Impact of lipid binding on the tertiary structure and allergenic potential of Jug r 3, the non-specific lipid transfer protein from walnut

Pawel Dubiela,Rebecca Del Conte,Tomasz Borowski,Stefano Alessandri,Christian Radauer,Merima Bublin,Roberta Aina,Karin Hoffmann-Sommergruber,Francesca Cantini

doi:10.1038/s41598-019-38563-1

Abstract

Plant non-specific lipid transfer proteins type 1 (nsLTP1) are small basic proteins with a hydrophobic cavity able to host a number of different ligands: i.e. fatty acids, fatty acyl-CoA, phospholipids, glycolipids, and hydroxylated fatty acids. However, ligand binding specificity differs among nsLTPs. Within this protein family, Jug r 3 from walnut has been identified as a major allergen. So far, data on the structural characterization of Jug r 3 and its lipid binding capacity are lacking. We report the results from a fluorescence-based ligand-binding assay and ligand-based NMR experiments, to study the binding interactions between Jug r 3 and the 18-carbon monounsaturated oleic acid. Furthermore, protein-based NMR experiments were employed to detect the oleate binding site of Jug r 3. The NMR data were used to dock the oleate molecule into the structural model of Jug r 3. Finally, the impact of the interaction on the allergenic potential of Jug r 3 was investigated by IgE ELISA with 6 sera from walnut allergic patients. Our data corroborate the hypothesis of direct impact of food-derived matrix on the IgE reactivity of nsLTPs.

Highlights

Allergens were found in only 2% of all sequence-based and 5% of all structural protein families suggesting common biochemical features such as lipid binding[3]
Tertiary structures of some Non-specific lipid transfer proteins (nsLTPs) have already been resolved, there is still a lack of experimental knowledge regarding the impact of ligands on the protein 3D structure
Since it has been shown that the ligand binding specificity can vary among protein family members[10,20] and that this is an important factor regarding nsLTP allergenicity[30], we investigated the ligand binding ability of the major allergen from walnut, Jug r 3

Summary

Introduction

Allergens were found in only 2% of all sequence-based and 5% of all structural protein families suggesting common biochemical features such as lipid binding[3]. Non-specific lipid transfer proteins (nsLTPs) have been discovered in 1975 and received their name from the ability to transfer lipids in plants[4] They are classified as pathogenesis-related proteins (PR-14) and in parallel belong to the prolamin superfamily[5], nsLTPs are small and soluble, cysteine-rich proteins[6]. The common feature of the structure is the presence of a cavity that can bind small hydrophobic molecules This function is relevant for several physiological roles within the plant, such as the stabilization of membranes, cell wall organization, and signal transduction[7]. The tunnel can host different kind of ligands, i.e. fatty acids, fatty acyl-CoA, phospholipids, glycolipids, hydroxylated fatty acids and prostaglandin B26,23–27 This binding capacity varies among different members of the nsLTP family, and depends on the specific characteristics of their tertiary structure. From a total of 46 patients with mild or severe clinical symptoms, 36 (78%) individuals revealed IgE reactivity to Jug r 328

Methods

Results

Conclusion