scholarly journals 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

2021 ◽  
Vol 12 ◽  
Author(s):  
Öykü Üzülmez ◽  
Tanja Kalic ◽  
Vanessa Mayr ◽  
Nina Lengger ◽  
Angelika Tscheppe ◽  
...  

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.

2004 ◽  
Vol 135 (2) ◽  
pp. 101-107 ◽  
Author(s):  
Belinda J. Hales ◽  
Anthony Bosco ◽  
Kristina L. Mills ◽  
Lee A. Hazell ◽  
Richard Loh ◽  
...  

2014 ◽  
Vol 133 (2) ◽  
pp. AB398
Author(s):  
Stephane Hazebrouck ◽  
Blanche Guillon ◽  
Marie-Françoise Drumare ◽  
Evelyne Paty ◽  
Jean-Michel Wal ◽  
...  
Keyword(s):  
Ara H 2 ◽  

2002 ◽  
Vol 109 (1) ◽  
pp. S300-S300 ◽  
Author(s):  
Moon Sen ◽  
Randall Kopper ◽  
Laurent Pons ◽  
EC Abraham ◽  
Wesley Burks ◽  
...  

2005 ◽  
Vol 100 (2) ◽  
pp. 153-158 ◽  
Author(s):  
Annick Barre ◽  
Jean-Philippe Borges ◽  
Raphaël Culerrier ◽  
Pierre Rougé

2020 ◽  
Vol 181 (5) ◽  
pp. 334-341 ◽  
Author(s):  
Federico Storni ◽  
Gustavo Cabral-Miranda ◽  
Elisa Roesti ◽  
Lisha Zha ◽  
Paul Engeroff ◽  
...  

2018 ◽  
Vol 141 (2) ◽  
pp. AB179 ◽  
Author(s):  
Stephane Hazebrouck ◽  
Blanche Guillon ◽  
Evelyne Paty ◽  
Karine Adel-Patient ◽  
Hervé Bernard

Allergy ◽  
2013 ◽  
Vol 68 (6) ◽  
pp. 803-808 ◽  
Author(s):  
R. A. Wood ◽  
S. H. Sicherer ◽  
A. W. Burks ◽  
A. Grishin ◽  
A. K. Henning ◽  
...  

2019 ◽  
Author(s):  
Seth Herzon ◽  
Alan R. Healy ◽  
kevin wernke ◽  
Chung Sub Kim ◽  
Nicholas Lees ◽  
...  

<div>The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal E. coli, and clb metabolites are thought to initiate colorectal cancer via DNA cross-linking. Precolibactin 886 (1) is one of the most complex isolated clb metabolites; it contains a 15-atom macrocycle and an unusual 5-hydroxy-3-oxazoline ring. Here we report confirmation of the structural assignment via a biomimetic synthesis of precolibactin 886 (1) proceeding through the amino alcohol 9. Double oxidation of 9 afforded the unstable α-ketoimine 2 which underwent macrocyclization to precolibactin 886 (1) upon HPLC purification (3% from 9). Studies of the putative precolibactin 886 (1) biosynthetic precursor 2, the model α-ketoimine 25, and the α-dicarbonyl 26 revealed that these compounds are susceptible to nucleophilic rupture of the C36–C37 bond. Moreover, cleavage of 2 produces other known clb metabolites or biosynthetic intermediates. This unexpected reactivity explains the difficulties in isolating full clb metabolites and accounts for the structure of a recently identified colibactin–adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 (1) to form a non-genotoxic pyridone, suggesting precolibactin 886 (1) lies off-path of the major biosynthetic route.</div>


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