人乳特异性糖蛋白寡糖结构与功能研究

This project is aimed to investigate the human specific milk glycomes in terms of structure and function, by comparing with bovine and formula milk glycomes. Breast-fed infants have been widely found to possess better health compared to formula-fed infants， which brought the milk oligosaccharides to the focus of research interest. Oligosaccharides is the third most abundant macro molecule in milk and it has been reported to function as prebiotics, anti-pathogenes and immunity regulators. Moreover, differences of structure and function have been found between human and bovine milk oligosaccharides. It is therefore believed that the oligosaccharides in milk is the main yet to be verified reason for the health difference between breast-fed and formula-fed babies. However, the comprehensive study on milk oligosaccharides has been hindered due to the lack of powerful technology for structure analysis. The existing research on milk oligosaccharides has been mainly focused on free oligosaccharides and very little has been done to glycans from glycoconjugates, especially glycoproteins in milk. Additionally, although the baby formula is made of bovine milk the composition change during the processing is unavoidable and no attention has yet been paid to the final oligosaccharides composition in baby formulas. This project will be the first study on the entire milk glycomes (including both N- and O-linked glycans)from all glycoproteins with comparison between human milk, bovine milk and baby formula. We will first attempt to establish a novel multi-dimensional UHPLC-MS-based platform for glycan structure analysis with less requirement for advanced equipment. With this platform, structure profiling of all the glycans from all glycoproteins in milk will be carried out, followed by the functional study including promotion of beneficial bacteria and prevention of pathogene using FRET-based method. Human specific milk glycans with unique structure and biological function will be thus elucidated and the result can be used as the reference for the attempt of improving the quality and functionality of baby formula.

本项目旨在从糖组学角度对乳中所有糖蛋白寡糖进行结构和功能研究。母乳喂养的婴儿其健康状况明显优于奶粉喂养的婴儿，这一现状使得乳寡糖的研究日益成为新的热点。寡糖是乳中含量第三的大分子物质，具有调节菌群平衡、抵抗有害微生物以及免疫调节等方面的作用，且人乳和牛乳寡糖在结构和功能上都有较大差异，这很可能是造成人乳和配方奶粉功能差异的主要原因。目前关于乳寡糖的研究更多的集中在游离寡糖上，对糖蛋白寡糖的研究则相对滞后。针对此现状，本项目将首先建立并优化以多维超高效液相色谱-质谱为核心技术的寡糖结构分析平台，对人乳、牛乳以及市售配方奶粉中所有糖蛋白寡糖组（包括N-和O-链寡糖）进行结构分析，并对其在益生和抗菌两方面的生物活性进行深入研究。研究结果将建立起适用于一般实验室的更为经济可行的寡糖分析方法，应用此方法筛选出人乳中的特异性功能寡糖，为提高婴儿配方奶粉的质量、缩小其与母乳之间的差异提供科学依据。

项目背景：母乳喂养的婴儿其健康状况明显优于奶粉喂养的婴儿，这一现状使得乳寡糖的研究日益成为新的热点。寡糖是乳中含量第三的大分子物质，具有调节菌群平衡、抵抗有害微生物以及免疫调节等方面的作用，且人乳和牛乳寡糖在结构和功能上都有较大差异，这很可能是造成人乳和配方奶粉功能差异的主要原因。但目前关于乳寡糖的研究更多地集中在游离寡糖上，对糖蛋白寡糖的研究寥寥无几。本项目旨在从组学角度出发对人乳和牛乳中的糖蛋白寡糖组进行系统的结构和功能解析与比较研究。. 主要研究内容：（1）寡糖结构解析新工具的挖掘和平台的建立。（2）乳中糖蛋白寡糖组的结构分析和比较。（3）乳糖蛋白寡糖组益生和抗致病菌功能研究。. 重要结果：（1）建立了以HILIC为主要分离模式、以2AB为荧光标记物和以组合外切糖苷酶处理为辅助的N-链寡糖检测和结构推测平台；建立了以MALDI-TOF-MS/MS为主要手段的寡糖结构分析方法；发现了功能更为强大的N-糖酰胺酶和其他外切糖苷酶，建立了更为快捷高效的N-糖解离方法。（2）全面解析了人乳、牛乳和市售配方奶粉中的N-链寡糖组的结构，发现奶粉与牛乳的结构基本相同。对人乳和牛乳N-链寡头组的结构差异进行了详细对比。（3）发现了人乳和牛乳糖蛋白寡糖都具有抗致病菌功能和益生功能，且功能与结构密切相关。. 关键数据：本课题在挖掘了多种新型糖苷酶和建立了方法平台的基础上详细解析了人乳和牛乳N-链寡糖结构分别为36和63种，共有结构20种。人乳和牛乳N-链寡糖对5种常见致病菌都具有较好的抗致病菌功能（包括抑制生长和抗黏附作用），但前者显著优于后者。当去掉岩藻糖修饰后寡糖组的抗菌功能显著下降，且二者间的差异显著性消失。人乳和牛乳N-链寡糖组都对婴儿长双歧杆菌具有益生功能，同样地人乳寡糖优于牛乳寡糖。实验结果进一步显示乳N-链寡糖可能首先被转运到细菌细胞内然后在细胞内被利用。. 科学意义：建立起了适用于一般试验室的更为经济可行的寡糖分析方法，并首次系统解析和比较了人乳和牛乳N-链寡糖组的结构差异，证明了二者所具有的生物学功能，为以后婴儿配方奶粉的开发升级打下了重要科学基础。同时发现了乳N-链寡糖的重要生物学功能，对于未来功能或医药产品的开发也具有重要意义。