【作者】 马青原；

【导师】 高宏伟；

【作者基本信息】 吉林大学 ， 基础兽医学， 2008， 博士

【摘要】 蓖麻毒素(ricin),又称为蓖麻毒蛋白。由A和B两条多肽链组成,其间通过二硫键相连接。A链(ricin A chain, RTA)是活性链,具有N-糖苷酶活性,可催化真核细胞核糖体28S rRNA第4324位腺嘌呤发生脱嘌呤作用,从而抑制蛋白质的合成。B链(ricin B chain, RTB)具有凝集素活性,能够识别和结合细胞表面末端含有半乳糖结构的受体,并协助A链进入细胞。一个毒素分子进入细胞内,就足以使整个细胞的蛋白质合成完全停止而死亡。一般认为,蓖麻毒素进入细胞涉及到一系列的步骤:1、通过B链与细胞表面含有β-1,4糖苷残基的糖蛋白和糖脂结合;2、通过内吞进入细胞;3、进入早期内涵体;4、通过囊泡转运,毒素进入高尔基体反式外侧网络(TGN);5、逆向转运进入内质网;6、RTA和RTB间的二硫键断裂;7、部分展开折叠的RTA通过Sec61p易位子穿过内质网膜;7与核糖体相互作用,催化脱嘌呤作用。近期研究表明,由于蓖麻毒素没有专一性的受体,故毒素可能通过多种途径进入细胞内,而且在细胞中的逆向转运的各个过程中,可能也存在不止一种机制起作用。本研究利用经典的MTS检测、免疫共沉淀和RNAi等技术研究了α甘露糖苷酶样内质网降解增强蛋白2(ER degradation enhancingα–mannosidase-like protein,EDEM2)、ADP-糖基化因子相关蛋白1(ADP-ribosylation factor-related protein 1,ARFRP1)和ERp29对于蓖麻毒素在细胞内逆向转运过程中的作用。通过研究我们发现:过量表达EDEM2后提高了细胞对蓖麻毒素的抵抗能力,检测发现细胞的蓖麻毒素的总量并没有改变。细胞转染EDEM2后,用嘌呤霉素处理的细胞对蓖麻毒素的敏感性明显提高。免疫共沉淀结果显示,嘌呤霉素处理后,更多的蓖麻毒素与EDEM2发生作用,也有更多的蓖麻毒素通过Sec61p蛋白通道进入胞浆。Kifunensine抑制EDEM2与错误折叠蛋白的作用后,显著地提高了蓖麻毒素A链的逆向转运。免疫共沉淀证明,kifunensine能够显著提高蓖麻毒素与EDEM2以及蓖麻毒素与Sec61α的相互作用。转染ARFRP1(wt)和ARFRP1(Q97/L)后,细胞对毒素的敏感性明显增强,转染ARFRP1(T31/N)后,细胞抵抗毒素的能力有一定的提高。同时,转染后的细胞中蓖麻毒素的总量没有明显变化。用BFA处理细胞后,蓖麻毒素对细胞的毒性显著降低,但转染ARFRP1和ARFRP1(Q97/L)的细胞对毒素的敏感性依然较高。转染后的细胞加入蓖麻毒素后低温诱导培养24h后,细胞毒性检测结果表明。低温诱导对表达ARFRP1和ARFRP1(T31/N)的细胞影响更大。过量表达ERp29蛋白后,CHO-K1细胞对蓖麻毒素的敏感性明显提高,内质网中糖基化的蓖麻毒素A链明显增加。而抑制细胞内ERp29蛋白的表达后,细胞抵抗蓖麻毒素的能力升高,与对照细胞相比,进入内质网的糖基化的蓖麻毒素的量相对减少。免疫共沉淀结果显示,过量表达ERp29蛋白后,有更多的蓖麻毒素与Sec61α相互作用,而干扰ERp29蛋白表达后,与Sec61α作用的蓖麻毒素相应减少。本研究首次证实EDEM2、ARFRP1及ERp29影响蓖麻毒素细胞内转运过程,尤其是国内外迄今仍未见有过ERp29参与蛋白质毒素逆向转运的相关报道。更多还原

【Abstract】 Ricinus communis is belong to the Euphorbiaceae Ricinus, which is the one of top ten oil crops. Our contry is one of major cultivated region. The area which cultiveated caster bean plant is seven million Mu, and harvests 300 thousand ton castor bean every year, which is second place in the world. According to statistics, the quantity which castor bean been usea to product castor oil is one million ton. There have 5% ricin in the waste left over from processing castor beans. Ricin is one of glycoprotein, and a prototype ribosome-inactivating protein. The tertiary structure of ricin was shown to be a globular, glycosylated heterodimer in which ricin A chain and ricin B chain are joined by a single disulphide bond. Ricin A chain is an RNA-specific N-glycosidase that hydrolytically cleaved the N-glycosidic bond of the adenine residue at position 4324 (A4324) within the 28S rRNA of eukaryotic cell and fatally disrupted protein synthesis in the end. Ricin B chain is an agglutinant, which can identify and bind terminal galactose residues on cell surface components. Ricin is one most toxin in the native poison up to now. It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition.Ricin can use many molecules, channels and mechanisms of target cells. The ready availability of ricin, coupled to its extreme potency when administered intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeutically, its cytotoxicity has encouraged the use of ricin in‘magic bullets’to specifically target and destroy cancer cells, and the unusual intracellular trafficking properties of ricin potentially permit its development as a vaccine vector.Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell surface glycolipids or glycoproteins havingβ-1,4-linked galactose residues; (ii) uptake into the cell by endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB; (vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum (ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that, once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically active conformation; and (x) interaction with the ribosome to catalyse the depurination interaction .For no special receptor on the cell surface, ricin can exploit several mechanisms of internalization. As endocytosis, more than one mechanisms are involved in the transport of ricin.In the studys, we investigated the role of EDEM2,ARFRP1 and ERp29 in the retrotranslocation of ricin in the cell by the classic MTS method, co-immunoprecipitation and RNAi.In research to the influence of EDEM2 in retrotranslocation of ricin, HEK293T cells was transiently transfected with pRK7-EDEM2-HA by Lipofectamine 2000 according to manufacturer`s procedure. Three days posttransfection, cells were grown in complete medium DMEM supplemented with 30μg/mL ricin under 5% CO2 in a 37℃incubator for 24h. Optionally, the cells were preincubated with inhibitors(lactose(0.1mol/L),Kifunensine(0.2mmol/L)or puromycin(0.5mg/L)respectively) for 30 min before ricin was added. After extracted total protein in the cells, the sample were detected by western Blot, and a part of sample were immunoprecipitated using mouse anti-HA, mouse anti-RTA or rabbit anti-Sec61αantibody. Moreover, we got RTA in the cytosol by cells` permeabilization using KOAc containing 3μg/mL digitonin, and detected the change of RTA quantity in the cytosol. For investigation the cytotoxicity change influenced by EDEM2, HEK293T cells(1×104 cells in each well) were seeded in 96-well plates and transfected with pRK7-EDEM2-HA vector, then added ricin(concetrations of ricin were 100,10,1,0.1 or 0.01μg/mL, respectively) as before. After the incubation period, added 10μL of the MTS to each well and incubated the microtiter plater for 4h in a humidified atmoshpere. Then measure the spectrophotometrical absorbance of the asmples using a microtiter plate (ELISA) reader. The wavelength to measure absorbance of the product is 490 nm.In research to the effect of ARFRP1 in the retrotranslocation of ricin, CHO-K1 cells was transiently transfected with pcDNA3-ARFRP1(wt)-HA, pcDNA3-ARFRP1(Q97/L)-HA or pcDNA3-ARFRP1(T31/N)-HA by Lipofectamine 2000 according to manufacturer`s procedure. Three days posttransfection, cells were grown in complete medium F12-K supplemented with 10μg/mL ricin under 5% CO2 in a 37℃incubator for 24h. there was one group was preincubated with 2μg/L brefeldin A for 30 min before ricin was added. Other group was incubated in a 4℃incubator for 24h after ricin was added. The detected of cytotoxicity was operated as before.In research to the interaction between the ERp29 and ricin in the retrotranslocation of ricin, we amplified ERp29 gene from the liver of Harlan Sprague-Dawley rat by RT-PCR, the primers using in the RT-PCR were : ERp29-1:5`-AAgCTTATggCCgCCgCCgCCggggTg-3`, ERp29-2:5`-CTCgAgCAgCTCCTC CTTCTCAgCCTC-3`. Then we cloned the gene into the pMD18-T vector, and got the pMD18-T-ERp29 vector. The vector was identified by PCR, double enzyme digestio with HindⅢand Xho I and sequencing. The final we further got pcDNA3-ERp29-HA expression vector using the pcDNA3-HA vector. The result of identification of vector was positive.CHO-K1 cells was transiently transfected with pcDNA3-ERp29-HA. The ricin was recombinant ricin which ricin A chain with C terminal glycosylation sites. Then we detected the cytotoxicity by MTS and the change of quantity of ricin in the ER or cytosol. For further affirmation the role of ERp29 in the retrotranslocation of ricin. We used RNAi in study. At first, transfected-cells were selected with 400μg/mL G418. we got the stably transfected ERp29 cells and used them as control in later study. Two siRNA duplexes(siRNA1: 5`-AGUUCGUCUUGGUGAAGUU-3`, siRNA2:5`- AGCUGAACAUGGAGCUGAG-3`) with symmetric dTdT overhand were chemically synthesized by GenePharma. Transfections were performed using Lipofectamine 2000 transfection reagent according to the manufacturer`s preotocol. ERp29 silencing achieved its maximum after 6 days by RT-PCR and Western Blot identity. Then we done the cytotoxicity assay and studied the quantity of ricin in the ER or cytosol.By the experiment, we found that:There was a protection against ricin in EDEM-transfected cells, the interaction did not influced by lactose, meanwhile the total quantity of ricin in the cell did not changed. Ppuromycin was found to sensitize both control and EDEM-transfected cells to ricin. Importantly, in EDEM-transfected cells the observed sensitization was higher than the sensitization seen in mock-transfected cells which could be observed by the rncreased retratranslocation of ricin to the cytosol. Treatment of HA-EDEM2 transfected cells with puromycin resulted in a increase in the amount of ricin co-immunoprecipitated with anti-HA antibody whereas puromycin had an even larger effect on the amount of ricin co-immunoprecipitated with the Sec61αantibody (compared with the amount of ricin found in untreated EDEM-transfected cells). When inhibited the interaction between the EDEM2 and misfolded proteins by kifunensine, more ricin translocated in cytosol. So EDEM2 could promote the retranslocation of ricin under the kifunesine. After kifunensine treatment, not only was there an increased interaction of ricin with EDEM, but more ricin was coimmunoprecipitated with the Sec61 translocon. The results of our data suggested that EDEM2 was also important for ricin retrotranslocation from ER to the cytosol in a different way than the interaction between the EDEM2 and misfolded proteins.The sensibility of ARFRP1(wt)-/ARFRP1(Q97/L)-transfected cells was higher than the control cells, but cells could against ricin which were overexpressed the ARFRP1(T31/N). In all experiment, the amont of ricin did not changed. The cytotoxicity of ricin decreased by BFA treatment. Otherwise the cells transfected ARFRP1(wt) or ARFRP1(Q97/L) were more sensitive to ricin than control. The sensibilit of ARFRP1(wt)-/ARFRP1(T31/L)-transfected cells were greater influenced by the subzero induction. The result of our date suggested that ARFRP1 could promote the retrotranslocation of ricin in the cell.CHO-K1 cells which overexpressed ERp29 were more sensitive with ricin, more glycosylated-RTA was observed in the ER and cotosol. Cells could against ricin after interfered the expresion of ERp29, and the amont of ricin in the ER and cotosol notably degraded. More ricin co-immunoprecipitated with Sec61αin the ERp29-transfected cells, but less ricin interaction with Sec61αif interfered the expresion of ERp29. With our date, we presumed that ERp29 could promote ricin into ER and assist the processing and transport of ricin in the ER. Because we did not detected the intereaction between the ricin and ERp29, the mechanism of ERp29 accelerated retrotranslocation of ricin need deeply research.In this study, we investigated the role of EDEM1, ARFRP1 and ERp29 in the retrotranslocation of ricin in the cell. The results suggested that EDEM2 could promot ricin translocated from ER to cotosol when the intereaction between the EDEM2 and misfolded proteins. ARFRP1 accelerated the ricin transport between the endosome and TGN, the intereaction was more influenced by low temperature than inhibitor. ERp29 could facilitate ricin into ER, and assist the processing and translocation of ricin in the ER.This study confirmed the intereaction between the EDEM2, ARFRP1 or ERp29 with ricin respectively, especially we have seen any report about the role of ERp29 in the retrotranslocation of protein toxin.更多还原