唾液酸糖基化修饰对骨骼肌分化的影响及其作用机制的研究

Sialic acids (SA) are a family of nine-carbon monosaccharides found predominately at the non-reducing end of oligosaccharide chains on glycoproteins and glycolipids. They play many important roles in many physiological and pathological processes, such as cell differentiation, virus infection, formation, progression and metastasis of tumors, and also the biological stability of glycoproteins. Though many proteins are found to be involved in the differentiation of skeletal muscle cells, our previous results have indicated that sialylated glycosylation also plays some role in the process. However, the mechanism of sialylated glycosylation in the differentiation of skeletal muscle cells still needs to be explored, for example, which sialylated glycosylated proteins display significant changes during the differentiation, what are the changes of glycans during the differentiation, and how sialylated glycosylation regulates the differentiation of skeletal muscle cells. Aiming at investigating the role of sialylated glycosylation in the differentiation of skeletal muscle and its possible mechanism, several steps will be taken in this proposal. First, we plan to optimize technologies for enrichment and analysis of intact sialylated glycopepeptides with mass spectrometry. Then, we will analyze the global glycoproteome changes during the differentiation of skeletal muscle cells by integrating SILAC-based quantitative strategy. After that, we will analyze the glycoproteome data with bioinformatics tools to seek for the key sialylated glycoproteins and glycans that involve in the differentiation of skeletal muscle cells. Then, myoblasts will be treated with sialyltransferase inhibitor to inhibit protein sialylation or with different sialidases to remove sialic acids from glycans during the differentiation of skeletal muscle cells, and expression of specific protein markers of skeletal muscle differentiation will be detected to investigate the possible mechanism of sialylated glycosylation on the cell differentiation. We hope that this study will not only provide some useful information to elucidate the mechanism of skeletal muscle cells differentiation, but also offer some insights into potential therapeutic targets for myopathy.

唾液酸（SA）是一系列含九个碳原子的单糖衍生物的总称，通常位于糖蛋白和糖脂的糖基化非还原末端，在细胞生理和病理活动中发挥重要作用，如细胞分化、病毒的感染、肿瘤的形成、进展和转移以及维持糖蛋白的稳定。尽管已经发现了一些蛋白质参与调控骨骼肌分化，我们前期工作提示唾液酸糖基化修饰在骨骼肌细胞分化过程中发挥一定的作用，但是唾液酸糖基化修饰参与调控骨骼肌分化的作用机理还不是很清楚，如骨骼肌分化前后哪些唾液酸糖基化蛋白和唾液酸糖链发生了变化以及唾液酸糖基化修饰如何调控骨骼肌分化等。为回答这些问题，本课题首先将优化唾液酸完整糖肽的富集和质谱分析方法，然后结合SILAC定量蛋白质组技术，从定量糖蛋白质组角度系统分析小鼠成肌细胞分化成肌管细胞过程中唾液酸糖基化蛋白质和唾液酸糖链的变化，进一步利用生物信息学方法发掘与骨骼肌分化相关的唾液酸糖基化蛋白和糖链。随后，我们将利用唾液酸转移酶抑制剂抑制蛋白质唾液酸糖基化和唾液酸酶处理细胞以除去糖苷上的唾液酸，通过检测骨骼肌分化不同阶段标志蛋白的表达来研究蛋白质唾液酸糖基化修饰参与骨骼肌分化的作用机理。本研究为了解骨骼肌分化的机理以及骨骼肌疾病的治疗提供了新的思路。

糖基化修饰是一种重要和复杂的蛋白质翻译后修饰，在细胞分化与发育、肿瘤生长和转移、精卵识别和免疫中发挥着重要作用。唾液酸是一种九碳酸性单糖结构，通常位于糖蛋白和糖脂的糖链末端，是真核细胞糖缀合物中丰度最高的末端单糖，在细胞粘附和细胞-细胞间的相互作用等细胞生理和病理活动中发挥重要作用。. 本项目旨在建立一套完整的唾液酸糖基化蛋白质组分析方法，包括唾液酸糖基化肽段富集、质谱分析和数据分析方法，进一步研究唾液酸糖基化修饰在骨骼肌分化中的作用。在项目支持下，我们建立了基于ZIC-HILIC微量富集糖基化肽段的方法，大大提高了糖基化肽段的回收率和富集特异性；比较了两种不同完整糖肽的质谱分析方法，找到糖基化肽段鉴定数目最佳的方法，实现唾液酸完整糖基化肽段高灵敏度的鉴定。随后，系统分析了骨骼肌C2C12前体细胞的蛋白质组、磷酸化蛋白质组和N-连接唾液酸糖基化蛋白质组，整合分析三个蛋白质组学数据发现骨骼肌分化信号通路的细胞膜表面蛋白，如Cdon、Cdh15、Cdh2、Boc和Neo1等蛋白都发生了N-连接唾液酸糖基化修饰，提示蛋白质唾液酸糖基化修饰参与骨骼肌细胞分化过程。随后的糖链表征分析发现骨骼肌分化前后唾液酸糖链丰度和糖链组成都发生了明显变化，进一步证实唾液酸糖基化修饰在骨骼肌发育分化过程中发挥一定的作用。最后，在细胞培养过程中加入唾液酸合成抑制剂3Fax-NeuAc抑制唾液酸的产生，利用免疫荧光检测骨骼肌分化标志蛋白MHC（myosin heavy chain），发现抑制唾液酸的产生会降低骨骼肌分化的水平，这些结果都提示唾液酸糖基化修饰在维持骨骼肌正常生理功能和骨骼肌发育分化过程中发挥重要作用。. 鉴于唾液酸糖基化修饰在骨骼肌正常生理功能和分化发育中的重要作用，该研究在糖蛋白质组水平深入研究了蛋白唾液酸糖基化修饰在骨骼肌分化中的作用，为深入了解骨骼肌疾病特别是骨骼肌损伤的治疗提供新的思路。

内容获取失败，请点击重试