Plant non-specific lipid transfer proteins type 1 (nsLTP1) are small basic proteins with a hydrophobic cavity able to host a number of different ligands: i.e. fatty acids, fatty acyl-CoA, phospholipids, glycolipids, and hydroxylated fatty acids. However, ligand binding specificity differs among nsLTPs. Within this protein family, Jug r 3 from walnut has been identified as a major allergen. So far, data on the structural characterization of Jug r 3 and its lipid binding capacity are lacking. We report the results from a fluorescence-based ligand-binding assay and ligand-based NMR experiments, to study the binding interactions between Jug r 3 and the 18-carbon monounsaturated oleic acid. Furthermore, protein-based NMR experiments were employed to detect the oleate binding site of Jug r 3. The NMR data were used to dock the oleate molecule into the structural model of Jug r 3. Finally, the impact of the interaction on the allergenic potential of Jug r 3 was investigated by IgE ELISA with 6 sera from walnut allergic patients. Our data corroborate the hypothesis of direct impact of food-derived matrix on the IgE reactivity of nsLTPs.

Nonspecific lipid transfer proteins (nsLTPs) are relevant food allergens. They have a compact 3D structure, with a hydrophobic lipid-binding cavity.1 It seems that the complex with a ligand can affect protein allergenicity.2 Recently, we have shown that the binding of oleic acid (OLE) to the peach nsLTP, Pru p 3, affects the conformation and IgE-binding activity of the allergen.3 We therefore investigated whether this observation can be extended to other homologous proteins. Thus, we analyzed nsLTPs with different allergenic potential, Mal d 3 from apple (high), Cor a 8 from hazelnut (intermediate), and Hel a 3 from sunflower seed (low),4, 5 and their interactions with 3 fatty acids: OLE, stearic (STE), and lauric (LAU) acids. We extracted natural nsLTPs and produced recombinant Mal d 3 and Cor a 8 using Pichia pastoris. Ligand binding of nsLTPs was assessed by ANS (1-anilinonaphthalene-8-sulfonic acid) displacement assay, measuring the decrease in ANS fluorescence. IgE reactivity of nsLTPs, alone or bound to fatty acids, was tested by IgE-ELISA using sera from subjects sensitized to peach and/or hazelnut nsLTP. For selected nLTPs/ligand complexes, molecular dynamic (MD) simulations were performed to analyze the impact of ligand binding on the protein conformation with a focus on specific IgE epitopes (details on methods and patients' data are presented in the Supporting Information). The different natural and recombinant nsLTPs were purified and characterized (Figure S1). rMal d 3, rCor a 8, and nHel a 3 bound the ANS probe to varying extent (Figure 1A). All proteins showed the lowest preference for STE (Figure 1B-D) with a maximum fluorescence reduction of 17% for rMal d 3/STE (50 μmol/L). LAU induced a dose-dependent reduction of ANS binding to all proteins, reaching values at 50 μmol/L of 53%, 7%, and 26% for rMal d 3, rCor a 8, and nHel a 3, respectively. In general, OLE induced a more pronounced reduction of ANS fluorescence, equal to 59%, 23%, and 46% at 50 μmol/L for rMal d 3, rCor a 8, and nHel a 3, respectively. Eighteen patients' sera containing specific IgE to Pru p 3 and/or Cor a 8 (Table S1) were tested by direct IgE-ELISA applying rMal d 3, rCor a 8, and nHel a 3 (Figure S2). We used selected sera (n = 6 for rMal d 3 and rCor a 8 each) to test if the IgE binding to the proteins is influenced by the interaction with OLE and STE, chosen as representatives of unsaturated and saturated fatty acid, respectively. Due to its very low IgE reactivity, nHel a 3 was excluded from this analysis. Preincubation of rMal d 3 with OLE significantly increased IgE-binding (P < 0.05), whereas STE did not affect rMal d 3 IgE-binding properties (Figure 1E). Regarding rCor a 8, both fatty acids induced an increase in IgE-binding, but it was statistically significant only for OLE (P < 0.01; Figure 1F). We performed computational calculations, detecting some differences in the cavity size of the apo nsLTPs: Mal d 3 had a larger pocket size (124 Å3) compared to both Cor a 8 and Hel a 3 (106 Å3 and 111 Å3, respectively). MD simulations were performed on Mal d 3 and Cor a 8 in complexes with either OLE or STE. For both proteins, the inclusion of ligands significantly increased the cavity volume, affecting the protein conformation. As shown in Figure 2, both allergens underwent conformational changes upon OLE binding, especially displacing loop 3 (I59 to N63) of Mal d 3, which moved toward the allergen surface, and affecting the C-terminal region in both allergens, which is consistent with our previous findings.3 Notably, this C-terminal region has been identified in Pru p 3 as a major IgE epitope,6 thus explaining the increased IgE reactivity observed for the nsLTP/OLE complexes. Interestingly, we observed similar conformational changes for the Cor a 8/STE complex, although to a minor extent, which was paralleled by a slight, albeit not statistically significant increase in IgE reactivity. On the contrary, binding of STE to Mal d 3 did not induce any structural change in the allergen, but rather increased its stability, yet without changing the IgE-binding properties. Considering differences in the allergenic potential of homologous proteins, we should take into account "the necessity of other matrix component to induce allergenic responses or differences and particularities in the epitope composition."7 Here, we focused on both food matrix and epitopes. With regard to the latter, the epitope sequences identified in Pru p 3 are well conserved in Mal d 3, but less in the other two nsLTPs, especially in Hel a 3 (Figure S1A)6. This could explain its very low IgE-binding, as in most cases, Pru p 3 is the primary sensitizer. Regarding the food matrix, we focused on how the interaction with fatty acids, commonly present in the food sources,8 could affect the allergenic properties of nsLTPs. Consistently with our previous observation for Pru p 3,3 all nsLTPs had the highest preference for OLE, whose binding induced major structural changes that affected IgE epitope orientation, and thus, their recognition by IgE, in both, Mal d 3 and Cor a 8. Recently, the direct immunomodulatory effect of food-derived lipids has gained increasing interest. Tordesillas et al showed the adjuvant activity of the natural ligand of Pru p 3 in the allergen sensitization.9 Summarizing the recent findings, including our data, it still remains to be fully elucidated whether lipids alone or in complexes can activate key players of both, the innate and adaptive immune system within an allergic response. In conclusion, this multidisciplinary study analyzed for the first time the ligand-binding capacities of Mal d 3, Cor a 8, and Hel a 3 and confirmed that these individual homologous allergens display different, higher and lower, IgE-binding activities, due to differences in their epitope structure. Furthermore, the volume and structural properties of the ligand-binding cavity of individual nsLTPs are critical for their ligand-binding activities. Upon ligand binding, the accessibility of these epitopes changed. Taken together our data provide molecular evidence how ligand binding to nsLTP affects IgE-binding activity. This work was funded by the Austrian Science Fund (FWF): DK W1248 to PD and PH, and SFB F4603 to KHS and SG. This research was also supported in part by the Marie-Curie project CARAMEL within the 7th European Community Framework Programme (Grant n 626572 to RAi) and by PL-Grid Infrastructure for TB. Computations were performed at the Academic Computer Centre Cyfronet AGH. RAi has received funding from the Marie-Curie project CARAMEL within the 7th European Community Framework Programme. KHS, PD, SG, and PH have been supported by the Austrian Science Fund (FWF). The remaining authors report no conflicts of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.

Methods: The binding of lipids to purified Pru p 3, Cor a 8, and Jug r 3 was monitored by adding 10 μM 1,8ANS and measuring the decrease of 1,8-ANS fluorescence. Furthermore, molecular dynamic analysis (MD) was applied to explore the nature of interaction between nsLTPs and tested ligands. Saturation transfer difference (STD) spectroscopy and W-LOGSY (Water-Ligand Observed via Gradient Spectroscopy) technique were applied to confirm results obtained by in silico approaches. Impact of lipid binding on the allergenicity of the proteins was investigated by ELISA.

BackgroundIn many areas of Europe, double sensitization to ragweed and mugwort is common, and because of the overlapping flowering periods of the 2 plants, it is not possible to diagnose the primary sensitizing allergen source and hence to determine the proper immunotherapy.ObjectivesTo elucidate whether double-sensitized patients are cosensitized or cross-sensitized and, in the latter case, to define the primary sensitizer.MethodsSerum samples from 34 patients with late summer respiratory allergy underwent skin prick testing with whole ragweed, and mugwort extracts were analyzed for their reactivity to recombinant Art v 1 and Amb a 1 by ImmunoCAP and then to Amb a 1, Art v 6, and Art v 1 isoforms by a proteomic approach. In double reactors, the primary sensitizing sources were detected by inhibition experiments.ResultsSerum samples from patients monosensitized to ragweed contained IgE to epitopes specific of all Amb a 1 isoforms. In contrast, serum samples from double reactors found to be primarily sensitized to mugwort reacted to Art v 1 and Art v 6 and cross-reacted to a few Amb a 1 isoforms. Finally, serum samples from double reactors found to be primarily sensitized to ragweed contained IgE reacting to all Amb a 1 isoforms, part of which cross-reacted to Art v 6. We did not find cosensitized patients.ConclusionThis study found that Art v 6 plays an important role in mugwort allergy and that the cross-reactivity between Art v 6 and Amb a 1 is frequent, bidirectional, and clinically relevant in the area of Milan.

The presence of genotoxic chemicals in soil can negatively affect crop yield and human health. In this study Trifolium repens was used as bioindicator to assess the impact of three different agriculture practices on soil genotoxicity. Three separate sites representative of the three different agricultural management systems were selected in the Pavia Province, Italy. Two biomonitoring experiments were performed during autumn 2004, after plant harvest and during summer 2005, just after soil preparation. Soil genotoxicity was evaluated with AFLP molecular markers. Results showed that all the three soils induced DNA damage in the indicator-plants. Nevertheless, on the base of the present findings, biodynamic agricultural management system seems the best farming approach to maintain soil quality with regard to genotoxicity.

In the present study, rice seedlings were exposed to a range of Cd concentrations (0.1 mu M, 1 mu M, 10 mu M, 100 mu M and 1 mM) for 15 days and a combination of different molecular approaches were used to evidence Cd effects and to assess the plants' ability to counteract metal toxicity. At a macroscopical level, only the highest Cd concentration (I mM) caused a complete plant growth inhibition, whereas the lowest concentrations seemed to stimulate growth. At genome level, the amplified fragment length polymorphism (AFLP) technique was applied to detect DNA sequence changes in root cells, showing that all the Cd concentrations induced significant DNA polymorphisms in a dose-dependent manner. Data also evidenced the absence of preferential mutation sites.Plant responses were analysed by measuring the levels of gluthatione (GSH) and phytochelatins (PCs), the thiol-peptides involved in heavy metal tolerance mechanisms. Results showed a progressive increase of GSH up to 10 mu M of Cd treatment, whereas a significant induction only of PC3 was detected in roots of plants exposed to 100 mu M of Cd. As suggested by the proteome analysis of root tissues, this last concentration strongly induced the expression of regulatory proteins and some metabolic enzymes. Furthermore, the treatment with 10 mu M of Cd induced changes in metabolic enzymes, but it mainly activated defence mechanisms by the induction of transporters and proteins involved in the degradation of oxidatively modified proteins. (c) 2006 Elsevier B.V. All rights reserved.

Polycyclic aromatic hydrocarbons (PAHs) are among the most dangerous environmental contaminants due to their toxic, carcinogenic and mutagenic effects. Although there are many data in literature that detail the effects of PAHs on animals, little is known about their action on higher plants which are often used as bioindicators.The aim of the present study was to evaluate the genotoxicity of two different PAHs, benzo[a]pyrene (BaP) and naphthalene (Naph), on Trifolium repens L. Clover plants were exposed to soil which had been artificially contaminated with three concentrations of BaP (5, 10 and 20μgg−1) or Naph (25, 50 and 100μgg−1). After 15 days, changes in the DNA content and sequence of roots and shoots were evaluated by flow cytometry (FCM) and amplified fragment length polymorphism (AFLP). Root and shoot dry weight were also determined to assess plant growth.Results showed that BaP and Naph were both genotoxic for white clover, inducing significant changes in root and shoot DNA sequence. Damage was more severe in the root than in the shoot suggesting that the translocation of these compounds and their genotoxic metabolites was limited. Ploidy alterations were not detected and the extent of damage caused by all the tested PAH concentrations was not sufficient to affect plant development.

Peach fruit quality consists of many characteristics among which flesh firmness is of great concern. Firmness and texture of ripe fruit are considered to result, in large part, from the disassembly of the primary cell wall (Rose and Bennett, 1999, Brummel and Harpster, 2001). This process involves several biochemical and physiological factors, among which proteins able to plasticize (expansins) or degrade (endopolygalacturonases [endoPGs], pectin methyl esterases, cellulases) the cell wall components are of great importance. The involvement of endoPG proteins in peach softening, was investigated by our group which demonstrated that both protein transcription and protein modification, in particular glycosylation, are mechanisms used by plant to regulate endo-PG activity (Ardolino et al., 2005). In this study, which is part of a project aimed to understand the molecular mechanisms regulating fruit softening, morphological analyses of peach mesocarp and esocarp have been carried out by producing semi-thin sections (0.5 μm). The following different cultivars have been analysed at different ripening stages: (i) “melting flesh” peaches (MF) showing a pronounced decrease in fruit firmness during the final stages of ripening; (ii) “non melting flesh” peaches (NMF) which remain relatively firm since the lack of the final melting phase of softening; (iii) “Stony Hard” peaches (SH) which maintain flesh firmness for several days after harvest even at room temperature; (iv) “Slow ripening” peach mutant characterized by the fruit development arrest in the pre-climateric phase with the absence of the typical maturation processes. Sections used for morphological analyses have been also probed with a rabbit polyclonal antibody specifically produced and tested for peach endopolygalacturonase (endoPG). Image analysis results showed a progressive loss of cell adhesion and turgor in parallel with softening and with endoPG localization in cell wall. The analysis of the different peach cultivar showed that the loss of cell adhesion and turgor are characteristics strictly associated with the loss of flesh firmness and with the endo-PG accumulation in the cell wall.