Structural and Functional Characterization of the Hazelnut Allergen Cor a 8

Abstract

Nonspecific lipid transfer proteins (nsLTPs) are basic proteins, stabilized by four disulfide bonds, and are expressed throughout the plant kingdom. These proteins are also known as important allergens in fruits and tree nuts. In this study, the nsLTP from hazelnuts, Cor a 8, was purified and its crystal structure determined. The protein is stable at low pH and refolds after thermal denaturation. Molecular dynamics simulations were used to provide an insight into conformational changes of Cor a 8 upon ligand binding. When known epitope areas from Pru p 3 were compared to those of Cor a 8, differences were obvious, which may contribute to limited cross-reactivity between peach and hazelnut allergens. Differences in epitope regions may contribute to limited cross-reactivity between Cor a 8 and nsLTPs from other plant sources. The structure of Cor a 8 represents the first resolved structure of a hazelnut allergen.

Keywords: allergen; food allergy; hazelnut; nonspecific lipid transfer protein; protein crystallization.

Figure 1

Purification of Cor a 8: (A) cation exchange chromatography of the hazelnut prolamin extract and Coomassie-stained 15% SDS-PAGE of Cor a 8-containing fractions; (B) 15% SDS-PAGE of purified Cor a 8 under nonreducing (NR) and reducing (R) conditions; (C) mass spectrum of Cor a 8.

Figure 2

Far-UV CD spectra of Cor a 8: (A) spectra of Cor a 8 at room temperature (dotted line), after heating to 95 °C (dark gray line), and after recooling to room temperature (light gray line) at pH 2.5 and 7.5, respectively; (B) change in molar ellipticity at 222 nm during heating and cooling.

Figure 3

(A) Overall structure of Cor a 8. Secondary structural elements are colored separately where α-helices are cyan, β-sheets are red, loops are magenta, and disulfide bonds are blue. (B) Surface representation of Cor a 8 with electrostatic potentials where blue indicates positive charge and red indicates negative charge in units of kcal/(mol·e). (C) Ribbon representation of Cor a 8 showing conserved residues derived from an alignment of 150 Cor a 8-related protein sequences selected by ConSurf. The most conserved residues are shown in blue, whereas the most variable residues are shown in red. (D) Sequence of Cor a 8, in which the most conserved residues are blue and the most variable residues are red. The cysteine residues participating in disulfide linkages are bracketed.

Figure 4

(A) Comparison of known epitope surface regions between Cor a 8, Pru p 3, and Zea m 14 (maize nsLTP); surface representation with electrostatic potentials where blue indicates positive charge and red indicates negative charge in units of kcal/(mol·e). (B) Primary sequence alignment of the mature protein sequences of Cor a 8, Pru p 3, and Zea m 14. Arrows indicate α-helices, and previously identified IgE-binding epitopes of Pru p 3 are marked with boxes.