Characterization of the biological activity of pru p 3 in plant and intestinal epithelium

Abstract

ABSTRACT Allergy is a serious social problem that impairs the quality of life of patients and is currently considered the most prevalent chronic disease in Europe. A large increase in its prevalence has occurred since the mid-twentieth century. However, the underlying causes for this increase and the factors that trigger allergy are still unknown. Knowing why a protein becomes an allergen would be essential in the prevention and treatment of allergy diseases. The main aim of this doctoral thesis was to study the molecular mechanisms involved in the allergic sensitization processes. Increasing evidence suggests that lipids transported by certain allergens can be recognized by cellular receptors of the immune system, and may play a prominent role in allergic sensitization. In this work, Pru p 3, the major allergen of the peach and a lipid-transfer protein (LTP), has been chosen as model. The lipid ligand of Pru p 3 has been structurally characterized for the first time using ESI-micrOTOF-QII. The ligand has thus been identified as a hydroxyl derivative of camptothecin bound to phytosphingosine, a hydrocarbon tail which is inserted into the hydrophobic tunnel of the protein. This result was validated by assays of inhibition of topoisomerase I as well as fluorescence emission at 254 nm, both features characteristic of camptothecin. Additionally, different in vitro techniques (such as RT-PCR and immunohistochemistry) were combined to characterize the possible role of the ligand in plant. The highest expression of Pru p 3 was detected in pollinated styles by contrast to its absence in non-pollinated flowers where the expression decreased after the antithesis. Besides, the highest expression of Pru p 3 was localized in the tricomas, but not in the pulp. These data, together with the inhibition of pollen germination by the ligand, suggest that Pru p 3 can inhibit a secondary pollination and keep herbivores away until seed maturation. Additionally, the immunological activity of the lipid ligand was evaluated using different in vitro models (moDC, PBMCs, THP1, and Caco2) and a mouse model of anaphylaxis. Thus, it was determined that the ligand was capable of modulating the immune system through a Th2 response, responsible for the activation of antigen presenting cells, and in increasing the capacity of allergic sensitization to Pru p 3 in a mouse model. Furthermore, the ligand was introduced by the CD1d receptor into the epithelial cells to activate iNKT cells. These results have been validated using the ex vivo model InTESTineTM with fragments of pig tissue. The ligand did not affect the transport of Pru p 3 through the intestinal barrier, but appears to remain accumulated in Peyer's Patches. The presence of the ligand was also observed to induce the expression of cytokines that are typical of a Th2 response like IL4 and IL13. Moreover, it was confirmed that Pru p 3 co-localized with the CD1d receptor in the presence of the ligand, as well as increasing the presence of iNKT cells. In summary, this thesis work represents a novel approach to characterize allergic response mechanisms and presents results which are useful for improving diagnosis and for providing a basis for new investigations related to allergy.