CD97胞外区的结构域特征及其与HeLa细胞粘附的研究

发布时间：2018-05-20 02:25

本文选题：CD97 + 胞外域　；参考：《中国科学院上海药物研究所》2016年硕士论文

【摘要】：CD97来源于粘附型G蛋白偶联受体(GPCR)的表皮生长因子(EGF)-七次跨膜(7TM)受体家族,并参与肿瘤细胞的聚集、迁移和侵入过程。CD97胞外域(ECD)由以下三部分组成:3至5个相连的EGF模块、1个诱导GPCR自动蛋白水解(GAIN)区域和1个GPCR蛋白水解位点(GPS)。根据EGF模块mRNA自动剪接方式的不同,CD97分为CD97(1,2,5),CD97(1,2,3,5)和CD97(1-5)三个亚型。本课题通过质粒构建和蛋白表达纯化的方法获得CD97(1-5)ECD野生型、截短型、GPS突变型和N-糖基化位点突变型共14个蛋白。其中只有GAIN蛋白无法表达,而EGF4-GAIN和EGF5-GAIN蛋白则成功表达。该现象暗示着GAIN区域的稳定存在可能至少需要与第5个EGF模块相互作用。结合Western blot和SDS-PAGE手段,首次发现CD97(1-5)ECD的GPS自动水解呈浓度依赖,提示其自动水解机理可能为分子间作用,而这不同于CD97(1,2,5)的分子内水解机理。这为粘附型GPCR的自动蛋白水解机制研究提供帮助。为研究CD97(1-5)ECD结构域间的相互作用关系,对野生型及去糖基的蛋白进行了X-射线小角散射(SAXS)检测和同源模建。结果表明,含糖基和去糖基的CD97(1-5)ECD的外形类似一个小铲子,而GAIN区域和EGF模块分饰“铲身”和“把手”的角色。其中,GAIN区域与第5个EGF模块之间存在潜在的相互作用。此外,我们建立了CD97(1-5)ECD蛋白与HeLa细胞粘附的模型,以研究该蛋白结构域对HeLa细胞粘附发挥的作用。运用此模型研究发现CD97(1-5)ECD的第5个EGF模块和GAIN区域均对HeLa细胞的粘附发挥重要作用;而GAIN区域的N-糖基化和GPS自动水解则并非HeLa细胞粘附所必需。在评价CD97(1-5)ECD的N-糖基化和GPS水解作用对HeLa细胞粘附功能的影响上,我们运用了糖苷酶PNGase F去糖基、N-糖基化位点突变和GPS断裂位点突变这三种人为调控方法,发现它们对CD97(1-5)ECD蛋白与HeLa细胞的粘附产生了不同的影响。三种方法中只有N-糖基化位点突变保持了蛋白与HeLa细胞粘附的活性,说明PNGase F去糖基和GPS断裂位点突变可能造成了CD97(1-5)ECD蛋白的功能损伤,进而影响其与HeLa细胞的粘附功能。因此,在研究糖基化和某些蛋白模块对蛋白功能的影响时,需注意选择合适的调控方法进行多方验证,才能真实准确地反映研究对象所发挥的生物作用。综上所述,本课题的研究揭示了CD97(1-5)亚型的胞外域蛋白与HeLa细胞粘附的分子基础,为进一步了解CD97在肿瘤疾病中发挥的作用提供了帮助,并为调控肿瘤的发生发展提供可能的靶点。
[Abstract]:CD97 is derived from the EGF-7TM7 receptor family of Adhesion G protein coupled receptor (GPCR) and is involved in the aggregation of tumor cells. Migration and invasion. CD97 ECD97 consists of three parts: 3 to 5 connected EGF modules, 1 GPCR automatic protein hydrolysate region and 1 GPCR protein hydrolysis site. According to the mRNA automatic splicing mode of EGF module, CD97 can be divided into three subtypes: CD97, CD97 and CD97, respectively. In this study, the wild type of CD97(1-5)ECD was obtained by plasmid construction and protein purification, and 14 proteins were obtained by truncated CD97(1-5)ECD mutant and N-glycosylation site mutation. Only GAIN protein could not be expressed, while EGF4-GAIN and EGF5-GAIN proteins were successfully expressed. This phenomenon suggests that the existence of stability in the GAIN region may require interaction with at least the fifth EGF module. In combination with Western blot and SDS-PAGE, it was found for the first time that the automatic hydrolysis of GPS of CD97(1-5)ECD was concentration-dependent, suggesting that the mechanism of automatic hydrolysis of CD97(1-5)ECD might be intermolecular, which was different from the mechanism of intramolecular hydrolysis. This will be helpful for the study of automatic proteolysis mechanism of adherent GPCR. In order to study the interaction between CD97(1-5)ECD domains, the wild type and desglycosylated proteins were detected by X- ray small angle scattering (SAXS) and homologous modeling. The results show that the shape of CD97(1-5)ECD containing sugar group and deglycosyl group is similar to that of a small shovel, while the GAIN region and the EGF module are divided into the roles of "shovel" and "handle". There is a potential interaction between the Gain region and the fifth EGF module. In addition, we established a model of CD97(1-5)ECD protein adhesion to HeLa cells to study the role of the protein domain in HeLa cell adhesion. Using this model, it was found that both the fifth EGF module and the GAIN region of CD97(1-5)ECD played an important role in the adhesion of HeLa cells, while the N-glycosylation of GAIN region and the automatic hydrolysis of GPS were not necessary for HeLa cell adhesion. In order to evaluate the effect of N-glycosylation and GPS hydrolysis of CD97(1-5)ECD on the adhesion function of HeLa cells, we used three artificial regulation methods: PNGase F deglycosylation site mutation and GPS break site mutation. It was found that they had different effects on the adhesion of CD97(1-5)ECD protein to HeLa cells. Among the three methods, only N- glycosylation site mutations maintained the activity of protein adhesion to HeLa cells, indicating that PNGase F deglycosylation and GPS break site mutations may cause functional damage of CD97(1-5)ECD protein and further affect its adhesion function to HeLa cells. Therefore, when studying the effects of glycosylation and some protein modules on protein function, we should select appropriate regulation methods to verify the biological function of the research object. To sum up, our study revealed the molecular basis of the adhesion of CD971-5) extracellular domain proteins to HeLa cells, which may be helpful to further understand the role of CD97 in tumor diseases. It also provides a possible target for the regulation of tumor occurrence and development.
【学位授予单位】：中国科学院上海药物研究所
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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