Sánchez-Monge R, Pascual C Y, Díaz-Perales A, Fernández-Crespo J, Martín-Esteban M, Salcedo G
Unidad de Bioquímica, Departamento de Biotecnología, E.T.S. Ingenieros Agrónomos, Madrid, Spain.
J Allergy Clin Immunol. 2000 Nov;106(5):955-61. doi: 10.1067/mai.2000.109912.
Lentils seem to be the most common legume implicated in pediatric allergic patients in the Mediterranean area. However, no lentil allergen has been isolated and characterized.
We sought to purify and characterize relevant IgE-binding proteins from boiled lentil extracts.
IgE-binding proteins from crude and boiled lentil extracts were detected with a pool of sera from patients with lentil allergy. Allergens were isolated by gel-filtration chromatography followed by cation- and anion-exchange chromatography or by reverse-phase HPLC. Their characterization included N-terminal amino acid sequencing, complex asparagine-linked glycan detection, specific IgE immunodetection with 22 individual sera from allergic patients, and immunoblot and CAP inhibition assays.
Heat treatment of lentils produced substantial changes in the SDS-PAGE patterns of whole extracts, mainly a strong increase of 12- to 16-kd bands and a decrease of 25- to 45-kd components. Major IgE-binding proteins from the boiled lentil extract were located in the 12- to 16-kd and 45- to 70-kd ranges. Two allergens of 16 kd, proteins L1 and L2, and another one of 12 kd, protein L3, were purified. N-terminal sequencing indicated that all 3 were related and allowed their identification as gamma-vicilin subunits. Protein L1 was recognized by 68% of the individual sera tested and inhibited 64% of the IgE binding by commercial lentil CAPs. A second type of allergen of 66 kd, named protein H, was also isolated and identified as a seed-specific biotinylated protein. Protein H reacted with 41% of the individual sera and produced 45% inhibition in CAP inhibition assays.
Two different types of allergens have been identified in boiled lentils. Those of 12 to 16 kd, called Len c 1, correspond to gamma-vicilin subunits, and those of 66 kd, designated Len c 2, correspond to seed-specific biotinylated protein. Homology with proteins from other legume species can explain potential cross-reactions among these foods.
小扁豆似乎是地中海地区儿科过敏患者中最常见的豆类。然而，尚未分离和鉴定出小扁豆过敏原。
我们试图从小扁豆煮提取物中纯化和鉴定相关的IgE结合蛋白。
用小扁豆过敏患者的血清池检测粗制和煮制小扁豆提取物中的IgE结合蛋白。通过凝胶过滤色谱，然后进行阳离子和阴离子交换色谱或反相高效液相色谱分离过敏原。其鉴定包括N端氨基酸测序、复杂的天冬酰胺连接聚糖检测、用22份过敏患者的个体血清进行特异性IgE免疫检测，以及免疫印迹和CAP抑制试验。
小扁豆的热处理使全提取物的SDS-PAGE图谱发生了显著变化，主要是12至16kd条带强烈增加，25至45kd成分减少。煮制小扁豆提取物中的主要IgE结合蛋白位于12至16kd和45至70kd范围内。纯化了两种16kd的过敏原，即蛋白L1和L2，以及另一种12kd的蛋白L3。N端测序表明这三种蛋白相关，并确定它们为γ-豌豆球蛋白亚基。68%的受试个体血清识别蛋白L1，它抑制了商业小扁豆CAPs 64%的IgE结合。还分离出了第二种66kd的过敏原，命名为蛋白H，并鉴定为种子特异性生物素化蛋白。蛋白H与41%的个体血清反应，在CAP抑制试验中产生45%的抑制作用。
在煮制小扁豆中鉴定出两种不同类型的过敏原。12至16kd的过敏原称为Len c 1，对应于γ-豌豆球蛋白亚基；66kd的过敏原称为Len c 2，对应于种子特异性生物素化蛋白。与其他豆类物种蛋白的同源性可以解释这些食物之间潜在的交叉反应。
