母乳蛋白的核心岩藻糖基化修饰选择性促进新生儿肠道乳酸杆菌优势生长的机制研究

The fucosylation level of human milk glycoprotein is significantly higher than that of bovine milk. The core fucosyltransferase (Fut8) catalyzed core-fucosylation is the main fucosylation pattern of human milk N-glycans. Previously, the applicant found that Lactobacillus spp. (L. casei, etc.) in the intestinal tract of infants fed by high core fucosylated breast milk was more abundant than those fed by mothers with low core fucosylated milk N-glycan; Compared with Fut8+/+ maternal mice, the relative abundance of Lactobacillus spp. in intestinal tract of neonates fed by Fut8+/- maternal mice was significantly lower. Lactobacillus spp. such as L. casei could metabolize fucose to produce lactate and propanediol, which in turn promote B cell activation. However, it is not clear how core fucose in human milk selectively promotes the growth of Lactobacillus spp. in gut of infants. The chemically synthesized core fucosylated trisaccharide could promote the growth of specific Lactobacillus spp. and up-regulate the expression of genes encoding endoglycosidase EndoL and fucosidase AflC, suggesting that these two enzymes are the key glycosidases expressed by Lactobacillus spp. to specifically hydrolyze the core fucose on milk N-glycan. In this study, by means of gene knockout and protein expression, L. casei will be used as an example to elucidate the molecular mechanism of EndoL and AflC involved in decomposition of core fucose on breast milk N-glycan by Lactobacillus spp., Fut8+/- mouse model will be adopted to study the regulation of fucosylated trisaccharide on intestinal Lactobacillus spp. and B cell activation in offspring. Results from this study will lay a foundation for the development of new microecological agents for infants.

母乳糖蛋白的岩藻糖基化水平显著高于牛乳，其中以核心岩藻糖基转移酶(Fut8)催化的核心岩藻糖基化为主。申请人前期发现：高核心岩藻糖基化母乳哺育的新生儿肠道乳酸杆菌(L.casei等)丰度较高；与Fut8+/+母鼠相比，Fut8+/-母鼠哺育的子鼠肠道乳酸杆菌丰度偏低；L.casei等可代谢岩藻糖产生乳酸、丙二醇并促进B细胞活化。但母乳核心岩藻糖基如何选择性地促进乳酸杆菌生长的机制不明。化学合成的核心岩藻糖基化三糖可促进L.casei生长并上调内切酶EndoL和岩藻糖苷酶AflC基因表达，提示该两种酶是乳酸杆菌特异性分解母乳核心岩藻糖基的关键。本课题以L.casei为例采用基因敲除和蛋白表达等手段阐明EndoL和AflC参与乳酸杆菌分解母乳核心岩藻糖基的分子机制；并以Fut8+/-小鼠模型深入研究核心岩藻糖基化三糖对子鼠肠道乳酸杆菌及B细胞活化的调节作用；为研发新型婴幼儿微生态制剂奠定基础。

母乳中的糖类是塑造生命早期肠道菌群定植及免疫发育的重要成分。研究表明母乳糖蛋白的岩藻糖基化水平显著高于牛乳，其中以核心岩藻糖基转移酶(Fut8)催化的核心岩藻糖基化为主。申请人前期研究发现：高核心岩藻糖基化母乳哺育的新生儿肠道乳酸杆菌丰度较高；与Fut8+/+母鼠相比，Fut8+/-母鼠哺育的子鼠肠道乳酸杆菌丰度偏低；L.casei等可代谢岩藻糖产生乳酸、丙二醇并促进B细胞活化。但母乳核心岩藻糖基如何选择性地促进乳酸杆菌生长的机制不明。化学合成的核心岩藻糖基化三糖可促进L.casei生长并上调内切酶EndoL和岩藻糖苷酶AflC基因表达，提示该两种酶是乳酸杆菌特异性分解母乳核心岩藻糖基的关键。在本项目中，我们首先利用体外实验研究证实了在无糖培养基中添加人源乳铁蛋白（hLF）、岩藻糖单糖、和利用PNGase酶对乳铁蛋白进行酶解所释放的N-聚糖可促进L. gasseri DM1718菌株及标准菌株L.casei ATCC334生长，并产生乳酸和1,2-丙二醇等物质。通过蛋白异源表达证实了糖苷酶EndoL和AflC作用参与乳酸杆菌特异性分解利用母乳蛋白N-聚糖上核心岩藻糖基的分子机制；以Fut8+/-母鼠模型探究了母乳蛋白N-聚糖的糖基化水平改变对子代肠道微生态及免疫平衡的影响；同时，也探究了岩藻糖基化修饰在致病菌感染中对机体免疫，特别是B-T细胞相互作用的影响及和内在机制。在此基础上，以核心岩藻糖基化三糖饲喂Fut8+/-母鼠哺育的子鼠模型，证实了核心岩藻糖基化三糖促进子鼠肠道乳酸杆菌生长及代谢产物的生成；进一步也证实了核心岩藻糖基化三糖对Fut8+/-母鼠哺育子鼠B细胞活化的影响；同时利用核心岩藻糖基化三糖进行体外实验研究证实了其对乳酸杆菌生长、代谢及基因表达的影响及相关机制。以上成果为研发基于核心岩藻糖基化低聚糖的新型婴幼儿微生态制剂奠定基础。