Enhanced Pru p 3 IgE-binding activity by selective free fatty acid-interaction
Authors: Pawel Dubiela, Roberta Aina, Dominika Polak, Sabine Geiselhart, Piotr Humeniuk, Barbara Bohle, Stefano Alessandri, Rebecca Del Conte, Francesca Cantini, Tomasz Borowski, Merima Bublin, Karin Hoffmann-Sommergruber
Journal: Journal of Allergy and Clinical Immunology (2017)

Nonspecific lipid transfer proteins (nsLTPs) are major cross-reactive allergens identified in most plant-derived foods as well as pollen from diverse plants, and are often associated with severe symptoms in food allergy.1Hauser M. Roulias A. Ferreira F. Egger M. Panallergens and their impact on the allergic patient.Allergy Asthma Clin Immunol. 2010; 6: 1Crossref PubMed Scopus (225) Google Scholar Currently, Pru p 3 (major food allergen from peach [Prunus persica]) is regarded as the primary sensitizer for nsLTP-caused allergies.2Vereda A. van Hage M. Ahlstedt S. Ibañez M.D. Cuesta-Herranz J. van Odijk J. et al.Peanut allergy: clinical and immunologic differences among patients from 3 different geographic regions.J Allergy Clin Immunol. 2011; 127: 603-607Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar Pru p 3 shares the physicochemical characteristics of the nsLTP family. It is a small (9187 Da) basic protein, with a highly conserved 3-dimensional structure provided by 8 conserved cysteine residues forming 4 disulfide bridges. The common feature of nsLTPs is a hydrophobic cavity throughout the whole molecule that can host ligands such as fatty acids. However, this lipid-binding capacity varies among different nsLTP members, and depends on the specific characteristics of their tertiary fold as revised by Liu et al.3Liu F. Zhang X. Lu C. Zeng X. Li Y. Fu D. et al.Non-specific lipid transfer proteins in plants: presenting new advances and an integrated functional analysis.J Exp Botany. 2015; 66: 5663-5681Crossref PubMed Scopus (171) Google Scholar There is limited knowledge about the binding capacity of Pru p 3. So far, interactions with lauric acid,4Pasquato N. Berni R. Folli C. Folloni S. Cianci M. Pantano S. et al.Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens.J Mol Biol. 2006; 356: 684-694Crossref PubMed Scopus (114) Google Scholar cis-parinaric, palmitic, and linoleic acids5Abdullah S.U. Alexeev Y. Johnson P.E. Rigby N.M. Mackie A.R. Dhaliwal B. et al.Ligand binding to an allergenic lipid transfer protein enhances conformational flexibility resulting in an Increase in susceptibility to gastroduodenal proteolysis.Sci Rep. 2016; 6: 30279Crossref PubMed Scopus (29) Google Scholar have been reported. Several structures of nsLTPs with and without ligands have been determined including peach4Pasquato N. Berni R. Folli C. Folloni S. Cianci M. Pantano S. et al.Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens.J Mol Biol. 2006; 356: 684-694Crossref PubMed Scopus (114) Google Scholar and hazelnut6Offermann L.R. Bublin M. Perdue M.L. Pfeifer S. Dubiela P. Borowski T. et al.Structural and functional characterization of the hazelnut allergen Cor a 8.J Agricultural Food Chem. 2015; 63: 9150-9158Crossref PubMed Scopus (28) Google Scholar and suggested a plasticity of the cavity when binding to ligands as compared with an unliganded molecule. However, whether the apo- or holoform of Pru p 3 has an impact on the IgE-binding activity is yet unknown. Therefore, we studied a range of saturated and mono-/poly-unsaturated fatty acids and their interaction with Pru p 3 and investigated whether Pru p 3-ligand interaction is able to affect IgE recognition in sera from peach-allergic patients. To address this question, we first performed nuclear magnetic resonance (NMR) experiments and molecular dynamic analyses. Subsequently, the IgE-binding activity was investigated by ELISA and basophil activation test (BAT) assays. Details for materials, methods, and patients' data (Table E1) are provided in this article's Online Repository at www.jacionline.org. Natural and recombinant Pru p 3 were purified and characterized (see Fig E1 in this article's Online Repository at www.jacionline.org). Preincubation of Pru p 3 with individual unsaturated and saturated free fatty acids induced a dose-dependent reduction of 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. Unsaturated fatty acids induced a larger reduction in ANS binding compared with saturated fatty acids at all tested concentrations (10, 20, 50, and 100 μM). The largest reduction in the signal was observed for oleic acid (OLE [72.6%]) followed by linoleic acid (72%) and elaidic acid (68.6%) at a protein:ligand ratio of 1:10. In contrast, the saturated fatty acid stearic acid (STE) induced a reduction of 39.1% in ANS binding. In addition, saturated fatty acids with shorter C chains, such as lauric acid (C12), bind to the internal cavity of Pru p 3 more effectively than do saturated fatty acids with longer chains, that is, STE (C18) (Fig 1, A; see Table E2 in this article's Online Repository at www.jacionline.org). To support the results from the ANS assay, we decided to perform water-ligand observed via gradient spectroscopy (W-LOGSY) experiments, a frequently applied 1-dimensional ligand-observation NMR technique for the detection of protein-ligand interactions. W-LOGSY experiments were acquired for OLE and STE because they showed the highest and the lowest binding capacity, respectively. NMR experiments confirmed the binding capacity observed in the ANS assay (see Fig E2 in this article's Online Repository at www.jacionline.org). Comparison of these 2 spectra indicates that recombinant Pru p 3 (rPru p 3) was able to bind OLE as shown by an inversion of the signal. In contrast, no interaction of rPru p 3 with STE was detected. Because pH changes influence the presence of the OLE protonation state, we decided to investigate whether both OLE and its anion (OLE−) affect the 3-dimensional structure of Pru p 3. Molecular dynamic analyses suggest changes in protein structure due to binding of OLE or OLE−. Binding of OLE− leads to a substantial conformational change in the C-terminal fragment of the protein. Because of the interaction between OLE− and Pru p 3, the C-terminal loop is moved out toward the surface of the molecule, while the same region of Pru p 3 alone is closer to the core of the molecule (Fig 2, A). These changes are caused by binding of OLE− between the third α-helix (h3) and the C-terminal loop of Pru p 3, which is stabilized by hydrogen bonds with Arg32 (Fig 2, A). Analysis of intramolecular contacts between Pru p 3 and OLE− along the stable part of the molecular dynamic trajectory (10-150 ns; see Figs E3, A, and E4, A, in this article's Online Repository at www.jacionline.org) shows that the ligand interacts with polar residues on the surface of the protein (Arg32, Asn35, Arg44) and hydrophobic side chains in the upper part of the hydrophobic cavity (Ile31, Pro70, Ile77; Fig E4, A). This interaction with OLE− affects residues 75 to 85, leading to an exposure of Pro78 and Tyr79 outside the hydrophobic cavity (Fig 2, A). The Pru p 3-OLE complex has a different structure, as the charge-neutral ligand immerses almost completely in the hydrophobic cavity (Figs E3, B, and E4, B) and the key residues making contacts with the ligand are now Leu10, Ile31, Asn35, Leu51, and Ile81. OLE penetrates the cavity in a more stretched conformation than does OLE−, and in consequence, the bottom of the cavity opens via the movement of helix 3 away from helix 4 (see Figs E4, B, and E5 in this article's Online Repository at www.jacionline.org). These changes, although not affecting the overall structure, could be important regarding B-cell epitope exposure. Because the region affected by conformational changes of Pru p 3 is the one that was identified as the major IgE epitope responsible for severe reactions,7García-Casado G. Pacios L.F. Díaz-Perales A. Sánchez-Monge R. Lombardero M. García-Selles F.J. et al.Identification of IgE-binding epitopes of the major peach allergen Pru p 3.J Allergy Clin Immunol. 2003; 112: 599-605Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar we decided to investigate whether conformational changes due to ligand binding lead to increased IgE-binding capacity. To test whether interaction of Pru p 3 with free fatty acids influences its IgE-binding capacity, we used sera from 10 peach-allergic patients sensitized to Pru p 3. Based on our results from ANS and W-LOGSY experiments, the 2 ligands with highest (OLE) and lowest (STE) binding activity to Pru p 3 were selected. Preincubation of rPru p 3 with OLE significantly (P = .002) increased the IgE-binding capacity of all sera tested by ELISA assay when compared with the allergen alone (Fig 2, B; see Table E3 in this article's Online Repository at www.jacionline.org). The largest differences were observed with sera nos. 2 and 6, with an increase of 118% and 89%, respectively. According to our data, STE did not have a considerable influence on the IgE-binding capacity of rPru p 3 (P > .05) (Fig 2, B; Table E3). Furthermore, we investigated whether the binding of a lipid ligand also increases the activation of effector cells. Pru p 3 preincubated with OLE significantly increased the number of CD63+ cells as compared with Pru p 3 alone (P = .031). The percentages of CD63+ basophils after stimulation with Pru p 3 preincubated with OLE were also significantly (P = .011) increased when compared with CD63+ basophils after stimulation of Pru p 3 preincubated with STE. In addition, comparison of Pru p 3 preincubated with STE and Pru p 3 alone showed that this ligand did not have any impact on basophils' activation (P > .05) (Fig 2, C; Table E3). In conclusion, our ligand-binding assays provided interesting results regarding the binding specificity of Pru p 3, preferably binding poly- and mono-unsaturated fatty acids as compared with saturated ones. In parallel, using an in silico approach, we showed conformational changes of the cavity and a shift in the tertiary structure of Pru p 3 as a consequence of OLE and OLE− binding. Notably, the IgE ELISA performed with sera from 10 peach-allergic patients confirmed our hypothesis that exposure of the C-terminal loop due to the OLE/OLE− binding could indeed enhance the IgE-binding activity. In contrast, binding of STE was unable to increase the IgE-binding capacity. This corroborates our idea that only those ligands that shift the tertiary structure of Pru p 3, thus exposing IgE epitopes, could increase its allergenic potential. This observation is consistent with previous studies indicating that no synthetic peptides covering areas of helix 3 or loop 3 were identified among the most reactive peptides in the SPOT analysis, probably because most of this region is mainly buried inside the compact 3-dimensional structure of Pru p 3.7García-Casado G. Pacios L.F. Díaz-Perales A. Sánchez-Monge R. Lombardero M. García-Selles F.J. et al.Identification of IgE-binding epitopes of the major peach allergen Pru p 3.J Allergy Clin Immunol. 2003; 112: 599-605Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Consistent with the data obtained by ELISA, OLE, but not STE, had a significant impact on the basophil activation. This is in line with a study on grape nsLTP where the presence of phosphatidylcholine in an in vitro digestion assay had not only a protective effect on the allergen but also increased the ability of the allergen to induce basophil histamine release and to elicit skin reactions in 4 patients with grape allergy.8Vassilopoulou E. Rigby N. Moreno F.J. Zuidmeer L. Akkerdaas J. Tassios I. et al.Effect of in vitro gastric and duodenal digestion on the allergenicity of grape lipid transfer protein.J Allergy Clin Immunol. 2006; 118: 473-480Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar It becomes clear that lipids, in addition to their role as direct immune modulators, can influence the allergenicity of proteins9Bublin M. Eiwegger T. Breiteneder H. Do lipids influence the allergic sensitization process?.J Allergy Clin Immunol. 2014; 134: 521-529Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar by modifying the allergen structure and biochemical properties. Among unsaturated fatty acids, OLE is abundantly found in peach fruits, but it is also endogenously present in the lipid bilayer of the human gut. Pru p 3 seems to be presented in its holoform to the allergic individual throughout exposure and digestion. This, in turn, explains that this immunodominant B-cell epitope is conserved and surface exposed. In this context, the application of Pru p 3 in diagnostic tests should be reconsidered, using Pru p 3 together with OLE for component-resolved diagnosis to increase assay sensitivity. Furthermore, for designing low allergenic Pru p 3 variants for immunotherapy, the amino acid residues relevant for OLE interaction are the targets for mutations and thus reduce the risk of side effects during immunotherapy. All reagents were purchased from Sigma-Aldrich (St Louis, Mo) unless stated otherwise. Natural Pru p 3 (nPru p 3) was extracted and purified from peach (P persica) peel as previously described by Gaier et al.E1Gaier S. Marsh J. Oberhuber C. Rigby N.M. Lovegrove A. Alessandri S. et al.Purification and structural stability of the peach allergens Pru p 1 and Pru p 3.Mol Nutr Food Res. 2008; 52: S220-S229PubMed Google Scholar Briefly, the peel of 1 kg of peach fruits was ground in liquid nitrogen and transferred in the extraction buffer (46 mM sodium phosphate, 2 mM EDTA, 20 mM sodium diethyldithiocarbamate, 3 mM NaN3, 3% w/v PVPP, pH 7.0). The mixture was stirred for 1 hour at 4°C and then centrifuged (10,000g for 10 minutes, 4°C). The supernatant was precipitated with 95% w/v ammonium sulphate, resuspended, and filtered through a 0.22 μM filter. nPru p 3 was then purified with cation exchange chromatography (Mono S column, GE Healthcare, Little Chalfont, United Kingdom) followed by gel filtration (Superdex 200 column, GE Healthcare). The recombinant counterpart of nPru p 3 was produced in the yeast Pichia pastoris. The protein sequence of mature Pru p 3 was retrieved from Genebank (Acc. no CAB96876) and optimization for P pastoris codon usage as well as prediction of glycosylation sites was performed. The plasmid construct pPICZαA-Pru p 3 (ThermoFisher Scientific, Waltham, Mass) was linearized with SacI (New England Biolabs, Ipswich, Miss) and used to transform GS115 P pastoris cells (ThermoFisher Scientific) by electroporation. Transformed cells were grown on yeast extract peptone dextrose medium plates containing 100 μg/mL zeocin (Invivogen, San Diego, Calif) at 28°C for 5 days. Multicopy screening was performed by replica plating of the positive cells on yeast extract peptone dextrose with increasing zeocin concentration (up to 2000 μg/mL). Positive transformants expressing rPru p 3 with highest yield were cultivated in 200 mL minimal glycerol medium containing histidine for 1 day at 28°C under shaking conditions until the culture reached an OD600 value of 2 to 3. Cells were harvested and transferred to minimal methanol + histidine medium (24°C, 140 rpm), containing 1% of methanol to induce protein expression. Methanol (at a final concentration of 1%) was also added to the liquid culture every 12 hours. After 5 days of incubation, the culture supernatant was collected by centrifugation (6000g for 20 minutes at 4°C) and stored at −20°C. For subsequent purification, 200 mL of supernatant was dialyzed against binding buffer (20 mM sodium acetate, pH 6.5). After filtration through a 0.22 μm filter, the protein solution was applied to a 1 mL Mono S column (GE Healthcare). Bound proteins were eluted by a linear concentration gradient of NaCl (0-0.5 mol/L) at a flow rate of 1 mL/min. In the second step, samples containing protein of interest were applied on an RP-HPLC Jupiter C5 analytical column (Phenomenex, Torrance, Calif). The column was equilibrated with the mobile phase (10% acetonitrile, 90% water, 0.1% trifluoroacetic acid) and eluted by increasing concentration of acetonitrile (up to 50%) with flow rate 1 mL/min. nsLTP-containing fractions were analyzed by 15% SDS-PAGE and the concentration of the purified rPru p 3 was determined using the bicinchoninic acid assay according to the manufacturer's protocol (ThermoFisher Scientific). Samples were stored at −20°C. The N-terminal sequences of both recombinant and natural Pru p 3 were determined using an Applied Biosystems Procise 491 sequencer (Applied Biosystems, Foster City, Calif). Purified proteins (100 pmol) were adsorbed onto a Prosorb cartridge and subjected to sequence analysis. For intact mass determination, nonreduced protein samples of purified nPru p 3 and rPru p 3 were spotted in a ratio of 1:1 with matrix (α-cyano-4-hydroxycinnamic acid) onto a ground steel matrix-assisted laser desorption & ionization target plate and measured in linear mode on a matrix-assisted laser desorption & ionization time-of-flight mass spectrometer (Microflex, Bruker Daltonics, Bremen, Germany). Secondary structure analysis of purified Pru p 3 was performed by CD spectroscopy (Jasco International Co., Hachioji, Tokyo). CD spectra of natural and recombinant Pru p 3 were measured from 190 to 260 nm at 25°C and pH 7.5 using 10 mM sodium phosphate buffer in 2-mm path length quartz cell. Spectra represent the average of 4 accumulations collected at 100 nm/min with a 2-second time constant, 0.5 nm resolution, and sensitivity of ±100 mdeg. Spectra are represented as molar circular dichroism (with respect to moles of amide bonds). The probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) is nonfluorescent in water but fluorescent when binding to the hydrophobic cavity of Pru p 3, with a maximum emission wavelength at 456 nm. Natural and recombinant Pru p 3 (10 μM) were incubated overnight at 4°C with different ligands: 3 unsaturated fatty acids, OLE (C18:1), elaidic (or trans-OLE; C18:1), and linoleic (C18:2), and 3 saturated fatty acids, stearic (STE; C18:0), lauric (C12:0), and palmitic (C16:0), at the following molar ratios: 1:1, 1:2, 1:5, and 1:10. In addition, to test whether the hydroxyl group influences the binding of free fatty acids to Pru p 3, 16-hydroxypalmitic acid (C16OH) was tested as described above. Binding of ligands was monitored by adding 10 μM 1,8-ANS and measuring the decrease in 1,8-ANS fluorescence. All samples were analyzed in triplicate. Purified nPru p 3 with ANS and ligands with ANS served as controls. As saturation-transfer difference approach, W-LOGSY is based on a transient nuclear Overhauser effect experiment, and implies transfer of magnetization via an intermolecular nuclear Overhauser effect and spin diffusion. Nonbinders and binders are easily discriminated from each other because they give W-LOGSY signals of opposite sign.E2Dalvit C. Fogliatto G. Stewart A. Veronesi M. Stockman B. WaterLOGSY as a method for primary NMR screening: practical aspects and range of applicability.J Biomolecular NMR. 2001; 21: 349-359Crossref PubMed Scopus (439) Google Scholar W-LOGSY NMR experiments were acquired for OLE and STE dissolved in H2O with 10% (v/v) of (CD3)2SO (hexadeuterodimethyl sulfoxide). rPru p 3 stock solution was prepared in 20 mM sodium acetate buffer, pH 6.5, containing 0.1 mol NaCl/D2O 9∶1. The allergen was mixed with tested ligands at a molar ratio of 1:20, to a final protein concentration of 5 μM. High-resolution NMR experiments were carried out using an Avance 700 Bruker spectrometer equipped with a cryo probe, operating at a proton resonance frequency of 700 MHz (11.7 Tesla) at 298 K. The experiments were performed with a 180° inversion pulse applied over the water signal at approximately 4.7 ppm by means of a Gaussian-shaped selective pulse of 10 ms. Each W-LOGSY spectrum was acquired with 512 scans. Initial coordinates of Pru p 3 were taken from PDB:2B5S, and the AMBER force field ff03.r1 was applied to the protein. Molecular force fields of OLE and its anion (OLE−) were described with the general AMBER force field. Ligands were first optimized in extended conformations in vacuum at the B3LYP/6-31G(d,p) level and then the electrostatic potential around them was computed at the level that is consistent with the general AMBER force field, that is, HF/6-31G(d). Quantum chemical computations were performed with Gaussian 09 suite of programs.E3Frisch M.J. Trucks G.W. Schlegel H.B. Scuseria G.E. Robb M.A. Cheeseman J.R. et al.Gaussian 09, Revision E.01. Gaussian, Inc, Wallingford, CT2016Google Scholar From the electrostatic potential RESP atomic charges were fitted with the use of antechamber and RESP programs from the AmberTools package.E4Case D.A. Darden T.A. Cheatham T.E. Simmerling C.L. Wang J. Duke R.E. et al.AMBER 12. University of California, San Francisco2012Google Scholar The ionization state of Pru p 3 residues was tested with the PROPKA 3.1 software and for pH 7 all residues were predicted to be in their standard ionization state (N- and C-termini all LYS, ARG, and ASP charged). The N-terminal Met residue of the Pru p 3 structure was removed, as it was added to the protein because of its heterologous expression in a bacterial host. The protein molecule was subsequently placed in a periodic box filled with explicit water molecules described with the TIP3P model and appropriate number of chloride anions (7 for the unliganded protein and its complex with OLE, 6 for the Pru p 3/OLE− complex) to obtain a charge-neutral system. Size of the box was such that its edge was 10 Å away from the protein surface in each direction. The system was first minimized in 3 steps: 5,000 steps with protein atoms restrained with 500 kcal/mol Å2 harmonic constant; 5,000 steps with 10 kcal/mol Å2 harmonic restraint on protein; and then 10,000 steps of an unrestrained minimization. After minimization, the system was heated up from 0 to 300 K during a 50 ps NV dynamics and then its density was equilibrated in a 0.5 ns NPT dynamics. Subsequent unrestrained NPT (T = 300 K, P = 1 atm) production dynamics simulation spanned 150 ns for each system, with a snapshot saved every 10 ps. Integration time step used in molecular dynamics simulations was 2 fs. The SHAKE algorithm was used to constrain bonds of hydrogen atoms, and temperature and pressure were controlled with the Langevin dynamics and isotropic position scaling algorithm, respectively. Snapshots of the last 10 ns of simulations were clustered with the average linkage algorithm applied to Cα carbons of the protein backbone. Representative structures of dominating clusters were used for structure comparisons. Serum samples were obtained from a well-defined group of 10 peach-allergic patients (7 females and 3 males with a mean age of 29.7 years) and 4 controls from nonatopic donors. The study was approved by the Ethic Committee of the Medical University of Vienna (EK1263/2014), and written informed consent was obtained from all subjects. Peach-specific and Pru p 3–specific IgE values were evaluated by means of CAP/RAST (ThermoFisher, Uppsala, Sweden). IgE values and clinical symptoms to peach of all patients are summarized in Table E1. After consumption of peach, all patients developed systemic reactions, such as urticaria, angioedema, or rhinitis and some of them also suffered from oral allergy syndrome. In addition, all patients positively responded to skin prick test with commercial peach extract as well as to prick-to-prick test with fresh peaches. To ensure the binding of the protein in the native state to the ELISA plate, Thermo Scientific Nunc Immobilizer Amino surface plates were used. Twenty micrograms of rPru p 3 (2 μM) in 100 mM sodium carbonate, pH 9.6, was incubated at room temperature for 1 hour to enable reaction with amine and thiol functions. After coupling to the surface, remaining Nunc Immobilizer electrophilic groups were quenched by reaction with 10 μM ethanolamine. A total of 60 μM of OLE or STE was added and incubated overnight with protein at 4°C. Subsequent steps were performed as described previously.E1Gaier S. Marsh J. Oberhuber C. Rigby N.M. Lovegrove A. Alessandri S. et al.Purification and structural stability of the peach allergens Pru p 1 and Pru p 3.Mol Nutr Food Res. 2008; 52: S220-S229PubMed Google Scholar As negative controls, normal human serum, STE, and OLE, respectively, were tested in parallel; the mean value of the negative controls was subtracted. PBMCs were isolated from peripheral blood of non–peach-sensitized donors by density gradient separation with Ficoll-Hypaque (ThermoFisher). Receptor-bound IgE was removed from basophils by incubation in lactic acid (pH 3.9) as previously described.E5Santos A.F. James L.K. Bahnson H.T. Shamji M.H. Couto-Francisco N.C. Islam S. et al.IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens.J Allergy Clin Immunol. 2015; 135: 1249-1256Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar Stripped basophils were then passively sensitized by incubation in human serum from 6 peach-sensitized donors for 60 minutes at 37°C. Subsequently, they were stimulated with titrated concentrations of rPru p 3 (10 μg/mL, 1 μg/mL, 100 ng/mL, 1 ng/mL, 100 pg/mL, 1 pg/mL, 0.1 pg/mL) that had been incubated with different dilutions of STE or OLE (ratio 1:1, 1:5, 1:10, 1:100) overnight at 4°C. The ratio of protein:ligand (1:10) with a final rPru p 3 concentration of 1 ng/mL or 1 μg/mL was selected after preliminary optimization, and only these data are shown. Basophils were labeled with CCR3 (eBioscience, Santa Clara, Calif) and CD123 (Biolegend, San Diego, Calif) and their activation was presented as percentage of CD63+ basophils.Fig E2Pru 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3RMSD 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Intermolecular 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Superimposed structures of Pru p 3-OLE− (in blue) and Pru p 3-OLE (in orange/yellow) complexes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Clinical data of the patient populationPatient no.SexAge (y)IgE (kU/L) specific for:Symptoms to peach∗G, Grasses; eg, eggplant; le, lettuce; to, tomato; A, Alternaria species; J, cypress; me, melon; wa, walnut; ar, apricot; M, mugwort; O, olive; ap, apple; ha, hazelnut; pe, pear; T, plane; f, fish; D, mites; or, orange; pn, peanuts.Other sensitizations∗G, Grasses; eg, eggplant; le, lettuce; to, tomato; A, Alternaria species; J, cypress; me, melon; wa, walnut; ar, apricot; M, mugwort; O, olive; ap, apple; ha, hazelnut; pe, pear; T, plane; f, fish; D, mites; or, orange; pn, peanuts.OASURTAERHINRespiratoryFoodPeachPru p 31Female1644.054.0++Geg, le, to2Female2326.029.6++A, Jle, me, to, wa3Female440.83.1++Gar, le, to, wa4Male1413.54.2+G, J, M—5Male460.60.5++G, J, M, Oap, ha, pe6Male302.91.3+J, T, Oha7Female266.44.0+G, Tf8Female410.80.4+J—9Female264.86.8++A, D, G, J, Mha, le, or, to10Female317.51.9+Gha, eg, pnAE, Angioedema; OAS, oral allergy syndrome; RHIN, rhinitis; URT, urticaria.∗ G, Grasses; eg, eggplant; le, lettuce; to, tomato; A, Alternaria species; J, cypress; me, melon; wa, walnut; ar, apricot; M, mugwort; O, olive; ap, apple; ha, hazelnut; pe, pear; T, plane; f, fish; D, mites; or, orange; pn, peanuts. Open table in a new tab Table E2Pru p 3 displays differential ligand bindingLigandANS fluorescence reduction (%)10 μM20 μM50 μM100 μMLAU (C12:0)30.641.658.566.5PAL (C16:0)45.958.959.358.8HYD (C16-OH)25.041.449.751.1STE (C18:0)27.014.634.439.1OLE (C18:1)56.574.173.972.6ELA (C18:1)41.262.568.168.6LIN (C18:2)52.270.476.972.0Concentration-dependent reduction in ANS binding to Pru p 3 preincubated with free fatty acid.ELA, Elaidic acid; HYD, 16-hydroxypalmitic acid; LAU, lauric acid; LIN, linoleic acid; PAL, palmitic acid. Open table in a new tab Table E3Impact of interaction between free fatty acids and Pru p 3 on IgE binding and basophil activationPatient serum no.ELISABATPru p 3 + OLEPru p 3 + STEPru p 3 + OLEPru p 3 + STE1+42.57+54.69+6.50−1.302+117.86+2.64+15.70−1.503+72.41+3.10+19.00−5.104+54.47+18.60+9.40+5.205+23.17−4.81+6.00−0.806+89.28+68.23+16.50+5.207+23.97+9.938+54.13+1.299+76.40−1.4310+57.96−39.28BAT, Basophil activation test.Values in the table correspond to differences in % between Pru p 3 preincubated with OLE or STE and Pru p 3 alone. (+) upregulation, (−) downregulation. Open table in a new tab AE, Angioedema; OAS, oral allergy syndrome; RHIN, rhinitis; URT, urticaria. Concentration-dependent reduction in ANS binding to Pru p 3 preincubated with free fatty acid. ELA, Elaidic acid; HYD, 16-hydroxypalmitic acid; LAU, lauric acid; LIN, linoleic acid; PAL, palmitic acid. BAT, Basophil activation test. Values in the table correspond to differences in % between Pru p 3 preincubated with OLE or STE and Pru p 3 alone. (+) upregulation, (−) downregulation.