Non-specific lipid-transfer proteins trigger TLR2 and NOD2 signaling and undergo ligand-dependent endocytosis in epithelial cells

Abstract

BackgroundAllergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. ObjectiveWe show the molecular details and the effects of three non-specific lipid transfer proteins (nsLTPs; Mal d 3, Cor a 8 and Pru p 3) upon epithelial cell uptake and transport. MethodsWe used fluorescent imaging, flow cytometry, proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. ResultsNsLTPs are transported across the epithelium without affecting cell membrane stability or viability and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis (CME). Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent upon these ligands in the protein complex.nsLTPs were found to initiate cellular signaling via TLR2 but not the CD1d receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the three allergens induced intracellular stress signaling through activation of the NOD2 pathway. ConclusionsOur work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. Additionally, we have developed a versatile toolbox to extend these investigations to other allergens and cell types.