Ahrazem Oussama, Ibáñez M Dolores, López-Torrejón Gema, Sánchez-Monge Rosa, Sastre Joaquin, Lombardero Manuel, Barber Domingo, Salcedo Gabriel
Unidad de Bioquímica, Departamento de Biotecnología, E.T.S. Ingenieros Agrónomos, UPM, Madrid, España.
Int Arch Allergy Immunol. 2006;139(2):96-103. doi: 10.1159/000090384. Epub 2005 Dec 15.
Orange allergens are virtually unknown, in spite of the large consumption of this fruit. Germin-like proteins, together with vicilins and legumins, form the cupin superfamily of plant proteins, which includes many seed allergens.
Twenty-nine patients with allergy to oranges were studied. A major IgE-binding protein from orange extracts was isolated by means of a two-step cation-exchange chromatographic protocol. The allergen was characterized by N-terminal amino acid sequencing and MALDI analysis, and its reactivity explored by specific IgE determination in individual sera, ELISA inhibition assays and in vivo skin prick tests (SPT). Chemical deglycosylation of the purified allergen was achieved by trifluoromethylsulfonate acid treatment.
The 24-kDa purified allergen, designated Cit s 1, was identified as a germin-like glycoprotein, based on its N-terminal amino acid sequence, molecular size and recognition by rabbit anti-complex N-linked glycan antibodies. Specific IgE to Cit s 1 was detected in 62% of 29 individual sera from orange-allergic patients, whereas positive SPT responses to the purified allergen were obtained in only 10% of such patients. Deglycosylation of Cit s 1 resulted in a loss of its IgE-binding capacity. Moreover, the unrelated plant glycoprotein horseradish peroxidase inhibited over 70% the IgE-binding to Cit s 1.
Over 60% of patients with allergy to oranges show specific IgE to Cit s 1. However, the purified allergen exerts a low in vivo reactivity. Complex N-linked glycans seem to play a prominent role in the IgE-binding capacity of Cit s 1. This characteristic of Cit s 1, as well as of other orange glycoproteins, could lead to false positives if the diagnosis of allergy to oranges is mainly based on in vitro specific IgE determination.
尽管橙子的消费量很大，但几乎没有人了解橙子过敏原。类萌发素蛋白与豌豆球蛋白和豆球蛋白一起构成了植物蛋白的杯蛋白超家族，其中包括许多种子过敏原。
对29例橙子过敏患者进行了研究。通过两步阳离子交换色谱法从橙子提取物中分离出一种主要的IgE结合蛋白。通过N端氨基酸测序和基质辅助激光解吸电离分析对该过敏原进行了表征，并通过个体血清中的特异性IgE测定、酶联免疫吸附测定抑制试验和体内皮肤点刺试验（SPT）来探究其反应性。通过三氟甲磺酸处理实现了纯化过敏原的化学去糖基化。
基于其N端氨基酸序列、分子大小以及兔抗复合N-连接聚糖抗体的识别，将24 kDa的纯化过敏原命名为Cit s 1，并鉴定为类萌发素糖蛋白。在29例橙子过敏患者的个体血清中，62%检测到针对Cit s 1的特异性IgE，而只有10%的此类患者对纯化过敏原的皮肤点刺试验呈阳性反应。Cit s 1的去糖基化导致其IgE结合能力丧失。此外，不相关的植物糖蛋白辣根过氧化物酶抑制了70%以上的IgE与Cit s 1的结合。
超过60%的橙子过敏患者显示出针对Cit s 1的特异性IgE。然而，纯化的过敏原在体内的反应性较低。复合N-连接聚糖似乎在Cit s 1的IgE结合能力中起重要作用。如果主要基于体外特异性IgE测定来诊断橙子过敏，Cit s 1以及其他橙子糖蛋白的这一特性可能会导致假阳性结果。
