超声波协同糖基化修饰β-乳球蛋白过敏表位的分子机制

Our previous research showed that ultrasound combined with glycation effectively reduced the allergenicity of β-lactoglobulin (β-Lg), which was related to modification of antigenic epitopes. However, the biochemical mechanism is not clear, and needs to be further studied. β-Lg and saccharides will be chosen as materials in this project. With the aid of methods such as enzyme-linked immunosorbent assay(ELISA) and Western blotting, the influences of ultrasound combined with glycation modification on the allergenicity of β-Lg will be researched, from intact protein and digestion products two aspects.Techniques including O-18 labeling and liquid chromatography-mass spectrometry, will be employed to investigate the influence of ultrasound combined with glycation modification on the linear epitopes of β-Lg allergen, and to establish the correlation among parameter conditions, glycation extent, allergenicity and linear epitopes. Moreover, techniques such as isotope labeling, chromatography and mass spectrometry, will be employed to accurately measure spatial conformation of β-Lg treated by ultrasound combined with glycation modification and analyze the change rule of the conformational epitopes of β-Lg. Futhermore, based on the antigen antibody response, protein chip, hydrogen deuterium exchange mass spectrometry and other techniques will be used to accurately screen and characterize the antigenic epitopes. Synthesize all of above and relevant theories including statistics and protein chemicobiology, the molecular mechanism of antigenic epitopes modification and decreased allergenicity of β-Lg treated by ultrasound combined with glycation modification, will be clarified from linear and conformational epitopes of allergens.

前期研究表明，超声波协同糖基化能有效降低β-乳球蛋白（β-Lg）的致敏性，这与其过敏表位修饰有关，但其生化反应机制还不清楚，有待深入研究。本项目拟以β-Lg和还原糖为研究对象，运用ELISA、Western blotting等方法，从完整蛋白和消化产物两方面研究超声波协同糖基化修饰对β-Lg致敏性的影响；借助氧-18标记、液质联用等方法，研究超声波协同糖基化对β-Lg过敏原线性表位的影响，建立参数条件-糖基化程度-致敏性-线性表位的相关性；利用同位素标记、色谱、质谱等技术，精确测定超声波协同糖基化修饰后β-Lg的空间构象，解析β-Lg构象性表位的变化规律；基于抗原抗体反应，采用蛋白质芯片、氢氘交换质谱等技术精确筛选和表征β-Lg过敏表位。结合统计学和蛋白质化学生物学等相关理论，从过敏原线性表位和构象性表位入手，探明超声波协同糖基化修饰β-Lg过敏表位及降低其致敏性的分子机制。

前期研究表明，超声波协同糖基化能有效降低β-乳球蛋白（β-Lg）的致敏性，这与其过敏表位修饰有关，但其生化反应机制还不清楚，有待深入研究。因此，本项目以β-Lg为研究对象，选用不同超声波条件（超声时间、功率）协同还原糖（半乳糖、甘露糖和乳糖）对其进行糖基化改性；采用ELISA、嗜碱性粒细胞、过敏小鼠模型等方法研究不同条件改性β-Lg的致敏性；运用小鼠和模拟胃肠道消化模型，研究改性β-Lg的消化稳定性、消化产物的致敏性及其对肠道菌群的影响；采用高分辨质谱技术鉴定改性β-Lg的糖基化位点、肽段的糖基化修饰程度，建立参数条件-糖基化程度-致敏性-线性表位的相关性，同时分析改性β-Lg的多级结构和空间构象的变化规律。结果显示：超声波协同糖基化修饰显著降低β-Lg及其消化产物的IgE/IgG结合能力以及嗜碱性粒细胞免疫因子（β-氨基己糖苷酶、组胺和白细胞介素-6等）的释放，改善了人和小鼠肠道菌群的结构，缓解小鼠的过敏症状。超声波协同糖基化修饰主要通过糖基化位点掩盖β-Lg的线性表位和破坏其构象表位来降低其致敏性，表现在糖基化程度的增加（结合率、糖基化位点和数量、肽段的糖基化程度）以及β-Lg二级、三级结构和空间构象的改变。同时，改性的β-Lg在胃肠道消化吸收过程中具有抗蛋白酶活性，其消化产物（抗消化肽、氨基酸等）可以改善与食物过敏有关的特定属/种的肠道菌群结构，从而缓解过敏性。研究结果为定向改性蛋白质的过敏表位降低其致敏性提供重要的理论基础。