NMR spectra typical for allergens with random coil structure, compared with a spectrum of an allergen with rigid tertiary structure.

<p>Two enlarged regions of the spectra (gradient-based water suppression NMR experiment) of purified casein fraction from cow (Bos d 8, 0.08 mM, 900 MHz cryoprobe, 40 mM phosphate, pH 7.4), and goat (whole caprine casein, WCC, 0.19 mM; 40 mM phosphate pH 7.4), and of the recombinant tropomyosin from shrimp (Pen a 1, 0.13 mM, 36.4 kDa, MOPS 20 mM, NaCl 0.5 M, pH 7.6) are shown. The 0.3–1.2 ppm region includes the extreme methyl signals while the 6.2–7.4 ppm region includes the resonances of aromatic ring protons, side chain HN, and part of backbone HN; These spectra are typical of random coil structures, as indicated by the absence of signal upfield 6.6 ppm and 0.7 ppm. The same enlarged regions of the spectrum of Pru p 3 (0.39 mM, 91 AA, 9.2 kDa) are shown (top) to underline the difference between spectra of folded and unfolded proteins.</p

NMR spectra of nsLTP from peach, Pru p 3 under thermal treatment in acidic and near-neutral pH conditions.

<p>Heating of Pru p 3 (0.39 mM, 91 AA, 9.2 kDa) under acidic (pH 3.0) and neutral (pH 7.0) conditions; pre-saturation NMR experiments. To improve readability, the spectra at T>298 K are aligned with the reference spectrum at 298 K (25°C), by shifting them upfield as indicated after the temperature. pH 3.0 bottom: NMR spectra of Pru p 3 scanned at 298 K before and after heating up to 358 K (85°C). pH 7.0, bottom: NMR spectra of Pru p 3 scanned at 298 K before and after heating up to 358 K. top: spectrum scanned at 298 K after 358 K heating. In neutral conditions the protein undergoes irreversible denaturation, in the 80°C–85°C range of temperature.</p

NMR Spectra of non specific lipid transfer protein, nsLTP, from peach (Pru p 3), apple (Mal d 3) and hazelnut (Cor a 8).

<p>The spectra (gradient-based water suppression NMR experiment) of nsLTPs from different sources are compared: Cor a 8 from hazelnut (115 AA, 11.8 kDa, 0.15 mM; 25 mM phosphate, NaCl 150 mM, pH 7.0), Mal d 3 from apple (115 AA, 11.4 kDa, 0.08 mM, 6504 scans; 20 mM phosphate, pH 7.4), Pru p 3 from peach (91 AA, 9.2 kDa, 0.39 mM; 25 mM phosphate, pH 7.0). These spectra indicate the presence of extended, rigid and ordered tertiary structure for all three molecules and display a high degree of similarity.</p

Modelled and experimental structures of 11S globulins from hazelnut and almond.

<p>Model of Cor a 9 (11S globulin from hazelnut), based on template PDB 3fz3 (11S globulin from almond) chain C (515 residues). Disulphide bonds are rendered as spheres. Both the monomer and the hexamer of the template are shown. The template's chain C is rendered in light gray, with the same orientation, either as monomer or in the hexamer. The presence of mobile parts is indicated by the many extended loops of the model, corresponding to similar extended loops or to unresolved regions of the template. Modelled residue range: 34 to 496. Sequence Identity [%]: 51. QMEAN Z-score = −3.084.</p

Structures of tropomyosin from brown shrimp (modelled) and pig (experimental).

<p>Model of Pen a 1, the tropomyosin from brown shrimp (284 residues) based on tropomyosin (PDB 1c1g chain A) from cardiac muscle of pig (<i>Sus scrofa</i>). Tropomyosins can pass from a coiled coil to molten-like structure before complete dissociation of the two chains. Chains are flexible and can be unfolded at physiological temperature. Modelled residue range: 1 to 283. Sequence Identity [%]: 56. QMEAN Z-score = −0.371. QMEAN score 4 = 0.755 (0.732 on tropomyosin from striated-muscle of <i>R. norvegicus</i> 2b9c chain A).</p

NMR Spectra of globulins from peanut and hazelnut.

<p>The spectra (gradient-based water suppression NMR experiment, 848 scans) of selected globulins from different sources are presented: Ara h 3/4 (mixture of isoforms of 11S globulins from peanut, 37.0 kDa, putative concentration 0.14 mM; 50 mM phosphate, NaCl 150 mM, pH 7.5), Cor a 9 (11S globulin from hazelnut, 40 kDa, 0.03 mM, 50 mM phosphate, NaCl 150 mM, pH 7.5), Ara h 1 (7/8 S globulin from peanut, 63.5 kDa, 0.03 mM, 50 mM MOPS, NaCl 200 mM, pH 7.8), Cor a 11 (7/8S globulin from hazelnut 48 kDa, 0.04 mM, phosphate 50 mM, NaCl 150 mM, pH 7.5), Signals, though basically unresolved due to high MW and possible aggregation, can be observed in the extreme methyl region of all the spectra (around 0.5 ppm) which indicate of regions of rigid tertiary structure. The presence of extended disordered regions must also be considered, due to the few broad peaks that can be observed in the whole spectral window, especially in the HN region between 6 and 8 ppm and especially for Cor a 9. On the contrary the same region of Cor a 11 is populated by several more resolved and narrow resonances, indicative of rigid and ordered regions. Both, Cor a 9 and Cor a 11 show a noticeable signal broadening in the backbone NH region (8.0–8.7 ppm), indicating the presence of the molten globule state in solution. In the Ara 3/4 spectrum, narrow, weak signals, can be observed on an envelope of broader peaks. They could be due to the lighter (non-aggregated) component of the mixture.</p

Modelled and experimental structures of nsLTPs from apple, peach and hazelnut.

<p>Models of Mal d 3 and Cor a 8 based on the experimental structure of Pru p 3 as template (PDB 2alg, chain B, 92 residues). Disulphide bonds are rendered as sticks and the internal hydrophobic cavity is rendered as a mesh surface. The modelled residue range is 25–115 for both models. QMEAN Z-score = −1.895 (Mal d 3) and −1.639 (Cor a 8).</p

The Major Birch Pollen Allergen Bet v 1 Induces Different Responses in Dendritic Cells of Birch Pollen Allergic and Healthy Individuals

<div><p>Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.</p></div

Competitive binding of allergens to iMoDCs of BP allergic (AD) and normal donors (ND).

<p>(A) Uptake of labeled Api g 1 (20 μg/ml) in the presence of unlabeled Bet v 1 (shown for donors AD3 and ND3). (B) Cross-competition of labeled Bet v 1 with Api g 1 (Bet v 1*/Api g 1) and self-competition of Bet v 1 (Bet v 1*/Bet v 1) using 20μg/ml of labeled allergen and 200 μg/ml of competitor (donor AD3). Results in (A) and (B) show uptakes after 45 minutes of chase and are representative of four independent experiments with similar outcomes for both donor groups. (C) Densitometric quantification of the competition assays measuring a 1 mm² area.</p

Kinetics of allergen uptake into iMoDCs of BP allergic and normal donors.

<p>Internalization of labeled Bet v 1 (Bet v 1–488) and labeled Api g 1 (Api g 1–610) by iMoDCs of allergic (A) and normal donors (B) was followed by live-cell fluorescence imaging. Results are representative of independent experiments of three different donors for each group (shown for donors AD1 and ND3). Exocytosis of fluorescent markers (panel A) and the absence of spillover of fluorescence (panel B) are depicted by framed display details.</p