干细胞标志物CD133蛋白的N-糖链结构解析、调控及其在细胞定向迁移中的功能

Glycosylation is one of the most abundant post-translational modifications, and at least 70 percent of membrane proteins in eukaryotes are glycosylated. The sugar chains of membrane proteins function as biological information molecules to regulate cell differentiation, development, immunity, aging. Exploring the structure and function of sugar chain in membrane proteins reveals a high-level phenomenon of life in multi-cellular organisms. CD133/Prominin-1, a pentaspan membrane glycoprotein, displays a broad range of expression in several epithelial cell types and is confined to the apical plasma domain. Recent years, CD133 has received considerable interest due to its widespread use as a marker of various normal and cancer stem cells. Increasing studies indicate that CD133 performs a variety of functions in cell proliferation, differentiation, metabolism and migration. CD133 protein is predicted to contain eight N-linked glycosylation sites. Increasing evidences indicate that the glycosylation status of CD133, rather than the expression of CD133 protein itself, can act as an the marker of stem cells. And, the dynamic glycosylation of CD133 might play a critical role in its biological functions. Thus, identification of CD133 glycosylation status, which might be different in cells at different stages of differentiation and in different tissues, means a crucial step in defining the potential role of CD133 in normal and cancer stem cells. In this project, we intend to clarify the structure alteration of the N-glycans of CD133 during neural stem cell differentiation, and to investigate the mechanism regulating CD133 N-glycosylation. Furthermore, we perform to investigate the interaction between CD133 and animal lectins. Finally, we use cell and animal models to explore the funcion and mechanisms of the interaction between CD133 and animal lectin in directional migration in response to microenvironment. Through these efforts, we hope to identify animal lectin/ sugar chains of CD133 signaling axis to explore the fundamental biological aspect of CD133 glycosylation in cell directional migration.

糖链作为生物信息分子在生命和疾病过程中起着识别与调控作用。阐释关键膜蛋白的糖链结构和功能是揭示多细胞生物中的高层次生命现象的关键环节。干细胞、肿瘤干细胞的标志物CD133糖蛋白是调控细胞迁移等过程的关键蛋白。然而CD133的糖链结构不清已成为其在干细胞分选、功能研究和临床应用的瓶颈之一。我们前期结果已鉴定CD133的N-糖基化位点和部分位点的糖链结构，发现CD133依赖于N-糖链和半乳凝素-3相互作用，下调CD133表达抑制半乳凝素-3促神经干细胞迁移。本项目拟利用已成熟的糖链结构分析平台，解析CD133的N-糖链结构特征和调控机制，基于其结构寻找和CD133糖链相互作用的动物凝集素，并利用细胞和动物模型阐释CD133和动物凝集素相互作用在干细胞定向迁移的功能、机制和调控因素。本项目首次揭示出凝集素-CD133糖链-胞内信号的信号轴，为阐释微环境调控干细胞定向迁移的机制提供糖生物学的思路。

五跨膜糖蛋白CD133，作为一个被广泛应用于鉴定和分离正常干细胞、胶质瘤等肿瘤干细胞的分子标志物，在肿瘤干细胞的筛选和靶向性杀伤中具有重要作用。近年来的研究进一步发现干细胞、肿瘤干细胞的标志物CD133糖蛋白是调控细胞迁移等过程的关键蛋白。然而CD133的糖链结构不清已成为其在干细胞分选、功能研究和临床应用的瓶颈之一。本项目研究发现:解析CD133的N-糖基化位点、部分位点的N-糖链结构；发现调控其功能的关键糖基化位点并阐释其影响CD133促进肿瘤细胞增殖的机制；发现CD133 的 Asn-548 位点突变抑制CD133 促进细胞增殖；CD133蛋白Asn-548位点突变，影响其与β-catenin相互作用；发现与CD133的N-糖链相互作用的动物凝集素，明确其相互作用的调控规律；阐释CD133和动物凝集素相互作用在干细胞定向迁移中的功能及机制,揭示出半乳凝素3-CD133糖链-胞内信号的信号轴。研究成果：研究成果发表J Biol Chem2篇、Oncotarget3篇；申请1项专利。培养博士研究生3名。学术交流：国内糖生物学术会议2次。