Enhanced Pru p 3 IgE-binding activity by selective free fatty acid-interaction

Nonspecific lipid transfer proteins (nsLTPs) are major crossreactive allergens identified in most plantderived foods as well as pollen from diverse plants, and are often associated with severe symptoms in food allergy. Currently, Pru p 3 (major food allergen from peach [Prunus persica]) is regarded as the primary sensitizer for nsLTP-caused allergies. Pru p 3 shares the physico-chemical 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. There is limited knowledge about the binding capacity of Pru p 3. So far, interactions with lauric acid, cis-parinaric, palmitic, and linoleic acids have been reported. Several structures of nsLTPs with and without ligands have been determined including peach and hazelnut 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 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. 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 2C-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. 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. It becomes clear that lipids, in addition to their role as direct immune modulators, can influence the allergenicity of proteins 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 pin 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.