interaction with pruritogens, including SP and compound 48/80, we evaluated LL-37-induced itch using the mouse cheek model. As opposed to pruritogens, which induce scratching in this model, LL-37 does not induce scratching (see Fig E8 in this article's Online Repository at www.jacionline.org).The data presented here demonstrate that SP and compound 48/ 80 activate native Mrgprs but fail to activate Mrgprs in which a single amino acid residue was mutated. In contrast, LL-37 activates both native receptor and mutant receptors. This finding indicates that the site(s) of Mrgpr activation for the pruritogens SP and compound 48/80 differs from the activation site(s) for LL-37. Furthermore, an Mrgpr antagonist, QWF, 2 inhibits the interaction of SP and compound 48/80 with MRGPRX2 but not that between LL-37 and the receptor.These findings have several implications. First, they reveal that the Glu and Asn residues at the end of the fourth TMD and beginning of the fourth extracellular loop of Mrgprs are necessary for the interaction of SP and compound 48/80 with members of this receptor family. Second, not all compounds that activate Mrgprs or induce mast-cell degranulation result in itch. It is possible that LL-37 may interact with other cells such as keratinocytes and additional receptors to induce the release of molecules with antipruritic properties such as semaphorin 3A. 11 Another possibility is that activation of MRGPRX2 on mast cells by SP versus LL-37 may result in differential release of granules and their associated mediators from mast cells. Third, at least some Mrgprs appear to have more than 1 site for signaling followed by distinct behavioral effects. Fourth, although MrgprB2 has been proposed as the mouse ortholog of human MRGPRX2, modeling demonstrates that mouse MrgprA1 and human MRGPRX2 also exhibit topological similarities (see Fig E9 in this article's Online Repository at www.jacionline.org). Taken together, these data provide further support for the possibility that antagonists of MRGPRX2 may be useful for the treatment of itch and inflammation while providing guidance with respect to the development of receptor antagonists.

ScopeAllergens from nuts frequently induce severe allergic reactions in sensitive individuals. The aim of this study was to elucidate the physicochemical characteristics of natural Cor a 14, the 2S albumin from hazelnut.Methods and resultsCor a 14 was purified from raw hazelnuts using a combination of precipitation and chromatographic techniques. The protein was analyzed using gel electrophoresis, MS, and far‐UV circular dichroism (CD) analyses. The immunoglobulin E (IgE) binding of native, heat‐treated, and in vitro digested Cor a 14 was studied. We identified two different Cor a 14 isoforms and showed microclipping at the C‐terminus. CD spectra at room temperature showed the typical characteristics of 2S albumins, and temperatures of more than 80°C were required to start unfolding of Cor a 14 demonstrating its high stability to heat treatment. In vitro digestion experiments revealed that Cor a 14 is resistant to proteolytic degradation. Native and heat‐treated protein was recognized by sera from hazelnut allergic patients. However, denaturation of the allergen led to significantly reduced IgE binding.ConclusionWe identified two different isoforms of Cor a 14 displaying high stability under heating and gastric and duodenal conditions. Data from IgE‐binding experiments revealed the existence of both, linear and conformational epitopes.

Nonspecific lipid transfer proteins (nsLTPs) are basic proteins, stabilized by four disulfide bonds, and are expressed throughout the plant kingdom. These proteins are also known as important allergens in fruits and tree nuts. In this study, the nsLTP from hazelnuts, Cor a 8, was purified and its crystal structure determined. The protein is stable at low pH and refolds after thermal denaturation. Molecular dynamics simulations were used to provide an insight into conformational changes of Cor a 8 upon ligand binding. When known epitope areas from Pru p 3 were compared to those of Cor a 8, differences were obvious, which may contribute to limited cross-reactivity between peach and hazelnut allergens. Differences in epitope regions may contribute to limited cross-reactivity between Cor a 8 and nsLTPs from other plant sources. The structure of Cor a 8 represents the first resolved structure of a hazelnut allergen.

Dendritic cells (DCs) are the most important antigen presenting cells to activate naïve T cells, which results in the case of Type 1 allergies in a Type 2 helper T cell (Th2)-driven specific immune response towards allergens. So far, a number of different subsets of specialized DCs in different organs have been identified. In the recent past methods to study the interaction of DCs with allergenic proteins, their different uptake and processing mechanisms followed by the presentation to T cells were developed. The following review aims to summarize the most important characteristics of DC subsets in the context of allergic diseases, and highlights the recent findings. These detailed studies can contribute to a better understanding of the pathomechanisms of allergic diseases and contribute to the identification of key factors to be addressed for therapeutic interventions.

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.

Walnuts are ranked high in the list of the culprit foods inducing severe allergic reactions. Jug r 2 has been identified as a major allergen in common walnut by cDNA cloning from a somatic cell line. So far, studies were performed on the allergenic activity of recombinant Jug r 2, yet there is still no evidence about the physicochemical characteristics of the natural allergen. Therefore, we aimed to purify and deeply characterize natural Jug r 2 and to assess IgE cross-reactivity among vicilins from different tree nuts. Extensive mass spectrometry analysis of the obtained purified vicilin allowed identification of the protein sequence that displayed only 44% identity to Jug r 2. The newly identified vicilin (Jug r 6) was recognized by IgE of 26% in walnut allergic patients’ sera tested. In contrast to Jug r 2, Jug r 6 displayed a remarkable level of cross-reactivity when tested with homologues from hazelnut, sesame and pistachio. It is the first report showing the necessity of proteomic studies to improve allergy component resolved diagnosis.

SummaryBackgroundBuckwheat (Fagopyrum esculentum) has become increasingly popular as a healthy food in Europe. However, for sensitized individuals, consumption can cause anaphylactic reactions. The aim of this study was to identify individual well‐characterized buckwheat allergens for component‐resolved diagnosis.MethodsPatients were selected by positive skin prick test to buckwheat and divided into two groups: (1) sensitized to buckwheat without clinical symptoms and (2) buckwheat allergy. Buckwheat proteins were extracted from raw buckwheat seeds, purified applying a combination of protein precipitation and chromatographic methods, and analyzed by IgE immunoblotting and ELISA.ResultsBuckwheat‐allergic patients had a significantly larger median skin prick test weal diameter for buckwheat than the sensitized group and the positive control. Also, IgE immunoblotting clearly showed a distinct pattern in sera from allergic patients when compared to sensitized individuals. Several IgE‐reactive proteins were purified from crude buckwheat extract, namely legumin (Fag e 1 plus its large subunit), Fag e 2 (2S albumin), and newly identified Fag e 5 (vicilin‐like) as well as hevein‐like antimicrobial peptides, designated Fag e 4. All four allergens showed superior diagnostic precision compared to extract‐based ImmunoCAP with high sensitivity as well as high specificity.ConclusionsPatients with clinical symptoms clearly show a distinct allergen recognition pattern. We characterized a buckwheat vicilin‐like protein as a new relevant marker allergen, designated Fag e 5. Additionally, another new allergen, Fag e 4, potentially important for cross‐reactivity to latex was added to the allergen panel of buckwheat. Further, our data show that the full‐length legumin comprising both, large and small subunit should be applied for component‐resolved diagnosis. Our data indicate that concomitant sensitization to legumin, Fag e 2 and Fag e 5, predicts buckwheat allergy.

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