蛋白质N-糖基化对人乳和牛乳微生物调节功能差异的影响研究

It has been proven that the oligosaccharide in milk poses critical effects on the health of newborns. Of which, one of the conjugated sugar, the glycan attached to proteins has attracted increasing attention of researchers worldwide. Our group found from our previous work that the N-glycome released from both human and bovine milk exhibited notable functions including the beneficial bacteria growth promotion and the antipathegenic activity, with the human milk N-glycome being more effective than the bovine milk N-glycome. This finding is assumed to be one of the reasons for the fact that the breast-fed babies possess better health, in both short and long term, compared to the formula-fed babies. However, these functions are from the released milk N-glycans and the sugars are normally attached to the proteins and present in a conjugated form. Whether the functions of the released and the conjugated glycans are different and whether this is the reason for the functional difference between breast milk and formula remains unclear. Moreover, how the glycans work in the environment, such as the gut, when both the beneficial bacteria and the pathogens present. . In order to unreveal the role of the protein-linked glycans in milk and the association between the structure and the function, we are going to compare the released and the attached glycans in terms of their ability of promoting the growth of beneficial bacteria and the antipathogenic activity. The same comparison will be also performed between human and bovine milk glycans. Additionally, the mechanism that how the glycan works on the microbiota in the gut will be another topic of this project. B. infantis and L. monocytogenes will be employed and a model system containing both types of bacteria will be established by co-culturing these two bacteria. The mechanism that how milk glycan play its role in this system will provide us the information that how the glycan works in the gut. From this we will have a better idea how to improve the health situation of the formula-fed babies through manipulating the milk glycan in the formula.

已有的研究结果表明乳中寡糖对婴儿的健康有重要作用，其中对含量较高的结合态的糖蛋白寡糖的研究是国际该领域的一个新兴热点。本课题组前期研究发现，从人乳和牛乳糖蛋白上释放下来的自由N-链寡糖组都具有较好的益生和抗致病菌活性，但人乳糖蛋白N-链寡糖组的活性显著高于牛乳，推测这可能是导致奶粉喂养的婴儿其健康状况不如母乳喂养的婴儿的原因之一。但是， N-链寡糖在乳中是以糖蛋白的形式存在的，这种以结合态形式存在的N-链寡糖的功能和作用机理是什么目前尚不十分清楚。本项目将从益生和抗致病菌活性两个方面出发，以婴儿长双歧杆菌和单增李斯特菌为模式菌，利用糖组学和微生物学技术开展糖基化对乳蛋白质微生物学功能特性的影响、微生物对糖蛋白寡糖的利用模式两方面的研究，对乳中蛋白质N-糖基化对人乳和牛乳营养功能差异的影响及其作用机理进行深入探索。研究结果将为婴儿奶粉的升级开发使其更加接近母乳的功能提供新的方向和科学参考。

临床数据显示母乳喂养的婴儿其健康状况显著优于奶粉喂养的婴儿。我们的前期研究发现乳中蛋白质分子上所连接的N-链寡糖在释放下来后具有较好的抗致病菌和对婴儿长双歧杆菌的益生作用，且人乳N-链寡糖显著优于牛乳N-链寡糖。由于N-链寡糖在乳中是连接在蛋白质分子上的，这种结合态的N-链寡糖的功能如何以及对所连接的蛋白质的功能有何影响尚不清楚。本项目着重研究了N-糖基化与否对乳中蛋白质抗致病菌和益生功能的影响，考察了在致病菌和益生菌的共培养体系中对不同菌的丰度的影响以及对小鼠肠道菌群结构的影响，从而揭示蛋白质N-糖基化是否是人乳和牛乳之间功能差异的原因之一。从消化吸收的角度研究了乳中的糖蛋白在肠道中的去向。此外，还首次在相同实验条件下比较了人乳N-链寡糖与乳游离寡糖（HMOs）之间抗致病菌功能的差异。研究结果表明：无论是人乳还是牛乳，当从蛋白质分子上去掉糖链分子后，裸蛋白的抗菌效果和益生效果都大幅下降，人乳与牛乳裸蛋白的抗李斯特菌效果无显著差异，而两种糖蛋白之间差异显著，显示糖基化是造成两种蛋白抗菌效果差异的原因。N-糖基化对乳蛋白质对婴儿长双歧杆菌的益生功能的影响也类似。因此证明糖基化对于乳蛋白质的微生物调节功能影响显著。在婴儿长双歧杆菌与李斯特菌的共培养体系中，乳N-链寡糖的加入本身促进了益生菌的生长而抑制了致病菌的生长，与此同时，益生菌丰度的升高又进一步抑制了致病菌的丰度，证明乳中的N-链寡糖在微生态体系中所发挥的双重的作用，预示了其对肠道菌群的作用。接下来的小鼠（饲喂人乳和牛乳N-链寡糖）实验也进一步证明，二者对小鼠肠道菌群结构的影响是差异显著的。在此基础上，我们进一步研究了乳中典型的糖蛋白乳铁蛋白被摄入机体后的消化吸收情况，发现完整的乳铁蛋白可以被肠上皮细胞吸收，其上所连接的N-糖也一起被吸收，而被消化的乳铁蛋白肽则分为两种情况：不带糖链的肽可以被细胞吸收，而带糖链的肽则无法被吸收而主要进入大肠，在那里被大肠的肠道菌群所利用。该发现进一步证明了乳中N-糖链对肠道菌群结构具有较大的影响。因此，蛋白质糖基化的不同是造成人乳和牛乳营养功能差异的重要原因之一。此外，我们也首次比较了乳N-链寡糖与游离寡糖在抗金黄色葡萄球菌方面的差异，证明N-链寡糖具有“抑菌/杀菌”和“抗黏附”的双重功效，而乳游离寡糖（HMOs）仅具有抗黏附的作用，且量效比要远远低于N-链寡糖。