质子化诱导芸豆凝集素蛋白构象中间态与致敏性的构效关系研究

Lectin is one kind of the storage protein in the bean, and the existence of the protein is the urgent limit for the application of legume proteins in the food industry because of its high allergic potential. It has been proven that there is a close connection between the allergenicity of lectin and its spatial structure. And evidences for an intermediate state formation in Phaseolus vulgaris lectin unfolding have been confirmed in our previous studies, however, the structural forming mechanism and the relationship with allergenicity are lack of information. Therefore, a systemic and detailed investigation of Phaseolus vulgaris lectin will be carried out in the project as follows: (1) the antigen epitope peptides will be synthesized via solid-state reaction, and the spatial conformations of the epitopes will be explicated in the protein macroscopic view by homology modeling method and the spectra characterizations; (2) the forming mechanism of the intermediate state induced by protonation will be elucidated through physical properties, chromatography, spectroscopy and thermodynamic analysis, while the effects of acidic pH on the structures of the antigen epitope peptides will be also investigated using the peptide synthesis method; (3) the western blot and ELISA analyses will be subsequently applied, and the influences of intermediate state on the lectin allergenicity will be illuminated through in vitro and in vivo digestion experiments as well as physical field treatments, respectively. The develop rules of the lectin intermediate state will be revealed in our research, as well as the relationship between intermediate state and allergenicity will be clarified, and our results will provide a theoretical basis for the low sensitization food processing utilizing the grain proteins as the materials.

凝集素是豆类籽粒中的储存蛋白之一，其高致敏性制约着豆类蛋白质在食品工业中的广泛应用。研究证实，凝集素的致敏性与蛋白质的空间结构关系密切。我们前期研究发现，质子化诱导可促芸豆凝集素蛋白构象中间态的形成，但中间态的形成机制及其与致敏性的构效关系尚不清楚。本项目拟以芸豆凝集素为研究对象，开展以下理论研究：（1）同源模建凝集素蛋白构象，固相合成抗原表位肽，结合光谱表征，在宏观尺度研究凝集素核心抗原表位的空间结构；（2）通过物性、色谱、光谱和热力学分析，在蛋白质分子层面揭示质子化诱导蛋白中间态的形成规律，利用多肽合成研究质子化诱导对抗原表位肽结构的影响；（3）采用免疫印迹和ELISA技术，通过体内、体外实验，分析凝集素蛋白中间态及在物理场协同作用下与致敏性消减的相关性。项目预期成果将揭示质子化诱导凝集素中间态形成规律，阐明中间态与致敏性之间的关系，为低致敏谷物原料蛋白质的食品加工提供一定的理论基础。

芸豆凝集素的高致敏性限制着豆类食品开发。项目以芸豆凝集素蛋白为研究对象，同源模建蛋白构象，预测鉴定抗原表位，提出质子化诱导处理，通过物性、色谱、光谱和热力学分析揭示质子化诱导蛋白构象变化与致敏性之间的关联性，基于体内动物实验、ELISA、光谱学和分子动力学模拟分析阐明凝集素空间蛋白状态及多物理场协同作用下致敏性消减的分子机制。项目研究发现，在酸性条件下，蛋白分子带上多余的正电荷，静电斥力使得凝集素蛋白四聚体向单体状态解聚，分子链舒张，蛋白结构呈现“柔性”状态；当质子化诱导8小时后，芸豆凝集素蛋白的解折叠过程进入稳态，此时，芸豆凝集素蛋白的免疫原性随着孵育pH值的下降而显著降低，体外消化性显著提高；体内实验和计算机模拟分析表明，在质子化诱导的蛋白解折叠过程中，不仅蛋白消化位点暴露，抗原表位在空间上也暴露出更多的酶切位点，可显著提高芸豆凝集素蛋白的肠道消化率，降低致敏可能性。本项目研究成果丰富和完善了蛋白过敏原的空间构效关系理论，可为我国低致敏芸豆蛋白质产业化技术升级提供一定的理论依据。