Gruber Patrick, Becker Wolf-Meinhard, Hofmann Thomas
Deutsche Forschungsanstalt für Lebensmittelchemie, Lichtenbergstr. 4, D-85748 Garching, Germany.
J Agric Food Chem. 2005 Mar 23;53(6):2289-96. doi: 10.1021/jf048398w.
The influence of thermal processing and nonenzymatic browning reactions on the IgE-binding activity of rAra h 2 was studied and compared to findings recently reported for the allergen's natural counterpart. ELISA experiments as well as inhibition assays revealed that thermal treatment of rAra h 2 in the presence of reactive carbohydrates and carbohydrate breakdown products induces a strong increase of the IgE-binding activity, thus collaborating with the data reported for the natural protein isolated from peanuts. To localize the Ara h 2 sequences responsible for the formation of highly IgE-affine glycation sites, model peptides have been synthesized mimicking sequences which contain possible targets for glycation as well as the immunodominant epitopes. Immunological evaluation of these peptides heated in the absence or presence of reducing sugars and carbonyls, respectively, revealed that neither the two lysine residues of Ara h 2 nor its N-terminus are involved in the formation of IgE-affine structures by Maillard reaction. Also, the cysteine-containing major epitope 3 (aa 27-36) was found to lose its IgE-binding capacity upon heating. By contrast, the overlapping major epitopes 6 and 7, which do not contain any lysine or arginine moieties, showed a distinct higher level of IgE binding when subjected to Maillard reaction, thus giving the first evidence that nonbasic amino acids might be accessible for nonenzymatic glycation reactions and that these posttranslational modifications might induce increased IgE binding of the glycated Ara h 2. Analogous experiments were performed with peanut agglutinin, considered in the literature as a minor allergen. ELISA experiments revealed that the majority of tested sera samples from peanut-sensitive patients showed a high level of IgE binding to the lectin even after heat treatment. In contradiction to published data, nonenzymatic browning reactions seem to deteriorate the IgE affinity of the lectin.
研究了热处理和非酶褐变反应对重组Ara h 2 IgE结合活性的影响，并与最近报道的该过敏原天然对应物的研究结果进行了比较。酶联免疫吸附测定（ELISA）实验以及抑制试验表明，在活性碳水化合物和碳水化合物分解产物存在的情况下对重组Ara h 2进行热处理会导致IgE结合活性大幅增加，这与从花生中分离出的天然蛋白质的相关报道数据相符。为了定位负责形成高IgE亲和力糖基化位点的Ara h 2序列，合成了模拟肽，这些肽模拟了包含糖基化可能靶点以及免疫显性表位的序列。分别在不存在或存在还原糖和羰基的情况下对这些肽进行加热后的免疫学评估表明，Ara h 2的两个赖氨酸残基及其N端均不参与美拉德反应形成IgE亲和力结构。此外，发现含半胱氨酸的主要表位3（氨基酸27 - 36）在加热后失去其IgE结合能力。相比之下，不包含任何赖氨酸或精氨酸部分的重叠主要表位6和7在进行美拉德反应时显示出明显更高水平的IgE结合，从而首次证明非碱性氨基酸可能可用于非酶糖基化反应，并且这些翻译后修饰可能会诱导糖基化的Ara h 2的IgE结合增加。对文献中认为是次要过敏原的花生凝集素进行了类似实验。ELISA实验表明，即使经过热处理，大多数来自花生过敏患者的测试血清样本对该凝集素仍显示出高水平的IgE结合。与已发表的数据相反，非酶褐变反应似乎会降低凝集素的IgE亲和力。
