Modifications to an Fcγ-Fcɛ fusion protein alter its effectiveness in the inhibition of FcɛRI-mediated functions Lisa Chan Allen, PhD, Christopher L.

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Presentation transcript:

Modifications to an Fcγ-Fcɛ fusion protein alter its effectiveness in the inhibition of FcɛRI-mediated functions Lisa Chan Allen, PhD, Christopher L. Kepley, PhD, MBA, Andrew Saxon, MD, Ke Zhang, MD, PhD Journal of Allergy and Clinical Immunology Volume 120, Issue 2, Pages 462-468 (August 2007) DOI: 10.1016/j.jaci.2007.04.019 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Schematic diagram of human GE proteins. The molecules are depicted with the N-terminus on the left and the C-terminus on the right. The Fcγ domains (γ hinge-Cγ2-Cγ3) are striped, the Fcε domains (Cε2-Cε3-Cε4) are dotted, the flexible linker is black, and the hemagglutinin tag is white. Amino acid substitutions are designated by their IgG1 EU index position. Journal of Allergy and Clinical Immunology 2007 120, 462-468DOI: (10.1016/j.jaci.2007.04.019) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Purified GE proteins visualized on SDS-PAGE. A, Two micrograms of nonreduced samples of IgE isoforms, GE proteins, and BSA were run on a 5% phosphate gel. B, One microgram reduced samples of GE proteins and BSA were run on a 12% Tris-glycine gel. Molecular weight (MW) is indicated in kilodaltons on the right. Journal of Allergy and Clinical Immunology 2007 120, 462-468DOI: (10.1016/j.jaci.2007.04.019) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 GE proteins and IgE bind a basophil-like cell line, Ku812. Cells were incubated with several concentrations of IgE or GE proteins, and protein binding was measured as the percentage of α-ε fluorescein isothiocyanate–positive cells detected by means of flow cytometry. Error bars represent the SD among 3 repetitions. The GE H mutant at 5 μmol/L had 1 measurement. Journal of Allergy and Clinical Immunology 2007 120, 462-468DOI: (10.1016/j.jaci.2007.04.019) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Effect of GE proteins on human basophil FcεRI-mediated degranulation. A, Basophils from a representative donor presensitized with 10 μg/mL anti-NP IgE, treated with 10 μg/mL protein, and activated with NP-BSA. Percentage of degranulation is taken from duplicate samples. Journal of Allergy and Clinical Immunology 2007 120, 462-468DOI: (10.1016/j.jaci.2007.04.019) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 GE proteins block PCA: A, GE2 versus E2G; B, GE2 versus GE S mutant; C, GE2 versus GE H mutant; and D, size exclusion chromatography-purified E2G monomers versus E2G. Mice were injected with serum from a patient with cat allergy or purified IgE plus GE proteins and then challenged after 4 hours with Fel d 1 in Evans Blue dye. ∗Serum with 5 μg/mL myeloma IgE. Journal of Allergy and Clinical Immunology 2007 120, 462-468DOI: (10.1016/j.jaci.2007.04.019) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions