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Enhanced Pru p 3 IgE-binding activity by selective free fatty acid-interaction  Pawel Dubiela, MSc, Roberta Aina, PhD, Dominika Polak, MSc, Sabine Geiselhart,

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Presentation on theme: "Enhanced Pru p 3 IgE-binding activity by selective free fatty acid-interaction  Pawel Dubiela, MSc, Roberta Aina, PhD, Dominika Polak, MSc, Sabine Geiselhart,"— Presentation transcript:

1 Enhanced Pru p 3 IgE-binding activity by selective free fatty acid-interaction 
Pawel Dubiela, MSc, Roberta Aina, PhD, Dominika Polak, MSc, Sabine Geiselhart, PhD, Piotr Humeniuk, MSc, Barbara Bohle, PhD, Stefano Alessandri, PhD, Rebecca Del Conte, PhD, Francesca Cantini, PhD, Tomasz Borowski, PhD, Merima Bublin, PhD, Karin Hoffmann-Sommergruber, PhD  Journal of Allergy and Clinical Immunology  Volume 140, Issue 6, Pages e10 (December 2017) DOI: /j.jaci Copyright © 2017 The Authors Terms and Conditions

2 Fig 1 Pru p 3 displays differential ligand binding. Concentration-dependent reduction in ANS binding to Pru p 3 preincubated with FFA. Positive control: natural Pru p 3; Negative control: FFA. ELA, Elaidic acid; FFA, free fatty acid; HYD, 16-hydroxypalmitic acid; LAU, lauric acid; LIN, linoleic acid; PAL, palmitic acid. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2017 The Authors Terms and Conditions

3 Fig 2 Pru p 3-FFA interaction and impact on the IgE binding. A, Molecular dynamic analysis. Pru p 3 with OLE− (upper left); Cα superposition of Pru p 3-apo (blue) and with OLE− (red). The white ribbon of the Pru p 3-OLE− complex represents the region affected by the OLE binding (upper right); residues 75 to 80 of Pru p 3 affected by OLE− binding (lower panel). B, ELISA; data are representative of 10 patients' sera tested in triplicate with rPru p 3 alone and with OLE or STE. C, Basophil activation test assay. Percentages of CD63+ basophils after stimulation with allergen at a concentration of 1 ng/mL and an allergen:FFA ratio of 1:10 in 6 patients sensitized to Pru p 3. The P value refers to the comparison of the median between allergen alone and in the presence of ligand. FFA, Free fatty acid; ns, nonsignificant. *P ≤ .05 (unpaired 2-tailed t test). **P < .01. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2017 The Authors Terms and Conditions

4 Fig E1 Comparison of purified nPru p 3 and rPru p 3. A, Coomassie stained 15% SDS-PAGE. B, Matrix-assisted laser desorption & ionization time-of-flight mass spectrum analysis. C, Amino acid sequences. Cysteines, responsible for disulfide bonds, are marked in red; additional residues from P pastoris cleavage marked in green; N-terminal amino acid residues verified by sequencing are underlined. D, Far-ultraviolet circular dichroism spectra. Intens., Intensity. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2017 The Authors Terms and Conditions

5 Fig E2 Pru p 3 displays differential ligand binding. 1-Dimensional W-LOGSY spectrum of 80 μM OLE and STE alone (upper), in the presence of Pru p 3 (8 μM) (middle); 1-Dimensional 1H spectrum of OLE and STE (lower). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2017 The Authors Terms and Conditions

6 Fig E3 RMSD versus time plot for molecular dynamic simulation A, Pru p 3–OLE− complex; B, Pru p 3–OLE complex. RMSD, Root-mean-square deviation of atomic positions. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2017 The Authors Terms and Conditions

7 Fig E4 Intermolecular contacts in Pru p 3-OLE− (A) and Pru p 3-OLE (B) complex from molecular dynamic simulation (10-150 ns). Color code from red (most frequent contacts), through green to blue (no contacts). Protein residues making most contacts are labeled. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2017 The Authors Terms and Conditions

8 Fig E5 Superimposed structures of Pru p 3-OLE− (in blue) and Pru p 3-OLE (in orange/yellow) complexes. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2017 The Authors Terms and Conditions

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