本文選題：CD97 + 胞外域��； 參考：《中國(guó)科學(xué)院上海藥物研究所》2016年碩士論文

【摘要】：CD97來(lái)源于粘附型G蛋白偶聯(lián)受體(GPCR)的表皮生長(zhǎng)因子(EGF)-七次跨膜(7TM)受體家族,并參與腫瘤細(xì)胞的聚集、遷移和侵入過(guò)程。CD97胞外域(ECD)由以下三部分組成:3至5個(gè)相連的EGF模塊、1個(gè)誘導(dǎo)GPCR自動(dòng)蛋白水解(GAIN)區(qū)域和1個(gè)GPCR蛋白水解位點(diǎn)(GPS)。根據(jù)EGF模塊mRNA自動(dòng)剪接方式的不同,CD97分為CD97(1,2,5),CD97(1,2,3,5)和CD97(1-5)三個(gè)亞型。本課題通過(guò)質(zhì)粒構(gòu)建和蛋白表達(dá)純化的方法獲得CD97(1-5)ECD野生型、截短型、GPS突變型和N-糖基化位點(diǎn)突變型共14個(gè)蛋白。其中只有GAIN蛋白無(wú)法表達(dá),而EGF4-GAIN和EGF5-GAIN蛋白則成功表達(dá)。該現(xiàn)象暗示著GAIN區(qū)域的穩(wěn)定存在可能至少需要與第5個(gè)EGF模塊相互作用。結(jié)合Western blot和SDS-PAGE手段,首次發(fā)現(xiàn)CD97(1-5)ECD的GPS自動(dòng)水解呈濃度依賴(lài),提示其自動(dòng)水解機(jī)理可能為分子間作用,而這不同于CD97(1,2,5)的分子內(nèi)水解機(jī)理。這為粘附型GPCR的自動(dòng)蛋白水解機(jī)制研究提供幫助。為研究CD97(1-5)ECD結(jié)構(gòu)域間的相互作用關(guān)系,對(duì)野生型及去糖基的蛋白進(jìn)行了X-射線(xiàn)小角散射(SAXS)檢測(cè)和同源模建。結(jié)果表明,含糖基和去糖基的CD97(1-5)ECD的外形類(lèi)似一個(gè)小鏟子,而GAIN區(qū)域和EGF模塊分飾“鏟身”和“把手”的角色。其中,GAIN區(qū)域與第5個(gè)EGF模塊之間存在潛在的相互作用。此外,我們建立了CD97(1-5)ECD蛋白與HeLa細(xì)胞粘附的模型,以研究該蛋白結(jié)構(gòu)域?qū)eLa細(xì)胞粘附發(fā)揮的作用。運(yùn)用此模型研究發(fā)現(xiàn)CD97(1-5)ECD的第5個(gè)EGF模塊和GAIN區(qū)域均對(duì)HeLa細(xì)胞的粘附發(fā)揮重要作用;而GAIN區(qū)域的N-糖基化和GPS自動(dòng)水解則并非HeLa細(xì)胞粘附所必需。在評(píng)價(jià)CD97(1-5)ECD的N-糖基化和GPS水解作用對(duì)HeLa細(xì)胞粘附功能的影響上,我們運(yùn)用了糖苷酶PNGase F去糖基、N-糖基化位點(diǎn)突變和GPS斷裂位點(diǎn)突變這三種人為調(diào)控方法,發(fā)現(xiàn)它們對(duì)CD97(1-5)ECD蛋白與HeLa細(xì)胞的粘附產(chǎn)生了不同的影響。三種方法中只有N-糖基化位點(diǎn)突變保持了蛋白與HeLa細(xì)胞粘附的活性,說(shuō)明PNGase F去糖基和GPS斷裂位點(diǎn)突變可能造成了CD97(1-5)ECD蛋白的功能損傷,進(jìn)而影響其與HeLa細(xì)胞的粘附功能。因此,在研究糖基化和某些蛋白模塊對(duì)蛋白功能的影響時(shí),需注意選擇合適的調(diào)控方法進(jìn)行多方驗(yàn)證,才能真實(shí)準(zhǔn)確地反映研究對(duì)象所發(fā)揮的生物作用。綜上所述,本課題的研究揭示了CD97(1-5)亞型的胞外域蛋白與HeLa細(xì)胞粘附的分子基礎(chǔ),為進(jìn)一步了解CD97在腫瘤疾病中發(fā)揮的作用提供了幫助,并為調(diào)控腫瘤的發(fā)生發(fā)展提供可能的靶點(diǎn)。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)院上海藥物研究所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R96

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