 1National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 2Indoor Biotechnologies, Inc., Charlottesville, VA. RATIONALE: The cockroach allergen Bla g 1 is a marker of cockroach exposure that has been widely used in epidemiologic studies of asthma. Bla g 1 is produced in the digestive tract and has a novel structure comprising tandem amino acid repeats. The goal was to determine the structure of Bla g 1 and to investigate its biological function. METHODS: The structure of Bla g 1 was determined by X-ray crystallography. Ligand-binding properties of the allergen were examined by mass spectrometry and NMR. Natural Bla g 1 and recombinant constructs expressed in Escherichia coli and Pichia pastoris were compared by ELISA using specific monoclonal and polyclonal IgG antibodies and IgE antibodies from cockroach allergic patients. RESULTS: The recombinant Bla g 1 construct used for crystallography had comparable IgE reactivity and dose-response curves by ELISA to the natural allergen. The Bla g 1 tandem repeat forms a novel fold with 6 helices. Two repeats encapsulate a large spherical hydrophobic cavity of ;3,750 A3. Lipids in the cavity varied depending on the source of Bla g 1: PG and PE from E. coli-expressed Bla g 1; PS, PC and PI from P. pastorisexpressed allergen; palmytic, oleic and stearic acids from cockroach frass. The lipid chains were predominantly saturated or mono-unsaturated. CONCLUSIONS: Bla g 1 has a novel fold with a capacity to bind various lipids. A digestive function associated with non-specific lipid transport can be attributed to Bla g 1. Lipid ligands could contribute to allergenicity by acting as adjuvants that activate innate immunity.
J ALLERGY CLIN IMMUNOL FEBRUARY 2013
De Novo Creation of an Antibody Binding Epitope On Group 1 Mite Allergens Jill Glesner1, Maksymilian Chruszcz, PhD2, Wladek Minor, PhD3, Martin D. Chapman, PhD, FAAAAI1, Anna Pomes, PhD, FAAAAI1; 1 Indoor Biotechnologies, Inc., Charlottesville, VA, 2University of South Carolina, Columbia, SC, 3University of Virginia, Charlottesville, VA. RATIONALE: A common epitope in Der_p_1 and Der_f_1, and a Der_p_1-specific epitope, both involved in IgE antibody binding, were identified by determining the X-ray crystal structures of each allergen in complex with a monoclonal antibody (mAb). The Der_p_1-specific mAb10B9 interferes with the binding of the cross-reactive mAb4C1 to Der_p_1, but not Der_f_1. The goal was to identify the amino acids that confer specificity for mAb10B9 by engineering a new mAb10B9 epitope on Der_f_1. METHODS: Site-directed mutagenesis was performed in Der_f_1 based on the analysis of allergen-antibody crystal structures. Mutants were expressed in yeast Pichia pastoris, purified by affinity chromatography and analyzed by antibody binding assays. RESULTS: The expected overlap between epitopes for mAb4C1 and mAb10B9 was partial. Der_f_1 residues in equivalent positions to those in Der_p_1 recognized by mAb10B9, but not involved in 4C1mAb binding, were selected for mutagenesis. Four serines in Der_f_1 were sequentially mutated into the equivalent Der_p_1 residues (Ala, Gln, Asn). The mutation of at least two residues was sufficient for antibody binding by ELISA, indicating the creation of a new epitope. Dose-response curves displaced to the left and an increase in saturation levels of antibody binding as more substitutions were present in Der_f_1 indicated an increase in antibody affinity for the allergen. CONCLUSIONS: Specific residues for mAb10B9 binding were identified by creating a de novo epitope on Der_f_1. Structural studies of allergen epitopes combined with mutagenesis analysis provide detailed information to understand determinants of specificity and cross-reactivity, and are a proof-of-principle for analyzing allergen-IgE interactions.
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Recombinant Major Ragweed Allergen Amb a 1: Physicochemical Characterization and Immunologic Comparison of Five Recombinant Ragweed Isoallergens Amb a 1.01 to Amb a 1.05 Andreas Nandy, PhD1, Steffen Augustin, PhD1, Martin Wald, PhD1, Linda Pump1, Alexis Hermann1, Sven Treder1, Susan Hagen1, Riccardo Asero, MD2, Steen Klysner, PhD1; 1Allergopharma Joachim Ganzer KG, Reinbek, Germany, 2Clinica San Carlo, Paderno Dugnano, Italy. RATIONALE: Five isoallergens of the major allergen Amb a 1 are present in short ragweed pollen. They share about 60-86% identical amino acids. The aim of this work is to provide evidence for the isoallergen composition of a future recombinant immunotherapeutic vaccine for the treatment of ragweed allergy. METHODS: The five Amb a 1 allergens were expressed in E. coli and in P. pastoris, subsequently purified and characterized by SDS-PAGE, UVvis spectroscopy and size-exclusion chromatography with multiple angle light scattering (SEC-MALS). Immunologic comparison of the purified recombinant allergens was performed by solid phase human IgE immunoblotting. RESULTS: Four isoallergens (Amb a 1.01, 1.02, 1.03 and 1.05) could be expressed in P. pastoris and purified as folded, soluble and monomeric proteins. All five isoallergens could be expressed in E. coli as insoluble inclusion bodies and have been subsequently purified as unfolded aggregated proteins. Amb a 1.01, Amb a 1.02 and Amb a 1.03 show the highest IgE-binding for most of the analyzed allergics sera from Italy and from the U.S.A.. In contrast Amb 1.05 (formerly known as Amb a 2) and Amb a 1.04 show only low IgE-reactivity. CONCLUSIONS: Amb 1.01, Amb a 1.03 and Amb a 1.02 are the most relevant Amb a 1 isoallergens based on IgE-immunoblots. For the selection of the isoallergen/s for an immunotherapy vaccine these results need to be further completed by analyzing isoallergen cross-reactivity in human IgEinhibition ELISA and by analyzing T-cell cross-reactivities of the different isoallergens.
SATURDAY
55
The Anionic Surfactant Lipid, Palmitoyl-Oleoyl-Phosphatidylglycerol (POPG), Suppresses Der p 2-Stimulated Inflammatory Responses in Lung Epithelial Cells and Macrophages Sasipa Tanyaratsrisakul, PhD1, Surapon Piboonpocanun, PhD2, Dennis R. Voelker, PhD1; 1National Jewish Health, Denver, 2Institute of Molecular Biosciences, Mahidol University, Thailand. RATIONALE: Der p 2 is the major allergen from house dust mite (HDM). More than 80% of HDM allergic patients have high Der p 2-specific IgE reactivity. Previous studies showed that the lung surfactant lipid, POPG, inhibits LPS-induced inflammatory responses by mouse and human macrophages. This study reports the protective effects of POPG on Der p 2-induced allergic responses in human bronchial epithelial cells (BEAS2B) and mouse alveolar macrophages (RAW264.7). METHODS: Recombinant Der p 2 (rDer p 2) was produced in Pichia pastoris. The structure and IgE reactivity were confirmed by Circular Dichroism (CD) and rDer p 2 inhibition of serum-IgE binding to natural Der p 2. Pro-inflammatory responses were determined using BEAS2B and RAW 264.7 incubated with rDer p 2, Pam3cys, or LPS in the presence, or absence of surfactant phospholipids. RESULTS: rDer p 2-stimulated IL-8 secretion from BEAS2B cells and TNFa secretion from RAW 264.7 cells. POPG suppressed TNFa secretion with IC50 of 13.5 mg/ml and suppressed IL-8 secretion from BEAS 2 B cell with IC50 of 11 mg/ml. A negatively charged phospholipid, palmitoyloleoyl-phosphatidic acid, which has a structure similar to POPG, was ineffective as an inhibitor of IL-8 and TNFa secretion. The site of action of POPG is upstream of JNK and p38 MAP Kinase and appears to be at the level of cell surface receptors for Der p 2. CONCLUSIONS: The Der p 2-TLR2 activated proinflammatory responses of macrophages and epithelial cells occur via JNK and p38 MAPK signaling pathways which can be inhibited by the lung surfactant lipid POPG.
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The Cockroach Allergen Bla g 1 Forms Alpha Helical Capsules with an Internal Lipid Binding Cavity: Implications for Allergenicity Geoffrey Mueller, PhD1, Lars Pedersen1, Fred Lih1, Jill Glesner2, Martin D. Chapman, PhD, FAAAAI2, Ken Tomer1, Robert London1, Anna Pomes, PhD, FAAAAI2; 1National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, 2Indoor Biotechnologies, Inc., Charlottesville, VA. RATIONALE: The cockroach allergen Bla g 1 is a marker of cockroach exposure that has been widely used in epidemiologic studies of asthma. Bla g 1 is produced in the digestive tract and has a novel structure comprising tandem amino acid repeats. The goal was to determine the structure of Bla g 1 and to investigate its biological function. METHODS: The structure of Bla g 1 was determined by X-ray crystallography. Ligand-binding properties of the allergen were examined by mass spectrometry and NMR. Natural Bla g 1 and recombinant constructs expressed in Escherichia coli and Pichia pastoris were compared by ELISA using specific monoclonal and polyclonal IgG antibodies and IgE antibodies from cockroach allergic patients. RESULTS: The recombinant Bla g 1 construct used for crystallography had comparable IgE reactivity and dose-response curves by ELISA to the natural allergen. The Bla g 1 tandem repeat forms a novel fold with 6 helices. Two repeats encapsulate a large spherical hydrophobic cavity of ;3,750 A3. Lipids in the cavity varied depending on the source of Bla g 1: PG and PE from E. coli-expressed Bla g 1; PS, PC and PI from P. pastorisexpressed allergen; palmytic, oleic and stearic acids from cockroach frass. The lipid chains were predominantly saturated or mono-unsaturated. CONCLUSIONS: Bla g 1 has a novel fold with a capacity to bind various lipids. A digestive function associated with non-specific lipid transport can be attributed to Bla g 1. Lipid ligands could contribute to allergenicity by acting as adjuvants that activate innate immunity.
J ALLERGY CLIN IMMUNOL FEBRUARY 2013
De Novo Creation of an Antibody Binding Epitope On Group 1 Mite Allergens Jill Glesner1, Maksymilian Chruszcz, PhD2, Wladek Minor, PhD3, Martin D. Chapman, PhD, FAAAAI1, Anna Pomes, PhD, FAAAAI1; 1 Indoor Biotechnologies, Inc., Charlottesville, VA, 2University of South Carolina, Columbia, SC, 3University of Virginia, Charlottesville, VA. RATIONALE: A common epitope in Der_p_1 and Der_f_1, and a Der_p_1-specific epitope, both involved in IgE antibody binding, were identified by determining the X-ray crystal structures of each allergen in complex with a monoclonal antibody (mAb). The Der_p_1-specific mAb10B9 interferes with the binding of the cross-reactive mAb4C1 to Der_p_1, but not Der_f_1. The goal was to identify the amino acids that confer specificity for mAb10B9 by engineering a new mAb10B9 epitope on Der_f_1. METHODS: Site-directed mutagenesis was performed in Der_f_1 based on the analysis of allergen-antibody crystal structures. Mutants were expressed in yeast Pichia pastoris, purified by affinity chromatography and analyzed by antibody binding assays. RESULTS: The expected overlap between epitopes for mAb4C1 and mAb10B9 was partial. Der_f_1 residues in equivalent positions to those in Der_p_1 recognized by mAb10B9, but not involved in 4C1mAb binding, were selected for mutagenesis. Four serines in Der_f_1 were sequentially mutated into the equivalent Der_p_1 residues (Ala, Gln, Asn). The mutation of at least two residues was sufficient for antibody binding by ELISA, indicating the creation of a new epitope. Dose-response curves displaced to the left and an increase in saturation levels of antibody binding as more substitutions were present in Der_f_1 indicated an increase in antibody affinity for the allergen. CONCLUSIONS: Specific residues for mAb10B9 binding were identified by creating a de novo epitope on Der_f_1. Structural studies of allergen epitopes combined with mutagenesis analysis provide detailed information to understand determinants of specificity and cross-reactivity, and are a proof-of-principle for analyzing allergen-IgE interactions.
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Recombinant Major Ragweed Allergen Amb a 1: Physicochemical Characterization and Immunologic Comparison of Five Recombinant Ragweed Isoallergens Amb a 1.01 to Amb a 1.05 Andreas Nandy, PhD1, Steffen Augustin, PhD1, Martin Wald, PhD1, Linda Pump1, Alexis Hermann1, Sven Treder1, Susan Hagen1, Riccardo Asero, MD2, Steen Klysner, PhD1; 1Allergopharma Joachim Ganzer KG, Reinbek, Germany, 2Clinica San Carlo, Paderno Dugnano, Italy. RATIONALE: Five isoallergens of the major allergen Amb a 1 are present in short ragweed pollen. They share about 60-86% identical amino acids. The aim of this work is to provide evidence for the isoallergen composition of a future recombinant immunotherapeutic vaccine for the treatment of ragweed allergy. METHODS: The five Amb a 1 allergens were expressed in E. coli and in P. pastoris, subsequently purified and characterized by SDS-PAGE, UVvis spectroscopy and size-exclusion chromatography with multiple angle light scattering (SEC-MALS). Immunologic comparison of the purified recombinant allergens was performed by solid phase human IgE immunoblotting. RESULTS: Four isoallergens (Amb a 1.01, 1.02, 1.03 and 1.05) could be expressed in P. pastoris and purified as folded, soluble and monomeric proteins. All five isoallergens could be expressed in E. coli as insoluble inclusion bodies and have been subsequently purified as unfolded aggregated proteins. Amb a 1.01, Amb a 1.02 and Amb a 1.03 show the highest IgE-binding for most of the analyzed allergics sera from Italy and from the U.S.A.. In contrast Amb 1.05 (formerly known as Amb a 2) and Amb a 1.04 show only low IgE-reactivity. CONCLUSIONS: Amb 1.01, Amb a 1.03 and Amb a 1.02 are the most relevant Amb a 1 isoallergens based on IgE-immunoblots. For the selection of the isoallergen/s for an immunotherapy vaccine these results need to be further completed by analyzing isoallergen cross-reactivity in human IgEinhibition ELISA and by analyzing T-cell cross-reactivities of the different isoallergens.