末端唾液酸化修饰对人乳和牛乳糖蛋白N-链寡糖组结构和功能差异的影响研究

This project is aimed to investigate the effect of sialylation on the structural and functional differences of whole milk N-glycomes between human and bovine. The clinincal data showed that the breast-fed infants have better health than the formula-fed infants, which indicated the functional differences between human milk and bovine milk. In order to reveal the reason for the difference between the two, researches was carried out to investigate the structure and anti-pathogenic function of both human and bovine milk N-glycomes by our group. The obtained results showed that the difference of whole milk N-glycome structures between human and bovine was very obvious, and sialylation was one of the key factors for this structural difference. Moreover, human milk N-glycomes exhibited significantly stronger anti-pathogenic activities compared to bovine milk N-glycomes. This functional difference was shown to be closely associated with N-glycome structures. The milk N-glycome is thus presumed to be related to the health differences between breast-fed and formula-fed babies. .Based on the findings above, this project is going to first elucidate the structures of sialylated milk N-glycome using UHPLC-MS coupled with MALDI-TOF-MS/MS. The role of sialylation of milk N-glycomes will be investigated through inter- and intra-speices comparison of the anti-pathogenic activities of milk N-glycome before and after de-sialylation. In addition, due to the exist of an immunogenic sialic acid Neu5Gc which is specific in bovine milk, the function of this special sialic acid will be also studied through re-sialylation after de-sialylation treatments and gene-knockout mouse modeling. The results of this project will help us to comprehensively understand the biological function of sialylation in milk N-glycomes, and therefore provide valuable information for development of bovine milk-based formula which is functionally closer to mother milk.

临床发现母乳喂养的婴儿其健康状况明显好于奶粉喂养的婴儿。为找出母乳和牛乳之间的功能差异，本课题组前期以研究较少的乳中糖蛋白N-链寡糖组为对象，对其进行了结构和功能的比较研究。结果表明：人乳和牛乳N-链寡糖组都具有较好的抗致病菌作用，但前者显著优于后者；二者在结构上差异很大，其中唾液酸化修饰是造成这种差异的主要原因之一。因此有必要进一步对乳N-链寡糖组中唾液酸化修饰的作用进行研究。首先本项目拟利用UHPLC-MS结合MALDI-TOF-MS/MS对人乳和牛乳唾液酸化N-链寡糖组进行结构解析，并通过比较去唾液酸化前后乳N-链寡糖组结构和抗菌功能的变化揭示唾液酸化对乳N-链寡糖功能的影响；其次，还将利用再唾液酸化以及基因敲除小鼠模型对牛乳中独有的免疫原性唾液酸Neu5Gc的影响及其机理进行探索。研究结果将有助于全面了解唾液酸化修饰对乳N-链寡糖组功能的作用，为婴儿配方奶粉的开发升级提供重要依据。

临床发现母乳喂养的婴儿其健康状况明显好于奶粉喂养的婴儿。为找出母乳和牛乳之间的功能差异，本课题组前期以研究较少的乳中糖蛋白N-链寡糖组为对象，对其进行了结构和功能的比较研究。结果表明：人乳比牛乳N-链寡糖组具有更好的抗致病菌作用，二者在结构上差异很大，其中唾液酸化修饰是造成这种差异的主要原因之一。因此本项目开展了对乳N-链寡糖组中唾液酸化修饰的功能研究。首先，利用UHPLC-MS结合MA LDI-TOF-MS/MS对人乳和牛乳唾液酸化N-链寡糖组进行结构解析，其次利用酶法合成技术自主合成了高度模拟唾液酸结构的糖苷标准品，建立起了更为准确可靠的唾液酸含量及存在形态的检测方法，测定了人乳和牛乳中唾液酸种类、丰度，证明人乳中只含有Neu5Ac，而牛乳中除了Neu5Ac外还含有较多的Neu5Gc，且人乳中总唾液酸的含量显著高于牛乳。其次，通过比较去唾液酸化前后乳N-链寡糖组结构和抗菌功能的变化揭示了唾液酸化对人乳N-链寡糖抗菌功能具有重要影响，且与岩藻糖基化一起形成了协调增强的效应，而该效应只存在于人乳N-链寡糖中。利用人工合成的唾液酸糖苷X-Gal-Neu5Ac和X-Gal-Neu5Gc进行抗菌功能的比较研究，结果证明前者具有显著高于后者的杀菌/抑菌功能，间接证明人乳N-链寡糖的抗菌功能显著优于牛乳的可能原因之一是人乳中只有X-Gal-Neu5Ac，而牛乳中还含有X-Gal-Neu5Gc。此外，还利用CMAH基因敲除小鼠模型对牛乳中独有的免疫原性唾液酸Neu5Gc的影响及其机理进行探索，结果表明，一出生就摄入Neu5Gc（模拟奶粉喂养）和一出生不摄入Neu5Gc（模拟母乳喂养）对机体的部分生理生化指标及肠道菌群结构有显著影响。本项目研究结果证明了唾液酸化修饰对乳N-链寡糖组功能和机体健康的影响，为通过调整唾液酸种类和含量的方式提高婴儿配方奶粉的营养和功能提供了重要依据。