Biophysical and biological impact on the structure and IgE-binding of the interaction of the olive pollen allergen Ole e 7 with lipids.

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways.This work was supported by SAF2014-53209-R and SAF2017-86483-R grants from the Ministerio de Economía y Competitividadto R.B. and M.V., and to M.V., respectively, RTI2018-094564-B-I00 from the Spanish Ministry of Science and Universities andP2018/NMT4389 from the Regional Government of Madrid to J.P-G. and A.C., and RIRAAF Network RD12/0013/0015 grant and ARADyAL Network RD16/0006 grant from the Instituto de Salud Carlos III (ISCIII) co-founded by Fondo Europeo de Desarrollo Regional -FEDER-for the Thematic Networks and Co-operative Research Centres.A.J. and C.M. acknowledge PI-01119-2016 from the Consejería de Salud (Junta de Andalucía) and the Alergosur Foundation. JC.L-R., P.SS-A. and C.G-M. are recipients of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. We also gratefully acknowledge the financial support from theAES-ISCIII for the PI17CIII/00045 grant to R.B. We also thank the excellent technical support of Sara Abián.S