肉、蛋、奶中糖蛋白寡糖对特殊肠道功能菌Akkermansia Muciniphila丰度的影响及其机理研究

Akkermansia muciniphila is a novel functional gut bacteria discovered recently. It has been shown that the approperiate abundancy of this bacterium in gut is essential to human health. Up to date, the study on Akkermansia muciniphila has been focused on its biological functions, whereas very little is known about the regulation of its abundancy, especially by regular foods. It is well known that Akkermansia muciniphila is able to digest mucin, the most predominant protein in mucus, to produce short chain fatty acids. However, mucin is a protein which is heavily glycosylated, whether the bacterium uses the protein part or the sugar attached onto the protein remains unclear. . The previou study carried out in our lab showed that the glycan is widely present in many foods, especially in the animal-resourced foods. N-glycome derived from milk showed strong effects on the growth and function of different bacteria. Moreover, we also obtained the Akkermansia muciniphila strain and performed certain experiments with it's gene. Several exo-glycosidases such as sialidase, hexoacetolsaminidase fucosidase with acitivities were discovered from Akkermansia muciniphila, indicating the ability of digesting sugar of this strain. Putting all of these together, we thus hypothesized that food-originated glycoproteins can pose great effect on the growth and biological functions of Akkermansia muciniphila. . To prove this, we are going to first investigate the effect of mucin and food glycoprotein, deglycosylated protein and glycans on the growth of Akkermansia muciniphila to see which if the sugar can be consumed by the bacteria. Next, the mechanism through which the bacterium utilizes the sugar will be investigated. Finally, the principle factors from whatever effective food component will be screened based on the glycomics or proteomics technologies. . The results of this project will show if and how Akkermansia muciniphila can utilize the food-resourced glycans for the first time. This will provide the possible approach through which to maintain the healthy level of Akkermansia muciniphila in gut.

Akkermansia muciniphila是新发现的一种特殊肠道功能菌，其合适的丰度对健康至关重要。目前关于影响其丰度的因素研究较少，而关于常规食物对其丰度的影响几乎未见报道。黏蛋白是该菌唯一已知的天然营养来源物质，但黏蛋白是糖蛋白，该菌利用的是蛋白本身还是其上的寡糖目前尚不清楚。本实验室前期从A. muciniphila基因组中克隆表达得到了多种活性外切糖苷酶，显示该菌有利用寡糖的能力。我们还发现食物如肉蛋奶中含有较多的糖蛋白寡糖，且乳中寡糖对微生物的生长有显著影响，因此推测饮食中的糖蛋白对A. muciniphila的丰度具有调节作用。基于此，本项目将利用糖组学和微生物技术首次考察黏蛋白寡糖和肉蛋奶中的糖蛋白寡糖对A. muciniphila丰度的影响及其作用机理，并对主效因子进行筛选。研究结果将阐明该菌对食物源糖蛋白的利用情况和机理，为通过饮食调节保持该菌的健康丰度提供科学依据。

Akkermansia muciniphila是近年来比较热门的肠道功能菌，该菌在肠道中的适宜丰度与机体健康关系密切。因此，研究并揭示食物中影响该菌丰度的功能成分具有重要意义。粘蛋白是目前已知的能够显著促进该菌生长的物质，由于粘蛋白是一种高度糖基化的蛋白，本项目拟首先研究粘蛋白的蛋白部分和糖链部分各自所起的作用，在此基础上研究食物中糖蛋白对该菌的影响。研究结果表明：糖分子要在有氮源存在的情况下才能促进A. muciniphila的生长，而蛋白质单独可以促进A. muciniphila的生长，但效果不如带有糖链的好，证明蛋白质和糖对于A. muciniphila的生长都很重要。在有碳源和氮源时，糖链对菌的促生长作用要强于蛋白质的作用。非糖蛋白BSA的促进菌生长作用显著低于粘蛋白，证明该菌对蛋白质的结构组成有要求，进一步分析发现，黏蛋白的氨基酸序列中富含苏氨酸，而苏氨酸是A. muciniphila唯一不能自身合成的必需氨基酸。在此基础上，我们分别对乳、肉、蛋中的糖蛋白组进行了研究，发现：肉和牛乳清中的N-糖组对于A. muciniphila 的生长具有显著的抑制作用，乳清糖蛋白及裸蛋白对目标菌具有一定的促进作用，蛋清和蛋黄样品及肉中的裸蛋白对A. muciniphila菌的生长基本上没有影响。小鼠实验也证实饲喂了牛乳清N-链糖组的小鼠肠道菌群中幽微菌门的丰度下调。单糖测定发现，Gal对A. muciniphila具有最佳的促生长作用，其次是GalNAc,Fuc,GlcNAc, 而唾液酸Neu5Ac则展示出抑制作用。糖苷酶活性测定结果与单糖的益生作用相符，且添加牛乳清N-糖组显著降低了菌体大部分外切糖苷酶的分泌，这解释了牛乳清N-糖组抑制A. muciniphila菌生长的原因。比较粘蛋白O-糖组和食物N-糖组的结构发现：食物中N-糖与粘蛋白的O-糖相比，前者不含有GalNAc而后者含有GalNAc，前者中Gal的链接键型大多为β1，4链接，而O-糖中β1，3链接的较多；前者中糖链的结构更复杂、空间位阻效效应更大，而O-糖结构更简单，空间位阻效应小；唾液酸的含量也有差异等等。因此，食物中糖蛋白可以作为A. muciniphila的丰度下调剂进行开发。本项目另外一个突出成果是通过生物方法成功合成了Core 1粘蛋白和Core 2糖链，为该菌的益生元开发打下了良好基础。