基于新型N-糖酰胺酶PNGase T的白果32K蛋白糖链结构及致敏机制研究

The object of this project is to study the glycan structures and allergenic mechasnims of ginkgo glycoprotein using a newly discovered N-glycanase PNGase T. .Ginkgo is a functional food of high economic value. However ginkgo contains several types of allergens which can cause serious allergy response, sometimes even death if taken too much. The identification and desensitization of these allergens becomes an urgent task in the product development of ginkgo. A previous study on protein allergens of ginkgo showed that a glycoprotein with 32KDa molecular weight is one of the major causers. Considering that not only polypeptides but also the carbohydrate moieties of glycoproteins are responsable for allergy response, especially in the IgE mediated allergy, it is necessary to study the structure and the allergenic mechanism of the carbohydrate part of ginkgo 32K glycoprotein. .In the preliminary work,our group has discovered a novel N-glycanase (PNGase T) which has more flexible substrate specificity compared to the currently available PNGase F and PNGase A, and thus is a more preferable enzyme for glycan study of glycoproteins, in particular for the plant-originated glycoproteins characterized as containing α1,3 fucose on the core structure of the attached glycans. In this study, the novel PNGase T will be used as the ideal tool for N-glycan releasing from ginkgo 32KDa glycoprotein. An UHPLC-MS-based strategy, coupling with sugar fluorescent derivitization and sequential glycosidase digestion, for detailed structure mapping of all released N-glycans will be subsequently established and optimized. .For the allergenic mechanism study, the 32kDa glycoprotein from Ginkgo and the corresponding enzymatically hydrolyzed peptides will be both investigated. PNGase F, which is not active to the glycoprotein containing core α1,3 fucose, will be also employed for deglycosylation of the protein to compare with the treatment of PNGase T. The glycoprotein and glycopeptides, together with the deglycosylated protein and peptides will be studied by the immunoassays of IgE binding and antibody titration. The glycan structures, especially the core structures of the target protein will thereafter be elucidated by the aforementioned newly established UHPLC-MS based method. Assembling all the results together, the active components of the Ginkgo 32KDa glycprotein for the allergy will be revealed with the detailed information of whether and which part of the glycans from the Ginkgo 32K protein are responsible for the IgE mediated allergy. .The outcome of this project will provide the theoretical basis and scientific technique for establishment of approaches to reduce or eliminate the allergenicity in ginkgo foods, thus enhance the safety of ginkgo foods and promote the development of ginkgo products.

本项目拟以新型N-糖酰胺酶PNGase T为工具对白果致敏糖蛋白的糖链结构和致敏机制进行研究。白果作为功能食品开发具有很高经济价值，然而其所含的多种致敏原限制了对它的开发，因此对致敏原的识别和脱敏研究成为白果食品开发急需解决的问题。以往的研究显示植物致敏蛋白除了多肽部分,糖链也是重要的致敏成分，尤其是具有β-1,2木糖α-1,3岩藻糖核心结构的N-糖链与IgE介导的过敏反应密切相关。白果32K糖蛋白作为白果致敏蛋白中的主要成分，有必要对其糖链结构和致敏机制展开深入研究。本项目以前期工作筛选得到的PNGase T为基础，建立以UHPLC-MS技术为核心的糖链结构分析平台，分析白果32K糖蛋白糖链结构，并利用PNGase T和F的底物差异性，系统的研究不同结构的糖链的作用和可能的致敏机制。研究成果将为寻找有效的白果蛋白脱敏方法提供理论基础，从而保证白果食品的安全性加速白果功能食品开发。

白果是我国的特色经济作物，具有很高的营养和药用价值，但由于白果及白果制品食用安全问题导致相关产业发展缓慢。白果中的蛋白成分是白果及白果制品中的重要过敏原之一，而以往的研究显示蛋白上的糖链结构是重要的交叉过敏原，因此研究白果蛋白上的糖链结构以及相应的致敏原理具有重要意义。本项目选择白果蛋白做为研究对象，使用新型N-糖酰胺酶PNGaseH+为工具，以糖链化学结构为基础，围绕白果蛋白上糖链的致敏机制研究展开工作。本项目首先建立了以N-糖酰胺酶H+为基础、超高效液相色谱为核心结合飞行时间质谱的植物糖蛋白糖链结构分析技术平台。利用此技术平台完善了白果蛋白N-糖组数据库，并对我国主要白果产地的白果蛋白N-糖组进行了定性定量比较。其次建立了以酶联免疫印迹为基础、以印记膜上糖链释放技术为核心、结合超高效液相色谱的快速植物蛋白过敏原糖链结构分析方法。明确了白果蛋白中主要致敏原为糖蛋白，并鉴定了其主要糖链结构为MMXF。本项目还利用N-糖酰胺酶H+，建立和完善了酶法白果蛋白体外去糖基化反应条件，并使用蛋白凝胶电泳对去糖基化前后白果蛋白的模拟胃肠液产物进行比较分析，发现糖基化能够在一定程度上提高白果蛋白对模拟胃肠液消化的耐受性。使用竞争抑制ELISA方法，使用白果蛋白致敏小鼠血清为一抗，比较了去糖基化前后白果蛋白模拟胃肠液产物的免疫原性，明确了模拟肠液消化可以大幅降低白果蛋白的免疫原性。明确了体外去糖基化、模拟胃液和模拟肠液消化对白果蛋白致敏性的影响，为降低白果蛋白免疫原性，提高食用安全性提供了理论指导。