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 DPYSPOHS 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.

IgE cross-inhibition between Ara h 1 and Ara h 2 is explained by complex formation of both major peanut allergens

“…There is quite some evidence that the proline-rich epitope on Ara h 2 between positions 40 and 70 is a dominant IgE epitope in which prolines are hydroxylated. 27 – 29 That stretch includes an alpha helix and a well-exposed extended loop. 30 Also in our experiments, a synthetic peptide spanning amino acids 42 to 68, with prolines in hydroxylated form, exhibited strong inhibitory potency, being able to reduce IgE binding to nAra h 2 by around 40%.…”

“…The most likely explanation here is that Escherichia coli –produced rAra h 2 does not have hydroxy prolines, which have been shown to be important for IgE binding. 9 , 27 , 29 For Ara h 2, therefore, a natural purified or a plant-expressed recombinant may be preferred. Indeed, it was recently reported that a recombinant Ara h 2 molecule produced in Nicotiana benthamiana contained hydroxylated prolines and bound IgE more efficiently than recombinant Ara h 2 produced in E coli .…”

Legumain Functions as a Transient TrkB Sheddase

“…Excess of BDNF 2 dimers will dilute and dissolve existing TrkB 2 -BDNF 2 tetramers into TrkB 1 -BDNF 2 trimers. Such a biphasic concentration-dependent receptor dimerization is well characterized in immunology, for instance, in rat basophile release assays [ 32 ].…”

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality

“…While there has been considerable effort in glycoengineering popular expression hosts such as Nicotiana benthamiana , N. tabacum , tobacco BY2 cells and the moss Physcomitrella patens towards humanized- and customized N-glycosylation ( Bakker et al, 2001 ; Koprivova et al, 2004 ; Strasser et al, 2008 ; Kallolimath et al, 2016 ; Limkul et al, 2016 ; Mercx et al, 2017 ; Jansing et al, 2019 ; Bohlender et al, 2020 ; Herman et al, 2021 ; Göritzer et al, 2022 ; Kogelmann et al, 2023 ) limited attention was cast towards modulating the plant endogenous O-glycosylation pathway ( Castilho et al, 2012 ; Yang et al, 2012 ; Parsons et al, 2013 ; Dicker et al, 2016 ; Ramírez-Alanis et al, 2018 ; Mócsai et al, 2021 ; Uetz et al, 2022 ). Plant-endogenous HyP and further modifications with pentoses (arabinoses) were found in several recombinantly produced proteins such as IgA1, MUC1, EPO-Fc, and Ara h 2 ( Karnoup et al, 2005 ; Weise et al, 2007 ; Pinkhasov et al, 2011 ; Castilho et al, 2012 ; Yang et al, 2012 ; Üzülmez et al, 2021 ). The impact of these plant-specific HyP residues and attached pentoses on the function and potency of therapeutic proteins is currently unknown.…”