López-Torrejón Gema, Salcedo Gabriel, Martín-Esteban Manuel, Díaz-Perales Araceli, Pascual Cristina Y, Sánchez-Monge Rosa
Departamento de Biotecnología, Ingenieros Agrónomos, UPM, Madrid, Spain.
J Allergy Clin Immunol. 2003 Dec;112(6):1208-15. doi: 10.1016/j.jaci.2003.08.035.
Lentils are among the main plant foods causing allergic reactions in pediatric patients in the Mediterranean area and in many Asian communities. However, very few reports have been devoted to identifying lentil allergens. Seed storage proteins of the vicilin family have been characterized as major allergens in several seed legumes and tree nuts.
We sought to evaluate the role of lentil vicilins as food allergens.
A serum pool and individual sera from 22 patients with lentil allergy were used in different IgE-binding assays. Mature lentil vicilin was isolated by means of cation-exchange chromatography, followed by reverse-phase HPLC, and characterized by means of N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) analysis, complex asparagine-linked glycan detection, specific IgE immunodetection with individual sera, and ELISA inhibition assays. Complete cDNAs encoding lentil vicilin variants were isolated by means of PCR with primers based on the amino acid sequence of the allergen.
A major IgE-binding component of approximately 50 kd was detected in lentil extracts. This component was isolated and characterized, showing a single N-terminal amino acid sequence homologous to those of legume vicilins and a broad peak (maximum at 48613 d) in MALDI analysis. The purified allergen was recognized by 77% (17/22) of the individual sera from patients with lentil allergy and reached up to 65% inhibition of the IgE binding to the crude lentil extract. The allergen showed 3 isoforms varying in their degree of N-glycosylation. Two cDNA clones encoding different allergen variants were isolated. The amino acid sequences deduced from both clones (415 and 418 residues; 47.4 and 47.8 kd) showed greater than 50% identity with major peanut (Ara h 1) and soybean (conglutinin subunits) allergens belonging to the vicilin family. Furthermore, these sequences included those of the previously characterized lentil allergen Len c 1.02 (108 amino acid residues of the C-terminal domain) and those of a novel lentil IgE-binding protein of 26 kd.
The mature 48-kd lentil vicilin, designated Len c 1.01, is a major allergen. Two of its processing fragments, corresponding to subunits of 12 to 16 kd (previously named Len c 1) and 26 kd, are also relevant lentil IgE-binding proteins. The sequence homology of Len c 1.01 to those of major allergens from peanut, soybean, walnut, and cashew can help to investigate potential cross-reactions among these plant foods.
小扁豆是地中海地区和许多亚洲社区儿科患者中引起过敏反应的主要植物性食物之一。然而，专门用于鉴定小扁豆过敏原的报告非常少。豌豆球蛋白家族的种子储存蛋白已被鉴定为几种豆类种子和坚果中的主要过敏原。
我们试图评估小扁豆豌豆球蛋白作为食物过敏原的作用。
在不同的IgE结合试验中使用了来自22例小扁豆过敏患者的混合血清和个体血清。通过阳离子交换色谱法，随后进行反相高效液相色谱法分离成熟的小扁豆豌豆球蛋白，并通过N端氨基酸测序、基质辅助激光解吸/电离质谱（MALDI）分析、复合天冬酰胺连接聚糖检测、用个体血清进行特异性IgE免疫检测以及ELISA抑制试验对其进行表征。通过基于过敏原氨基酸序列的引物进行PCR，分离编码小扁豆豌豆球蛋白变体的完整cDNA。
在小扁豆提取物中检测到一种约50kd的主要IgE结合成分。分离并表征了该成分，其显示出与豆类豌豆球蛋白同源的单一N端氨基酸序列，并且在MALDI分析中呈现一个宽峰（最大为48613d）。纯化的过敏原被77%（17/22）的小扁豆过敏患者个体血清识别，并对IgE与粗制小扁豆提取物的结合产生高达65%的抑制作用。该过敏原显示出3种N-糖基化程度不同的异构体。分离出两个编码不同过敏原变体的cDNA克隆。从这两个克隆推导的氨基酸序列（分别为415和418个残基；47.4和47.8kd）与属于豌豆球蛋白家族的主要花生（Ara h 1）和大豆（凝集素亚基）过敏原具有大于50%的同一性。此外，这些序列包括先前表征的小扁豆过敏原Len c 1.02（C端结构域的108个氨基酸残基）的序列以及一种新的26kd小扁豆IgE结合蛋白的序列。
成熟的48kd小扁豆豌豆球蛋白，命名为Len c 1.01，是一种主要过敏原。其两个加工片段，对应于12至16kd（先前命名为Len c 1）和26kd的亚基，也是相关的小扁豆IgE结合蛋白。Len c 1.01与花生、大豆、核桃和腰果主要过敏原序列的同源性有助于研究这些植物性食物之间潜在的交叉反应。
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