SARS-CoV N蛋白与MASP2的相互作用及生物意义

【作者】 高婷；

【导师】 曹诚；

【作者基本信息】 中国人民解放军军事医学科学院 ， 遗传学， 2011， 博士

【摘要】 SARS即严重急性呼吸综合症,自2002年底我国广东报道首例患者以来,在不到半年时间内已在全球范围内造成8000余人感染,700余人死亡,致死率约10%。临床数据显示,SARS冠状病毒可以引起机体免疫系统过度激活,从而导致急性炎症。严重的急性呼吸窘迫症引起的呼吸衰竭是病人致病致死的主要原因。目前对SARS冠状病毒研究的进展多集中于细胞受体、抗体和疫苗开发等方面,但并未找到SARS导致炎症过激,尤其是严重肺部急性炎症的主要原因。本室前期工作通过酵母双杂交筛选到可能和SARS-CoV N蛋白相互作用的MASP2蛋白。MASP2是补体系统凝集素途径的主要丝氨酸蛋白酶,当MBL识别病原糖基并被激活后,MBL通过改变构象使MASP2自激活。活化形式的MASP2先后水解C4、C2形成C3转化酶,启动补体活化,发挥杀伤靶细胞的作用。补体活化中产生的补体片段具有趋化和调理作用,激活免疫细胞,释放炎症因子,引起局部和全身炎症。本研究试图通过研究SARS-CoV N蛋白和MASP2的相互作用,从分子水平解释SARS对机体免疫系统、尤其是炎症发生发展方面的影响,进而揭示部分SARS致病机理,为病毒防治和药物研究提供理论依据。SARS-CoV N蛋白可以被释放至患者血清,是SARS病人患病早期的诊断指标之一。本研究首先通过免疫共沉淀证实SARS-CoV N蛋白和人MASP2在体外存在相互作用,且其相互作用依赖于Ca2+。免疫共沉淀实验进一步证实,SARS-CoV N蛋白可以和人血清以及小鼠血清中的MASP2结合。之后我们构建了相关蛋白的截短突变体,通过免疫沉淀确认了SARS-CoV N蛋白和MASP2结合的结构域为SARS-CoV N蛋白的RNA结合结构域和MASP2的CUB1-EGF-CUB2结构域,后者与MASP2自激活密切相关,由此推测SARS-CoV N蛋白可能影响MASP2的自激活过程。为了在体外直接研究SARS-CoV N蛋白对MASP2功能的影响,本研究通过强阳离子交换和分子筛纯化了SARS-CoV N蛋白,通过原核表达和包涵体复性获得了MASP2蛋白,从人血浆中使用亲和层析获得了MBL蛋白。通过免疫沉淀发现SARS-CoV N蛋白和MASP2的相互作用促进了MASP2同源二聚体的形成,尤其在MBL和甘露聚糖共同参与的情况下,SARS-CoV N蛋白显著促进MASP2的二聚化。通过酶联免疫吸附实验(ELISA),使MBL结合于微孔板上,检测与之结合的MASP2,发现SARS-CoV N蛋白促进了MASP2和MBL结合。利用体外生化体系,使纯化的MASP2和SARS-CoV N或甘露聚糖活化的MBL在特定离子环境中反应,使用western blot检测MASP2自激活产生的水解片段,发现SARS-CoV N蛋白促进了MBL依赖的MASP2自激活。将纯化的补体C4加入MASP2激活体系,用western blot检测C4水解片段,发现SARS-CoV N蛋白可以促进MASP2水解C4,在MBL和甘露聚糖存在时尤为明显。之后使用C4b沉积法分析MBL-MASP2复合物的活性,发现SARS-CoV N蛋白可以加强血清来源的MBL-MASP2的活性。为了证实SARS-CoV N蛋白在人血清中也能发挥上述生化反应中的作用,本研究使用体外人血清体系作为MBL、MASP2和补体的原料库和反应场所,通过使用C1q-depleted血清排除经典途径,通过凝集素途径和替代途径对离子需求的不同来分离这两个途径,利用补体沉积实验发现SARS-CoV N蛋白可以通过凝集素途径促进补体成分C3活化和C5b-9生成,而且比正常的活化程度提高一倍以上。为了进一步证实SARS-CoV N蛋白在加强炎症方面的作用,本研究构建了致炎小鼠模型。用腺病毒载体在动物体内表达SARS-CoV N蛋白,检测动物血清中N蛋白含量可以达到SARS患者的相当水平。通过补体成分沉积实验确认LPS可以使补体C4、C3活化,补体抑制剂C1INH和抗MASP2抗体对其有抑制作用。因此LPS可以激活补体途径,并以凝集素途径为主。LPS可以诱导小鼠血清中的补体系统尤其是凝集素途径活化,从而引起急性炎症。通过尾静脉给小鼠注射LPS,并对同一只动物连续割尾取血,发现动物血清中补体活化后下游产生的炎症因子LTB4的含量在1~2小时内明显升高,并于3小时后开始回落,5小时后降至初始值以下,C1INH和水飞蓟宾(silybin)对LTB4升高有抑制,且后者在连续用药后效果显著。在动物体内提前表达SARS-CoV N蛋白并通过尾静脉注射LPS激活动物补体系统,发现表达SARS-CoV N蛋白的动物,其血清中LTB4的含量较对照动物提高至少一倍,C1INH和水飞蓟宾对此现象有抑制作用。将动物分组后分别注射表达SARS-CoV N蛋白的腺病毒和腺病毒,并用LPS诱导急性炎症,定时观察动物存活情况,发现表达SARS-CoV N蛋白的小鼠被LPS诱导急性炎症后,14小时内全部死亡,而注射空病毒的动物70%存活,水飞蓟宾对动物有保护作用。肺部病理切片证实表达SARS-CoV N蛋白的小鼠在LPS诱导急性炎症后发生了更严重的肺损伤,肺泡壁增厚,多形核白细胞计数显示白细胞浸润明显加强,而单表达SARS-CoV N蛋白或单用LPS刺激都没有如此严重的损伤,水飞蓟宾对症状有缓解作用。本研究发现了SARS冠状病毒核衣壳蛋白可以和血清中的免疫系统相关蛋白相互作用并影响其功能,并从分子水平部分解释了SARS引起过激炎症反应、尤其是包括肺损伤在内的全身炎症的结构基础和反应机制,为研究SARS致病机制及天然免疫系统与病毒的相互作用提供了依据,对于SARS的防治药物研究具有参考价值。更多还原

【Abstract】 Since the first case was reported in late 2002 in Guangdong Province, China, Severe Acute Respiratory Syndrome (SARS) infected more than 8000 people and caused more than 700 deaths in the world in the following months with a mortality rate of nearly 10%. Clinical presentation showed that the SARS coronavirus (SARS-CoV) induced severe activation of the host immune system and acute inflammation. Respiratory insufficiency leading to acute respiratory distress syndrome (ARDS) and respiratory failure was the main cause of death among fatal cases of SARS. The majority of recent studies have focused on SARS-CoV-associated cellular receptors, which mediate the infection of target cells via the surface protein of SARS-CoV. Research on virucidins and vaccines for SARS has also progressed rapidly. However, the main cause of severe acute inflammation, especially severe pulmonary inflammation, remains unclear.In our previous study, we showed with yeast two-hybrid experiments using SARS-CoV nucleocaspid (N) protein as bait protein that MBL-associated serine protease 2 (MASP2 )potentially interacts with SARS-CoV N protein. MASP2 is a main serine protease of the lectin pathway in the complement system. After mannan-binding lectin(MBL) identified surface carbohydrates of pathogens, MBL undergoes a conformation change that results in the auto-activation of MASP2. Then, activated MASP2 is able to hydrolyze complement C4 and C2, successively. C4 and C2 subsequently generate C3 convertase, thereby initiating the downstream reaction of complement activation.We endeavored to reveal the effects of SARS-CoV on the severe immune response and acute inflammation of the host by studying the interaction between SARS-CoV N protein and MASP2. With these studies, we would be able to make a contribution to antiviral drug development.We found that SARS-CoV N protein interacts with MASP2 in vitro by immunoprecipitation, and this interaction depends on the presence of Ca2+. We hypothesize that SARS-CoV N protein binds to MASP2 in the serum of SARS patients because SARS-CoV N has been found high levels in serum, and could be used as an early diagnostic marker for SARS. We confirmed this result by immunoprecipitation in both human serum and mouse serum; therefore, we could establish the mouse as an animal model for subsequent functional studies. Next, we constructed shortened mutants, and we found that the RNA-binding domain of SARS-CoV N protein and the CUB1-EGF-CUB2 domains of MASP2 interacted with one another, and the latter was important for MASP2 autoactivation. As a result, SARS-CoV N might play a role in MASP2 autoactivation.To directly study the effect of SARS-CoV N protein on MASP2 function in vitro, we purified the SARS-CoV N protein by strong acidic cation exchange and molecular sieve chromatography. We obtained pure MASP2 protein by prokaryotic expression and inclusion-body protein renaturation, and we obtained MBL protein from human plasma by affinity chromatography. We found that the SARS-CoV N protein helps MASP2 engage in homodimerization by its interaction with MASP2, especially in the presece of MBL and mannan. We detected MASP2 binding to MBL-coated plates by Enzyme-linked immunosorbent assay(ELISA), and we determined that the SARS-CoV N protein induced binding between MASP2 and MBL. We added purified MASP2, SARS-CoV N protein, and mannan-activated MBL to an in vitro biochemical reaction system with the necessary ions, detected the hydrolyzed MASP2 by western blotting, and found that the SARS-CoV N protein helps MBL activate MASP2. Next, we added purified C4 to the reaction system, and in the same way, we found that C4 hydrolysis was enhanced by the SARS-CoV N protein, especially in the presence of MBL and mannan. Then, we detected serum MBL-MASP2 function by C4b-deposition assay, and we confirmed that the SARS-CoV N protein had an enhancing effect on MBL-MASP2 activity.To extend the findings regarding the SARS-CoV N protein to the human serum system, we used C1q-depleted human serum as a complement pool to eliminate the classic pathway, and we used different reaction buffers to separate the MBL pathway and the alternative pathway. Then, we detected activated C3 and C5b-9 in the solid phase by a complement deposition assay and found that activation of the MBL pathway was almost twice that of its normal activity in the presence of SARS-CoV N protein.To confirm the effects of the SARS-CoV N protein on the host immune system, we used an inflammatory mouse model. We used an adenoviral vector to express SARS-CoV N protein in mice and found that the level of foreign SARS-CoV N protein in mouse serum was nearly the same as in SARS patient serum by western blotting and ELISA. Furthermore, we demonstrated that LPS could activate complement C4 and C3, while both C1INH and MASP2 antibodies had inhibitory effects. After activating the mouse complement system by tail vein injection of LPS, we obtained blood samples by cutting the tail each hour. We quantified the levels of the inflammatory factor LTB4, which is produced by the downstream reaction of complement activation. We found that LTB4 levels peaked in the first 1~2 hours and began to diminish after 3 hours. After 5 hours, LTB4 was at a level lower than normal concentrations. C1INH and silybin inhibited the generation of LTB4, and silybin was more effective when used more than once. After expressing the SARS-CoV N protein and injecting LPS through the tail vein, we found that the levels of LTB4 in mouse serum doubled when we expressed SARS-CoV N protein, while C1INH and silybin still inhibited the production of LTB4. Then, we grouped the animals, injected adenovirus-SARS-CoV N or adenovirus-null (as a control), used LPS as a activator of the complement system, recorded the number of live mice, and found that LPS-treated mice that expressed SARS-CoV N protein died within 14 hours, while 70% of the LPS-treated mice previously infected by adenovirus-null were still alive after 24 hours; in addition, silybin could protect animals from death. Pulmonary histopathological examinations showed that mice expressing SARS-CoV N protein suffered severe pulmonary injury, with thickened alveolar walls and increased white blood cell count. These symptoms were much more serious than in mice treated only with LPS or with adenovirus-SARS-CoV N, while silybin could relieve the symptoms.We found for the first time that the SARS-CoV nucleocaspid protein interacts with serum proteins associated with the immune system and affects the activation of complement system. Our study revealed one mechanism by which SARS causes severe inflammation and pulmonary injury at the molecular level. Furthermore, we obtained intriguing results with our serum reaction system and animal models. This study may offer clues to the pathogenicity of SARS and the interaction between the host innate immune system and the virus while also contributing to knowledge regarding major infectious disease control and prevention. .更多还原