Regina Selb, PhD, Julia Eckl-Dorna, MD, PhD, Teresa E

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Critical and direct involvement of the CD23 stalk region in IgE binding Regina Selb, PhD, Julia Eckl-Dorna, MD, PhD, Teresa E. Twaroch, PhD, Christian Lupinek, MD, Andrea Teufelberger, MSc, Gerhard Hofer, MSc, Margarete Focke-Tejkl, PhD, Barbara Gepp, PhD, Birgit Linhart, PhD, Heimo Breiteneder, PhD, Adolf Ellinger, MD, Walter Keller, PhD, Kenneth H. Roux, PhD, Rudolf Valenta, MD, Verena Niederberger, MD Journal of Allergy and Clinical Immunology Volume 139, Issue 1, Pages 281-289.e5 (January 2017) DOI: 10.1016/j.jaci.2016.04.015 Copyright © 2016 The Authors Terms and Conditions

Fig 1 Overview of CD23 constructs. A, Amino acid sequence of CD23. Indicated are the constructs CD23A, CD23B, CD23C, and CD23D; peptides (P1-P7) used to raise specific antisera (brown bars); N-linked glycosylation sites (red letters); and secondary structure and surface exposure (blue bars) of amino acids, as derived from the crystal structure PDB:2H2T (Wurzburg et al12). A region in which no data are available (R253-E257) is indicated by a gray bar. Amino acids involved in IgE binding according to Hibbert et al13 and Dhaliwal et al15 are shown in green and blue, respectively. Residues involved in IgE binding according to both publications are shown in orange. Cysteine residues involved in disulfide bond formation are numbered. CD23 domains are shaded (intracellular domain: red; transmembrane domain: yellow; stalk domain: green; head domain: purple). B, Schematic representation of the known (Wurzburg et al12) and predicted 3-dimensional structure of CD23. CD23 peptides are indicated. Data for region R253-E257, as well as for the C-terminal end, are not available. C, Sketch of the recombinant CD23 constructs (CD23A, CD23B, CD23C, and CD23D), as cloned into plasmid pTM1 with a C-terminal hexa-histidine tag (6xHis) and an N-terminal melittin signal sequence. Amino acids at the N- and C-terminal ends of CD23 and one mutated in CD23B are indicated in black. Journal of Allergy and Clinical Immunology 2017 139, 281-289.e5DOI: (10.1016/j.jaci.2016.04.015) Copyright © 2016 The Authors Terms and Conditions

Fig 2 Biochemical, biophysical, and immunologic characterization of recombinant CD23 constructs. A, Coomassie-stained gel containing aliquots of purified CD23 constructs. B, Gel filtration profile of the 4 CD23 constructs. Adsorptions at 210 and 280 nm (y-axes, milliabsorbance units [mAU]) recorded during elution (x-axes, elution volumes in milliliters and corresponding protein size ranges in kilodaltons) are shown. C, Circular dichroism analysis of the 4 CD23 constructs. An overlay of the 4 circular dichroism spectra of constructs CD23A to CD23D is shown. CD23A, red; CD23B, pink; CD23C, blue; CD23D, yellow. X-axis, wavelength [in nanometers]; y-axis, [θ]mr (mean-residue molar ellipticity). Journal of Allergy and Clinical Immunology 2017 139, 281-289.e5DOI: (10.1016/j.jaci.2016.04.015) Copyright © 2016 The Authors Terms and Conditions

Fig 3 Binding of IgE to CD23. A, Varying binding of IgE to the CD23 constructs. ELISA plates were coated with recombinant CD23 constructs A through D or a control protein and incubated with either IgE alone (IgE) or IgE-Bet v 1 complexes (IgE+Bet v 1). Absorbance values (A 405 nm) corresponding to bound IgE are indicated on the y-axis. B, Binding of the IgE–Bet v 1 complexes to CD23A and CD23C. For control purposes, IgE was omitted (Bet v 1 only) or rabbit antibodies without specificity for Bet v 1 (control antibody) were used. Absorbance values (A 405 nm) corresponding to bound Bet v 1 detected with rabbit anti–Bet v 1 antibodies are indicated on the y-axis. Error bars show SDs of triplicates. C and D, Binding of monoclonal IgE to recombinant CD23 constructs can be blocked with α-CD23 (Fig 3, C) or omalizumab (Fig 3, D). In Fig 3, C, ELISA plates with bound CD23A and CD23B were preincubated with α-CD23 (black bars) or with an isotype control (white bars) and incubated with IgE. In Fig 3, D, IgE was preincubated with omalizumab (black bars) or an isotype control (white bars) before incubation with CD23 constructs (x-axis: CD23A, CD23B, CD23C, and CD23D). Absorbance values (A 405 nm) corresponding to bound IgE are indicated on the y-axis. Error bars show SDs of triplicates. *P < .05 and **P < .01, 2-tailed test. Journal of Allergy and Clinical Immunology 2017 139, 281-289.e5DOI: (10.1016/j.jaci.2016.04.015) Copyright © 2016 The Authors Terms and Conditions

Fig 4 Blocking ability of CD23 peptide-specific antibodies. A, Ability of monoclonal anti-CD23 to bind to different anti-CD23 peptides. CD23A was used as a positive control. B, Influence of anti-CD23 peptide antibodies on binding of monoclonal anti-CD23. CD23A was preincubated with anti-peptide antibodies or the corresponding preimmune sera (x-axis) and then incubated with the anti-CD23 antibody. Absorbance values (A 405 nm: y-axes) correspond to bound anti-CD23 antibody. Error bars show SEs of at least triplicates. The experiments were repeated 3 times with similar results. C and D, Influence of anti-CD23 peptide antibodies on the binding of IgE to CD23A (Fig 4, C) and CD23B (Fig 4, D). CD23 constructs were preincubated with anti-peptide antibodies or the corresponding preimmune sera (x-axes) and then incubated with IgE. Absorbance values (A 405 nm: y-axes) correspond to bound IgE. Error bars show SDs of triplicates. E, Influence of antibodies on IgE binding to CD23-expressing B cells. Shown is the percentage blocking of IgE binding (y-axis) to CD23-expressing B cells by an anti-CD23 mAb and by antibodies specific for CD23 peptides (x-axis), as determined by using flow cytometry. Error bars show SDs of triplicates. The experiment was performed twice with similar results. *P < .05 and **P < .01, 2-tailed test. Journal of Allergy and Clinical Immunology 2017 139, 281-289.e5DOI: (10.1016/j.jaci.2016.04.015) Copyright © 2016 The Authors Terms and Conditions

Fig 5 Negative stain electron microscopy of recombinant CD23 in complex with IgE and allergen. Shown are 3 representative negative stain electron microscopy images and interpretive sketches of IgE alone (A), an anti-His IgG Fab fragment (B), a mix of IgE plus anti-His IgG Fab fragment (C), IgE-CD23 complexes marked with an anti-His IgG Fab fragment (D), and IgE-CD23-allergen complexes marked with an anti-His IgG Fab fragment (E). Black bars represent 25 nm. Journal of Allergy and Clinical Immunology 2017 139, 281-289.e5DOI: (10.1016/j.jaci.2016.04.015) Copyright © 2016 The Authors Terms and Conditions

Fig E1 Expression in insect cells and purification. A, Time course of expression (x-axes) of CD23 constructs, as detected in nitrocellulose-blotted insect cell supernatants with an anti−His-tag antibody. Molecular weights are indicated on the y-axes. B and C, Coomassie (Fig E1, B) and glycan (Fig E1, C) staining of purified constructs (CD23A, CD23B, CD23C, and CD23D), as well as of a nonglycosylated (N) and glycosylated (P) control protein. Journal of Allergy and Clinical Immunology 2017 139, 281-289.e5DOI: (10.1016/j.jaci.2016.04.015) Copyright © 2016 The Authors Terms and Conditions

Fig E2 Binding of rabbit antisera specific for CD23 peptides to recombinant CD23 constructs: A, CD23A; B, CD23B; C, CD23C; and D, CD23D. CD23 constructs were incubated with anti-CD23 antibodies with different specificities (x-axes: rabbit anti-CD23 peptide antibodies anti-P1, anti-P2, anti-P3, anti-P4, anti-P5, anti-P6, and anti-P7; monoclonal anti-CD23 antibody). Absorbance values (A 405 nm: y-axes) correspond to bound antibodies. Error bars show SDs of triplicates. Journal of Allergy and Clinical Immunology 2017 139, 281-289.e5DOI: (10.1016/j.jaci.2016.04.015) Copyright © 2016 The Authors Terms and Conditions