Final Report Summary - CARAMEL (Impact of food matrix interaction and post-translational modifications on the allergenicity of Mal d 3, a major apple allergen) CARAMEL - Publishable Summary Fruits are regarded as a healthy part of our diet. However, unfortunately, a lot of fruits can trigger allergic reactions, even severe ones, and, due to the large cross-reactivity between related species, the avoidance of a specific fruit allergen often leads to the elimination of many foods, which can result in an unbalanced diet. In Europe, perceived fruit allergy is estimated to affect about 0.4-6.6% of the adult population, a value that is even higher in children (2.2-11.5%). Typical examples are fruits from the Rosaceae family (i.e. peach, apple, plum, apricot and cherry), which show a similar allergenic pattern and are highly cross-reactive. Among them, apple is the most frequently consumed fruit in Europe, eaten both as a fresh fruit and processed food. To date, 4 apple allergens have been officially registered in the IUIS Database (www.allergen.org). Among those, Mal d 3, a non-specific Lipid Transfer Protein (nsLTP), are known to induce rather severe, potentially life-threatening, systemic reactions. For this reason Mal d 3, as well as its homologues from other fruits, has attracted the interest of the scientific community.The research project CARAMEL dealt with in depth characterisation of food allergens and their interaction with the food matrix. It focused on the non-specific Lipid Transfer Protein (nsLTP) from apple, the allergen Mal d 3. Like other nsLTPs, Mal d 3 is a small, basic protein with a compact 3D structure, which is highly resistant to degradation and is able to reach the gut only slightly modified, inducing severe, potentially life-threatening, systemic reactions in allergic patients.Despite the efforts made for the physicochemical, biochemical and immunological characterisation of nsLTPs, the protein structure by itself does not full justify the allergen’s ability to produce an immuno-response and other factors must be taken into consideration, such as the immune-modulating properties of both, food matrix interactions and post-translational modifications (PTMs), as well as their effects on the allergen structure. Notably, there is a lack of knowledge about the impact of such interactions/modifications on nsLTPs.The aim of CARAMEL is to shed light on this important aspect of nsLTPs allergenicity, studying apple as a model food and using a multidisciplinary approach that includes both, experimental and in silico methods. Production and purification of Mal d 3 for improved in vitro allergy diagnosisThe important apple allergen Mal d 3 was purified from apple peel, and in parallel. the recombinant protein was produced in the yeast expression system to obtain the target protein in high quantities for further experiments. Next, both protein batches, the naturally derived protein and the recombinant counterpart were compared: they showed high agreement with regard to their molecular mass and their structural features. Subsequently, purified recombinant Mal d 3 was tested for its application as a diagnostic tool to detect specific IgE antibodies in apple allergic patients’ sera. Based on these studies the recombinant Mal d 3 displayed a high sensitivity in the diagnostic tests and contributes to a molecule based patient-specific sensitisation profile which, allows to identify patients at risk to develop severe allergic reactions (Mal d 3-sensitized) versus apple allergic patients without Mal d 3 sensitization, with a concomitant low risk of severe food allergic reactions. This in turn helps to fine tune the dietary recommendation for a fruit allergic patient. Does Mal d 3 interact with food matrix components? Subsequently, the interaction of the allergenic protein, Mal d 3, with other components from the apple fruit were investigated on a molecular level. In a first approach, the nsLTP was tested for its binding activity towards a range of fatty acids. Detailed binding studies showed that Mal d 3 indeed showed a differential binding activity towards certain fatty acids, unsaturated fatty acids being the best binding partners. The binding to a specific fatty acid, also present in the apple fruit was also affecting the 3D structure of the protein and thus affecting its allergenic activity. In parallel, the interaction of Mal d 3 with pectin, an abundant component of apple fruits, was investigated with regard to changes in the structure of the protein and potential effects on the allergenic activity. Finally, nitration of the protein was performed and the effect on the structure and stability of the protein analysed. In summary, the interaction of Mal d 3 with neither pectin nor nitration of the protein was affecting its stability. The structure of Mal d 3 and its changes when binding to a ligand could be followed by performing molecular modelling simulation, that is a refined up-to-date method that measures potential changes of a protein structure when adding binding partners or modifying amino acid residues chemically. In summary, the detailed studies combining highly sensitive analytical methods, refined structure simulation methods and applying cellular and antibody-based assays, provided deeper insight into the allergenicity of a relevant fruit allergen. Taken together this panel of methods enabled a better understanding of the protein food matrix interaction. Furthermore, it helped to define the regions of the protein interacting with ligands and to assess whether these regions are also immune relevant areas, defined as epitopes. Indeed, we were able to prove that Mal d 3 interacts with certain fatty acids and thus changes its surface exposed structure, which in turn affects its allergenicity. Based on these molecular data, new strategies how to modify certain allergens can be developed. Depending on the application for diagnostic purposes we can add allergen in its liganded form to the test panel. For immunotherapeutic aspects we can design protein variants low in allergenicity that reduce the risk of side effects during therapy.
