蛋白特异性糖基化修饰的标记影像以及利用糖链为肿瘤细胞提供免疫治疗

发布时间：2018-10-16 11:33

【摘要】：糖基化修饰在生物体内具有极大的普遍性和重要性,糖基化修饰的蛋白占细胞内蛋白总量超过了70%,蛋白表面的糖链修饰与其结构、功能等均存在密切的关系。在以前的糖生物学研究中,较多的关注于蛋白整体的糖基化水平,而对于蛋白特异性的糖基化修饰关注的较少。目前,越来越多的研究显示,在生物学进程中,特定蛋白上的特异性糖基化比整体水平的糖基化更具有生物学意义。然而,如何能够直接在细胞水平标记特定蛋白的特异性糖基化修饰的技术手段还有待不断地开发。因为这存在两大难点：1、相比于磷酸化、乙酰化等蛋白翻译后修饰,糖基化修饰的糖链结构更加的复杂；2、糖链的低免疫原性限制了针对特定位点糖基化修饰的高效抗体的开发。为了进一步克服这些限制,我们利用代谢标记和邻位连接技术联合应用(METPLA)的策略来标记和影像特定蛋白的特异性糖基化修饰。这个策略中,糖链和靶标蛋白分别被连接上两条寡核苷酸,当这两条寡核苷酸的距离40nnm时,两者之间发生滚环复制反应,最终使标记的荧光达到在显微镜下可见的量,从而影像特定蛋白上的特异性糖基化修饰。我们利用该策略提供了一种新的方法,能够直接在细胞水平对各种不同类型的糖基化进行影像,并且在细胞动态变化过程中检测一些细胞膜受体蛋白的二聚体状态。利用机体的免疫系统去靶向和清除肿瘤细胞具有特异性和高效性,是一种理想的治疗策略。该策略通过招募人血清中天然存在的抗体到肿瘤细胞表面,从而引起补体依赖的细胞毒性(CDC)、抗体依赖的细胞毒性(ADCC)或抗体依赖的细胞吞噬(ADCP)去清除肿瘤细胞。但是这种传统的方法存在一些固有的缺陷：相比于共价结合的方式,配体-受体的非共价结合方式相对较弱并且不稳定；另外,肿瘤细胞表面本身的受体数量是有限的,这会导致受体-配体的结合达到饱和而导致靶向性的降低；最后,配体-受体的结合,可能会诱导细胞对结合在其表面的分子进行内吞作用,也将导致免疫应答效率的下降。对此,我们提出了一种新型的策略,联合糖代谢工程和双正交化学反应,通过控制糖链代谢的量,从而控制在肿瘤细胞表面带上的鼠李糖(Rha)表位的量。肿瘤细胞表面的Rha表位的存在有效地招募人血清中的Rha抗体,高效地引发补体介导的细胞毒性。
[Abstract]:Glycosylation modification has great universality and importance in organism. The amount of glycosylated protein is more than 70%. The sugar chain modification on the surface of protein is closely related to its structure and function. In previous studies of glycosylation, more attention was paid to the glycosylation level of the whole protein, but less attention was paid to the glycosylation modification of protein. At present, more and more studies have shown that the specific glycosylation of specific proteins has more biological significance than the whole level of glycosylation in the biological process. However, how to label specific glycosylation modification of specific proteins directly at the cell level has yet to be developed. There are two difficulties: 1. Compared with post-translational modification of proteins such as phosphorylation and acetylation, glycosylation modifies the structure of sugar chains more complex; 2. The low immunogenicity of sugar chains limits the development of highly efficient antibodies for glycosylation of specific sites. In order to overcome these limitations, we used the strategy of (METPLA) to label and image the specific glycosylation modification of specific proteins. In this strategy, the sugar chain and the target protein are connected to two oligonucleotides respectively. When the distance between the two oligonucleotides is 40nnm, a rolling loop replicating reaction occurs between the two oligonucleotides, resulting in the labeled fluorescence reaching the level visible under the microscope. Thus, the specific glycosylation modification on a specific protein is imaged. We use this strategy to provide a new approach to image different types of glycosylation directly at the cellular level and to detect the dimer status of some cell membrane receptor proteins during cell dynamics. It is an ideal therapeutic strategy to use the immune system to target and clear tumor cells. This strategy can induce complement dependent cytotoxic (CDC), antibody dependent cytotoxic (ADCC) or antibody dependent cell phagocytosis of (ADCP) to remove tumor cells by recruiting natural antibodies in human serum to the surface of tumor cells. However, the traditional method has some inherent drawbacks: the non-covalent binding of ligand-receptor is relatively weak and unstable compared with covalent binding, and the number of receptors on the surface of tumor cells is limited. This may lead to saturation of receptor-ligand binding and decrease in targeting. Finally, ligand-receptor binding may induce endocytosis of molecules binding to its surface, and decrease the efficiency of immune response. A new strategy is proposed to control the amount of rhamnose (Rha) epitopes on the surface of tumor cells by controlling the amount of sugar chain metabolism in combination with glycometabolism engineering and biorthogonal chemical reaction. The presence of Rha epitopes on tumor cells can effectively recruit Rha antibodies from human serum and efficiently trigger complement mediated cytotoxicity.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.51

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