The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis

Abstract

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSP^OHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON^® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.

Keywords: Ara h 2; Nicotiana benthamiana; PTM; hydroxyproline; peanut allergy; transient expression.

Figure 1

Amino acid sequences of the Ara h 2 proteins used in this study. (A) Mature nAra h 2.0201. Naturally occurring hydroxyproline residues within the immunodominant IgE epitopes (underlined) are highlighted in gray. The canonical N-glycosylation site (NQS) is boxed. (B) rAra h 2.0201 used for expression in N. benthamiana, including a C-terminal hexa-histidine tag and an ER retention signal (SEKDEL, boxed). The canonical N-glycosylation site NQS was mutated to QQS (boxed), and the single amino acid substitution is marked with an asterisk. Mass spectrometry-detected and manually confirmed hydroxyproline residues are highlighted in gray. (C) rAra h 2.0201expressed in E. coli, including an N-terminal hexa-histidine tag (boxed). The canonical N-glycosylation site is boxed.

Figure 2

Biochemical characterization of nAra h 2, rAra h 2 (N. benthamiana), and rAra h 2 (E. coli). (A) Circular dichroism spectra showing double minima at 208 and 222 nm indicated exclusively alpha helical content for all three proteins. (B) Dynamic light scattering measured monomeric states for all three proteins, except for rAra h 2 from E. coli, which contained 1% aggregates.

Figure 3

Characterization of nAra h 2, rAra h 2 (N. benthamiana), and rAra h 2 (E. coli). (A) The purity of nAra h 2, rAra h 2 (N. benthamiana), and rAra h 2 (E. coli) was visualized by CBB-stain after running 2 μg protein per lane on SDS-PAGE under reducing (red) and non-reducing (non-red) conditions. (B) All three proteins were detected using a monoclonal anti-Ara h 2 antibody, loaded as in (A). (C) A pool of sera of peanut-allergic patients (n = 5) was used to detect all Ara h 2 proteins, loaded as in (A).

Figure 4

IgE-binding of Ara h 2 proteins compared by ELISA. (A) IgE-binding to rAra h 2 from N. benthamiana was measured and compared with the nAra h 2 and the rAra h 2 from E. coli, using sera from 19 peanut-allergic patients. Data are the means of technical duplicates (n = 2). ^****P ≤ 0.0001, ^**P ≤ 0.01, ns: P > 0.05. (B) IgE-binding to all three Ara h 2 proteins was determined for each patient. Error bars represent the standard deviation from technical duplicates (n = 2).

Figure 5

RBL assays were performed to investigate the IgE cross-linking capacity of rAra h 2 from N. benthamiana in comparison with the nAra h 2 and the rAra h 2 from E. coli. (A) Three patients (P9, P18, and P20) were selected to perform the allergen titrations. Data are the means of technical triplicates (n = 3). Data are represented as stimulation indices (SI) calculated by dividing the luminescence values from antigen-treated cells with the ones from non-stimulated cells. A value above 2 was considered positive (indicated by dotted lines). (B) Data obtained from 17 peanut-allergic patients were used to compare the IgE-cross-linking performances of all Ara h 2 proteins at 0.1 ng/mL allergen concentration. Median values and interquartile range are indicated. ^****P ≤ 0.0001, ^*P ≤ 0.05. (C) As in (B) at 1 ng/mL concentration. ns: P > 0.05.