BackgroundThe search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties.ObjectiveWe assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1.MethodsBy exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model.ResultsFold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment.ConclusionsOur data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

More than 70% of birch pollen-allergic patients develop allergic cross-reactions to the major allergen found in apple fruits (Malus domestica), the 17.5 kDa protein Mal d 1. Allergic reactions against this protein result from initial sensitization to the major allergen from birch pollen, Bet v 1. Immunologic cross-reactivity of Bet v 1-specific IgE antibodies with Mal d 1 after apple consumption can subsequently provoke severe oral allergic syndromes. This study presents the three-dimensional NMR solution structure of Mal d 1 (isoform Mal d 1.0101, initially cloned from ‘Granny Smith’ apples). This protein is composed of a seven-stranded antiparallel β-sheet and three α-helices that form a large internal cavity, similar to Bet v 1 and other cross-reactive food allergens. The Mal d 1 structure provides the basis for elucidating the details of allergic cross-reactivity between birch pollen and apple allergens on a molecular level.

Background Most birch pollen-allergic patients develop allergic cross-reactions to the major allergen found in apples Mal d1, known as pollen-related food allergy (prFA). This is due to a strong clinically relevant homology between the major allergen in birch Bet v 1 and Mal d 1. Daily apple consumption induces oral tolerance in prFA, but its effect on the inhalational allergy has not been investigated. Objectives As continuous apple consumption might also mitigate the inhalational allergy, this study aimed to uncover apple cultivars suitable for treatment of birch pollen rhinoconjunctivitis and apple allergy in a controlled and established dosage. Methods Patients (n = 52) with birch pollen allergy and prFA to apples were subjected to a prick-to-prick test (SPT) with 23 cultivars (red-fleshed, old traditional and new commercial). By SPT, the apple parts flesh, peel equatorial and peel apical near the stalk were compared for their reactivity. One apple cultivar of each allergenicity class (low, middle and high) was subsequently tested in an oral provocation test (OPT). Results According to the SPTs, we provide a ranking of all 23 cultivars. Red-fleshed apples displayed the lowest reactivity, followed by old and new cultivars. Four cultivars showed disagreement from their allergenicity class: Santana and Pink Ladyâ, new cultivars that provoked only low to moderate. In contrast, White Rosemary and Goldparm€ ane, two old cultivars, induced strong reactions. Skin reactivity increased from flesh to peel to stalk, and SPT results could predict the severity of prFA of each allergenicity class. Conclusions Herein, we propose a treatment protocol for allergen immunotherapy to birch pollen and prFA with daily apple consumption. Red-fleshed, old and the new cultivars Santana and Pink Ladyâ provoke less allergic reactions and are suitable for initial induction. After a controlled and well-tolerated increase of intake, also moderate and finally high allergenic apple cultivars should be integrated into treatment of birch pollen allergenic patients.

The protein Bet v 1 represents the main cause for allergic reactions to birch pollen in Europe and North America. Structurally homologous isoforms of Bet v 1 can have different properties regarding allergic sensitization and Th2 polarization, most likely due to differential susceptibility to proteolytic cleavage. Using NMR relaxation experiments and molecular dynamics simulations, we demonstrate that the initial proteolytic cleavage sites in two naturally occurring Bet v 1 isoforms, Bet v 1.0101 (Bet v 1a) and Bet v 1.0102 (Bet v 1d), are conformationally flexible. Inaccessible cleavage sites in helices and strands are highly flexible on the microsecond-millisecond time scale, whereas those located in loops display faster nanosecond-microsecond flexibility. The data consistently show that Bet v 1.0102 is more flexible and conformationally heterogeneous than Bet v 1.0101. Moreover, NMR hydrogen-deuterium exchange measurements reveal that the backbone amides in Bet v 1.0102 are significantly more solvent exposed, in agreement with this isoform’s higher susceptibility to proteolytic cleavage. The differential conformational flexibility of Bet v 1 isoforms, along with the transient exposure of inaccessible sites to the protein surface, may be linked to proteolytic susceptibility, representing a potential structure-based rationale for the observed differences in Th2 polarization and allergic sensitization.

The major apple allergen Mal d 1 is the predominant cause of apple (Malus domestica) allergies in large parts of Europe and Northern America. Allergic reactions against this 17.5 kDa protein are the consequence of initial sensitization to the structurally homologous major allergen from birch pollen, Bet v 1. Consumption of apples can subsequently provoke immunologic cross-reactivity of Bet v 1-specific antibodies with Mal d 1 and trigger severe oral allergic syndroms, affecting more than 70 % of all individuals that are sensitized to birch pollen. While the accumulated immunological data suggest that Mal d 1 has a three-dimensional fold that is similar to Bet v 1, experimental structural data for this protein are not available to date. In a first step towards structural characterization of Mal d 1, backbone and side chain 1H, 13C and 15N chemical shifts of the isoform Mal d 1.0101 were assigned. The NMR-chemical shift data show that this protein is composed of seven β-strands and three α-helices, which is in accordance with the reported secondary structure of the major birch pollen allergen, indicating that Mal d 1 and Bet v 1 indeed have similar three-dimensional folds. The next stage in the characterization of Mal d 1 will be to utilize these resonance assignments in solving the solution structure of this protein.