U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, New Orleans, LA, USA.
Mol Nutr Food Res. 2014 Apr;58(4):884-93. doi: 10.1002/mnfr.201300299. Epub 2013 Dec 5.
The stability of food allergens to digestion varies. We characterized the stability of cashew allergens to digestion by pepsin and trypsin and identified IgE-binding epitopes that survive digestion.
The ability of pepsin and trypsin to digest cashew allergens was assessed with an in vitro digestion model. Samples were evaluated by SDS-PAGE, MS, ELISA, and immunoblotting to compare IgE binding. Increasing amount of protease resulted in greater degradation of higher molecular weight cashew proteins. Among cashew proteins, the 2S albumin, Ana o 3, was most resistant to digestion by both pepsin and trypsin. MS identified digestion resistant Ana o 3 protein fragments that retained reported IgE-binding epitopes. Pretreatment of extracts or purified Ana o 3 with reducing agent increased the sensitivity of Ana o 3 to protease digestion. Circular dichroism revealed the structure of purified Ana o 3 was largely alphahelical and was disrupted following reduction. Ana o 3 reduction followed by protease digestion decreased binding of serum IgE from cashew allergic patients. Our results indicate that the Ana o 3 disulfide bond dependent structure protects the protein from proteolysis.
Ana o 3 is the cashew allergen most likely to survive gastrointestinal digestion intact.
食物过敏原对消化的稳定性各不相同。我们通过胃蛋白酶和胰蛋白酶来确定腰果过敏原的稳定性，并鉴定出能在消化过程中存活的 IgE 结合表位。
采用体外消化模型评估胃蛋白酶和胰蛋白酶消化腰果过敏原的能力。通过 SDS-PAGE、MS、ELISA 和免疫印迹来比较 IgE 结合情况，对样品进行评估。蛋白酶用量的增加导致更高分子量的腰果蛋白降解程度更大。在腰果蛋白中，2S 白蛋白 Ana o 3 对胃蛋白酶和胰蛋白酶的消化均具有最强的抗性。MS 鉴定出消化抗性 Ana o 3 蛋白片段保留了报告的 IgE 结合表位。用还原剂预处理提取物或纯化的 Ana o 3 增加了 Ana o 3 对蛋白酶消化的敏感性。圆二色性显示纯化的 Ana o 3 的结构主要为α螺旋，还原后结构被破坏。Ana o 3 还原后再用蛋白酶消化会降低来自腰果过敏患者的血清 IgE 的结合。我们的结果表明，Ana o 3 的二硫键依赖结构使其免受蛋白水解的影响。
Ana o 3 是最有可能完整地通过胃肠道消化而存活下来的腰果过敏原。
