Download PP133 Impact of free fatty acids binding to nsLTP on their tertiary

PP133
Impact of free fatty acids binding to nsLTP on their tertiary structure and allergenic
activity
Pawel Dubiela, Roberta Aina, Piotr Humeniuk, Sabine Pfeifer, Merima Bublin, Francesca Cantini,
Tomasz Borowski, Stefano Alessandri, Karin Hoffmann-Sommergruber
Medical University of Vienna, Austria
Aim: The main aim of this study was to investigate potential influence of binding of free fatty acids on the
tertiary structure of nsLTP from peach (Pru p 3), hazelnut (Cor a 8) and walnut (Jug r 3). Moreover, we
explored effect of conformational changes on the allergenic activity of the proteins.
Methods: The binding of lipids to purified Pru p 3, Cor a 8, and Jug r 3 was monitored by adding 10 µM 1,8ANS and measuring the decrease of 1,8-ANS fluorescence. Furthermore, molecular dynamic analysis (MD)
was applied to explore the nature of interaction between nsLTPs and tested ligands. Saturation transfer
difference (STD) spectroscopy and W-LOGSY (Water-Ligand Observed via Gradient Spectroscopy) technique
were applied to confirm results obtained by in silico approaches. Impact of lipid binding on the allergenicity
of the proteins was investigated by ELISA.
Results: Due to pre-incubation of Pru p 3 with lipids a reduction of ANS binding was observed. Pru p 3
incubated (1:1; 1:10) with lauric acid showed 19% and 66% of ANS fluorescence reduction respectively,
compared with Pru p 3 without lipids. For oleic acid (1:1; 1:10) reduction was 57% and 77%, respectively.
MD analysis suggests changes in protein structure due to binding to certain ligands. Interaction between
oleic acid and Pru p 3, moved the C-terminal loop out towards the surface of the molecule. NMR based
experiments confirmed binding capacity observed in MD analysis. Pre-incubation of oleic acid with Pru p 3
significantly increased IgE binding in ELISA as compared to stearic acid and allergen alone.
Discussion: nsLTPs are relevant food allergens with a conserved fold and characteristic internal cavity. Our
study indicates that Pru p 3 displays some binding specificity as compared to Cor a 8 and Jug r 3. It prefers
to bind unsaturated versus saturated fatty acids and short carbon chains ligands (C12 vs C18). Moreover,
certain ligands can influence tertiary structure of protein. Interestingly, the region affected by
conformational changes is one that was shown in the literature as the main epitope of Pru p 3.
Conclusions: In this study, we observed differences in binding capacity of Pru p 3. MD simulation showed
that interaction between Pru p 3 and tested ligands can lead to conformational changes that influence
allergenic activity of nsLTPs. The allergen–lipid interaction may act as a potential danger signal during the
allergic sensitization phase or increase allergenicity during the effector phase.
POSTER SESSION 4:
Food allergens