High-throughput NMR authentication of food allergens belonging to the Lipid Transfer Protein family

For in vitro allergy diagnosis, purified food allergens have to meet high standard quality criteria. 1D 1H-NMR analysis can play a significant role to assess the conformation and to contribute to the authentication of allergens. The results of the application of a high throughput, low cost, NMR method to allergens of the nsLTP family (Cor a 8, Mal d 3 and Pru p 3) are presented and discussed. Purification methods were developed to obtain highly pure and biologically active allergens. Cor a 8 was expressed in yeast as non-fusion protein, Mal d 3 and Pru p 3 were purified from fruit peel. Primary and secondary structure were verified by N-terminal sequencing, MALDI-TOF-MS analysis and far-UV CD-spectroscopy. IgE binding capacity was tested in ELISA and immunoblotting assays using allergic patients’ sera. The presence and extent of the tertiary structure was assessed by two 1D-1H-NMR experiments, using a 700MHz field: an H2O presaturation experiment and an experiment including gradient-based H2O suppression by excitation sculpting . The experiments were done at 298K and variable buffer conditions. The consistency of the allergen’s spectra with structural information available either in the literature or in the PDB was checked. The structure of Pru p 3 was determined by X-ray crystallography. It consists of 4 helices, connected by short loops, packed against a C-terminal region and stabilized by 4 S-S bridges and by main-chain and side-chain intramolecular H-bonds. The structures of Mal d 3 and Cor a 8 are not experimentally known, but their sequences are and in silico structures could be modeled, including residues 25-115 , using the structure of Pru p 3 as template, with a sequence identity of 81.3% and 59% respectively. The 1D-1H-NMR spectra show the features of three rigid and extended tertiary structures. There are narrow and resolved peaks in the whole spectral window, in the methyl (-0.5-1.5 ppm), in the amide-aromatic (6–10 ppm) regions and dispersed signals in the 3.5–6ppm region, due to alpha- H. The spectra are very similar, confirming the structural similarity of the models of Mal d 3 and Cor a 8, and the experimental structure of Pru p 3, as expected from the high degree of sequence homology. The experiments described above are simple, no time-consuming, require low protein concentrations, do not require 15N and 13C labeling and make possible the structural authentication of nsLTPs.