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Anna Gieras, PhD, Birgit Linhart, PhD, Kenneth H

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1 IgE epitope proximity determines immune complex shape and effector cell activation capacity 
Anna Gieras, PhD, Birgit Linhart, PhD, Kenneth H. Roux, PhD, Moumita Dutta, PhD, Marat Khodoun, PhD, Domen Zafred, PhD, Clarissa R. Cabauatan, PhD, Christian Lupinek, MD, Milena Weber, MSc, Margarete Focke-Tejkl, PhD, Walter Keller, PhD, Fred D. Finkelman, MD, Rudolf Valenta, MD  Journal of Allergy and Clinical Immunology  Volume 137, Issue 5, Pages (May 2016) DOI: /j.jaci Copyright © 2015 The Authors Terms and Conditions

2 Fig 1 Conversion of horse heart myoglobin into an IgE-reactive protein. A, Schematic overview of myoglobin derivatives (MB1N, MB2N, MB1N1aa46-47, MB1N1C, MB2N1C, and MB4N) containing 1 to 4 identical copies of an IgE-reactive peptide (orange) in different proximity in the myoglobin sequence (arrows). Spacers and hexahistidine tags are indicated with gray and yellow, respectively. B, Ribbon and space-fill representations of the 3-dimensional structure of horse heart myoglobin showing the positions of the 3 N-terminal (blue) and 4 C-terminal amino acids (red) as well as of amino acids 46 and 47 (green). Fig 1, B, was made with the MacPyMOL Molecular Graphics System using PDB coordinate file 1wla. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

3 Fig 2 Coomassie-stained SDS-PAGE gels showing the mobility of purified myoglobin derivatives (lanes 1-6: MB1N, MB2N, MB1N1aa46-47, MB1N1C, MB2N1C, MB4N) separated under nonreducing (A) and reducing (B) conditions. Molecular weights (kDa) are displayed. C, Monomeric behavior of the myoglobin derivatives demonstrated by gel filtration (x-axis: elution volumes in mL; y-axis: milli absorbance units at 280 nm). Arrows indicate peaks of protein standards (thyroglobulin, 670 kDa; bovine gamma globulin, 158 kDa; chicken ovalbumin, 44 kDa; equine myoglobulin, 17 kDa; vitamin B12, 1.35 kDa). D, Far ultraviolet CD analysis showing mean residue ellipticities (Ө, y-axis) of purified myoglobin and myoglobin derivatives recorded at different wavelengths (x-axis). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

4 Fig 3 Visualization of complexes consisting of myoglobin derivatives and bound IgE antibodies by negative-stain EM. Representative micrographs and interpretive diagrams showing complexes of IgE with MB1N (A), MB2N (B), MB1N1aa46-47 (C), MB1N1C (D), MB2N1C (E), and MB4N (F). Bars represent 50 nm. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

5 Fig 4 Allergenic activity of myoglobin derivatives analyzed in vitro by RBL mediator release assays and in a murine model of passive anaphylaxis. A and B, RBL cells were loaded with different dilutions (Fig 4, A, 1:500; Fig 4, B, 1:5000) of the peptide-specific monoclonal IgE antibody and stimulated with increasing concentrations (  nM/mL) of myoglobin and the myoglobin derivatives (x-axes). C, Detailed analysis of basophil degranulation by myoglobin derivatives containing 2 IgE epitopes in different proximity. β-hexosaminidase release is displayed as percentage of total release on the y-axis (SDs: error bars). D and E, BALB/c mice (n = 4/group) were primed with peptide-specific monoclonal IgE antibody and challenged 10 hours later with either (Fig 4, D) 4 μg or (Fig 4, E) 40 μg of myoglobin derivatives. Rectal temperatures were determined every 5 minutes for the next 1 hour. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

6 Fig E1 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

7 Fig E2 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

8 Fig E3 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

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