酶促糖基化修饰下原肌球蛋白结构变化与过敏原性的关系

With recent developments in the food manufacturing practices and worldwide trade of food materials, food allergy has been encountered as an important health issue. Reduction in allergenicity as the priority, empirical approach is necessary to protect the consumers bearing food allergies. Current attempts in food allergy studies have revealed the cause of significant influence in the allergenicity of food due to its epitopes and modifications of amino acid residues. In this project, tropomyosin from crustacean seafood was selected to illustrate the effect of amino acid modification on allergenicity. Acknowledging, glutamine is one of the most abundant amino acids in crustaceans, TG enzyme was employed with amino-polysaccharide as acyl acceptor to transfer acyl groups. Based on this mechanism, tropomyosin glycosylation can be realized. Furthermore, using different amino-polysaccharides under various reaction conditions, regular patterns of glycosylation catalyzed by TG enzyme were then studied consistently. The linear and conformational structure changes in tropomyosin were analyzed subsequently. Whereas, the surface characters changes were also illustrated of glycosylated epitopes of tropomyosin. In addition, apart from structure changes, the allergenicity was also evaluated with a series of IgE binding-mast cell release-T lymphocyte-dendritic cell. Hence, above results reveal the inter-relationship between molecular changes and its allergenicity during enzymatic glycosylation modification in tropomyosin. In conclusion, this research can provide a novel basis to better understand the interrelationship mechanism involved in the tropomyosin and its allergenicity, which can scope to regulate/reduce the food allergy risk in future.

现代食品工业的发展和国际食品贸易的不断深化使得食品过敏已经成为重要的食品安全问题，降低食品过敏原性是控制过敏风险的首要选择。现有研究表明食品过敏原性主要取决于过敏蛋白的抗原表位，而表位中氨基酸残基的修饰能够显著影响过敏原性。本课题拟以甲壳类海产品过敏原——原肌球蛋白为研究对象，利用其表位中富含谷氨酰胺的特点，根据TG酶催化谷氨酰胺酰基转移的原理，以氨基多糖为酰基受体实现原肌球蛋白的糖基化，研究不同氨基多糖对原肌球蛋白谷氨酰胺残基的糖基化修饰规律；分析酶促糖基化对原肌球蛋白分子结构的影响，探讨其线性及构象性过敏表位表面结构特征的变化；在此基础上从IgE结合-肥大细胞释放-T淋巴细胞分化-树突状细胞递呈四个方面层层深入的对酶促糖基化产物的过敏原性进行评价，揭示糖基化修饰后原肌球蛋白分子结构变化与过敏原性的内在关系和规律，为调控/消减食品过敏风险提供新的理论依据。

食物过敏已经成为重要的食品安全问题，而医学上尚没有安全有效有效的根治办法，消费者只能采取避食的方式进行，但由于大部分过敏原是人类的营养来源，严格避食又会影响到营养安全，因此降低食物过敏原性已经成为成为控制过敏风险的首要选择。因此本项目旨在通过对甲壳类海产品过敏原原肌球蛋白的表位进行酶促定向修饰，分析修饰后过敏原结构和致敏性的变化，并探究这种变化的机理，建立可控性高，对食品品质影响小的降低食物过敏性的方法。通过系统研究酶促糖基化对过敏原的影响，证明了以壳聚糖等氨基多糖能够在谷氨酰胺转氨酶的作用下与原肌球蛋白中的谷氨酰胺残基结合，并诱发原肌球蛋白发生分子内和分子间交联，降低了原肌球蛋白结构中α-螺旋的含量，质谱的结果显示，部分修饰位点存在于原肌球蛋白的B细胞表位。在对结构分析的基础上，研究了修饰后原肌球蛋白过敏原致敏性的变化，结果显示修饰后的过敏原其IgE和IgG的结合能力降低可达70%以上，特别是诱发肥大细胞释放活性介质的能力得到显著下降。进一步的研究显示，这种致敏性的变化不仅仅存在于过敏效应的激发阶段，而且能影响到致敏阶段的能力，经过酶促修饰的的过敏原能够改变T淋巴细胞的分化途径，使得T细胞向Th1分化。在此基础上，系统研究了谷氨酰胺转氨酶、多酚氧化酶、漆酶等食品中常用酶对过敏原结构的影响，可以减少传统降低过敏原性方法的盲目性和不确定性，为采用定向酶促作用降低食品过敏原性提供了理论依据和方法学基础。