Data_Sheet_1_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.XLSX

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.

花生过敏症是一种可能危及生命的疾病，其发生机制涉及特异性过敏原免疫球蛋白E（IgE）抗体。主要花生过敏原Ara h 2，一种2S清蛋白种子储存蛋白，是危险性最大且效力最强的植物过敏原之一。Ara h 2在翻译后经过修饰，形成四个二硫键和三个羟脯氨酸。这些羟脯氨酸残基对于与DPYSPOHS基序（代表免疫优势IgE表位）的最佳IgE结合至关重要。迄今为止，重组Ara h 2已在大肠杆菌、乳酸杆菌、鳞翅目昆虫细胞和绿藻衣藻叶绿体表达系统中生产，但这些系统均无法进行脯氨酸羟化。然而，花生过敏的分子诊断是通过使用天然或大肠杆菌生产的花生主要过敏原来进行的。由于大多数患者的IgE针对Ara h 2，因此重组Ara h 2保留其所有的真核翻译后修饰至关重要。我们利用基于植物病毒的magnICON®瞬时表达系统，在烟草花叶植物叶片细胞的内质网中生产了含羟脯氨酸且正确折叠的Ara h 2，产量达到200 mg/kg新鲜生物质。为了比较原核与真核表达方法，Ara h 2在大肠杆菌中与二硫键异构酶DsbC共同表达，从而携带二硫键但没有羟脯氨酸。来自N. benthamiana和E. coli的重组过敏原被表征并与其从烤花生中分离的天然Ara h 2进行了比较。天然Ara h 2在IgE结合和通过IgE交联激活肥大细胞方面优于两种重组蛋白，后者表明了过敏原的效力。有趣的是，与大肠杆菌产品相比，N. benthamiana生产的过敏原引起的IgE交联效率显著更高。由于存在羟脯氨酸残基，N. benthamiana植物中的Ara h 2在肥大细胞激活方面与天然过敏原表现出更高的相似性，这支持了关于其贡献于免疫优势IgE表位的已发表数据。我们的研究倡导使用N. benthamiana植物而非原核表达宿主来生产主要花生过敏原Ara h 2。