Research & Pharmaceutical Development, Stallergenes, Antony, France.
Research & Pharmaceutical Development, Stallergenes, Antony, France.
J Allergy Clin Immunol. 2015 Oct;136(4):1055-64. doi: 10.1016/j.jaci.2015.03.001. Epub 2015 Apr 10.
Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe.
We sought to investigate the presence of undescribed allergens in ragweed pollen.
Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins.
High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding.
We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11.
短豚草（Ambrosia artemisiifolia）花粉过敏是美国和欧洲一个严重且不断扩大的健康问题。
我们试图研究豚草花粉中是否存在未被描述的过敏原。
将豚草花粉蛋白进行高分辨率凝胶电泳，并使用 92 位来自美国或欧洲的豚草过敏供体的血清进行免疫球蛋白 E（IgE）反应性检测。应用花粉转录组测序、质谱（MS）和重组 DNA 技术来鉴定新的 IgE 结合蛋白。
高分辨率 IgE 免疫印迹实验显示，92 位豚草过敏患者中有 50 位（54%）对一种 37 kDa 的过敏原发生了致敏反应，该过敏原与 Amb a 1 不同。通过 MS 测序联合豚草花粉 RNA 测序对该蛋白进行分析后，利用 PCR 克隆获得该分子的全长 cDNA 序列。该纯化过敏原被命名为 Amb a 11，通过 MS 进行了充分鉴定，并证实与 66%患者的 IgE 发生反应。该分子是木瓜蛋白酶家族的一种 262 个氨基酸的硫基蛋白酶，在从前体蛋白上切割 N 端和 C 端前肽后，以同工型和糖型的形式表达。三维建模显示与已知的半胱氨酸蛋白酶具有高度结构同源性，包括螨类过敏原 Der p 1。证实了 Amb a 11 的蛋白酶活性及其激活豚草过敏患者嗜碱性粒细胞的能力。在大肠杆菌中生产非糖基化的重组 Amb a 11 形式，证明糖基化不是 IgE 结合所必需的。
我们鉴定了豚草花粉中的半胱氨酸蛋白酶 Amb a 11 为一种新的主要过敏原。鉴于 2 种主要过敏原具有相似的理化性质，我们假设先前归因于 Amb a 1 的部分过敏原活性实际上由 Amb a 11 承担。
