Toxic Effect Of Ricin And Screening For Interaction Components With Intestinal Brush Border Membrane Proteins

Ricin (RT) is the most toxic natural poisons of plant proteins up to now. One molecule ricin enters into cell can kill it as a consequence of protein synthesis inhibition. The toxicity of ricin relies on its unique structure, which contains two chains, RTA and RTB. RTA is the active chain with N-glycoside activity, it can cleave the N-glycosidic bond of a single adenine residue in a defined position of 28S rRNA (A4324) within 60S large subunit in the ribosome of eukaryotic cell, and hydrolyzes N-glycosidic bond of A4324 to remove adenine. 28s rRNA will be degraded because of the loss of RNase resistance and can not combine with EF-2, thereby deterring the formation of ribosome, EF-2, GTP complex to lead to the inhibition of protein synthesis. RTB with lectin activity is able to identify and combine the end of the receptors containing galactose structure on the cell surface to assist the RTA to enter into the cell. The present study found that ricin toxicity on cells is not only manifested as inhibition of protein synthesis, but also induced apoptosis of immune cells and played an extremely significant role in intestinal injury. In order to further reveal the toxicity mechanism of ricin, our subject start from the basic biological characteristics of ricin and discuss in-depth on the toxic effects and its related mechanism.Firstly, isoelectric point (PI) and molecular weight (MW) of purified ricin were analyzed by the capillary electrophoresis. It indicated that the PI and MW were 6.49 and 61 900, respectively. Due to the different places of production and growing conditions possibly, they differed slightly from the former reported results 7.1 and 64,000. Secondly, detected amino acid sequence of ricin, and 24 aa mutated compared with the sequence of former reported. Thirdly, detected the toxicity of ricin in mice, median lethal dose (LD50) was 5.0mg/kg by oral administration and 4.6μg/kg by intraperitoneal injection. At last, detected its cytotoxicity, lymphacytes were treated with different concentrations (0, 0.3, 3, 30 and 300μg/ml) of ricin and different handling durations (3, 6, 12 and 24h), which indicated that the cells showed proliferative response obviously at the first 3 hours and the viability was higher than the control groups, but the viability began to decrease after infecting 4 hours. At 0.3μg/ml of ricin when treated with higher concentration of ricin, the viability decreased obviously without the progress of proliferative response and the reduction extent was enhancing along with the lengthen of handling time. That showed the cytotoxic effect had concentration-depended and tine-depended. While, the IC50 of lymphacytes was 3μg/ml.The most obvious pathological changes were found in mice after oral administrated 72 hours. They showed depression, anorexia, lethargy shock and coma in clinical, with swollen stomach, obvious congestive reaction in duodenum, hydroabdomen, and intestinal lesions. HE staining showed that necrosis and falling of the intestinal epithelial cells, mesocaval fibrin leaked out the duodenum and jejunum, mucosa intrinsic layer and the central lymphatic lacteal expanded, there was the existence of thrombosis on the lamina propria and the grass-roots level, but there were no lesions in ileum. In addition, atrophy of spleen and thymus was apparent. Under the transmission electron microscope, apoptosis debris of lymphocyte could be seen clearly, in addition, the color of splenic corpuscle in spleen changed light, and some parts of the splenic corpuscle were intumescent, lymphocyte proliferated, multinucleated giant cells (hematopoietic stem cells) came out, and the thymus bleed. In the lesioned immune cells, heterochromatin showed the phenomena of margination, edge were thicker, the membrane structure came out inside of the nuclear, the chromatin around the nuclear presented crescent-shaped. Duodenal intestinal almost completely lost by observing with scanning electron endoscopic, it indicated the ricin had a strong role in intestinal injury.In the second experiment we could see the apoptotic immune cells, but do not make a decisive judgment to determine that this apoptosis was caused by ricin. So in this experiment, we began with the basic characteristics of apoptosis to further study the mechanism of apoptosis induced by ricin. It did dot contradict with the tradition theory that ricin as a protein synthesis inhibitor, and most likely, it might be the crucial complement to the existing apoptotic theories. Firstly, we used flow cytometry to detect mitochondrial membrane potential with DePsipherTM as a detection probe. Results showed 3μg/ml ricin treated cells for 3 h, and 0.3% H2O2 treated cells for 5 min as positive control. Generally, change of MMP determined by FCM couold be expressed by the ratio of red fluorescence intensity to green (Fig.3). In the present study, ratio of treated cells (2.37) was significantly lower than control cells (3.31) (p<0.5). Ratio deflection illustrated the beginning of the apoptosis. Mitochondrial membrane potential cells were an important indicator of apoptosis, once the membrane potential change, the entire apoptotic process was irreversible. At the 3μg/ml ricin treated cells for 6h and after Hoechst33258 staining, the treated cells shrinkage, chromatin condensation, nuclear brokedown and produced apoptotic bodies, membrane became blurred, incomplete, etc. In the DNA "ladder" detection, ricin-treated cells (3μg/ml ricin for 0h, 24h and 36h) showed typical internucleosomal DNA fragmentation or''ladder''formation at the two time points tested , which indicated that cell genomic DNA degradation, and the intensity of banding was more prominent at 36h, control cells showed a clear band without DNA fragmentation. Several reports had suggested that high ROS concentrations significantly inhibited cell growth and induced apoptosis, so we observed the ROS level in ricin-treated lymphocytes. 3μg/ml ricin treated cells for 15-120 min, and 0.3% H2O2 treated cells for 5min as positive control. After treated for 15min, ROS level increased about 3-fold compared with control groups. After 30min, it reached the maximum level, and no further increase at longer treating durations. Results showed that ROS level had intimated relationship with the treating durations. There was evidence indicated that ROS regulated metabolic processes by acting as intracellular messenger molecules and that ROS mediate specific signaling pathways by modulating gene expression through regulation of several transcription factors and activation of mitogen-activated protein kinases (MAPKs), in view of the cascade relationship between them, we exploited Western-blot to study the signal transpathway about apoptosis. MAPKs contained three major transpathways, extracellular-regulated kinase (ERK1/2), stress-activated protein kinase (P38), c-jun N-terminal kinase (JNK1/2). In order to identify the process of apoptosis was conducted by which transpathway, we did the blocking analysis by using their specific inhibitors (SP600125, SB-202190, U0126), and final result showed that ricin-induced lymphocyte apoptosis was possibly by activating P38/MAPKs.For the severe damage of ricin to small intestinal, we developed a series of research to further study this intoxic effect. On passive and/or active transport, most toxins or drugs developed for oral administration were absorbed in the gastrointestinal tract followed by systemic circulation. Because in vivo investigations were, time-consuming and often difficult to quantify the results, it was evident that the predicative in vitro modle was needed. In a present study, we described a novel method to estimate binding events occurring on intestinal membranes based on the technical platform of proteomics (2-DE, SPR, HPLC, HPCE-ESI-MS). After the extraction and purification of mouse intestinal brush border membrane proteins, we did the kinetics analysis based on SPR technology, it could rapidly offer valuable information on the rate and extent of adsorption, association/dissociation kinetics, and the affinity constants of specific ligand-receptor interactions. Results showed that the affinity kinetics (KA) was 1.5×107 , disassociation kinetics(KD)was 6.64×10-8 . This result provided a strong theoretical basis for the explanation of intestinal injury leaded by ricin. In the following test, we analyzed the complexity of brush border membrane protein according to 2-DE technology. Though it had been purified, its composition was still very complicated, so in order to avoid blind identification of a single point, we used SPR recovery unit to recover the interacted components with real-time detection by HPLC. Then, the mixed composition was identified by LC-MS-MS. Results showed that 28 proteins were interacted with ricin in brush border membrane, including the KDEL endoplasmic reticulum retention signal receptor, annexin, original upstream regulatory binding protein, vacuolar protein, etc. This result not only provide a new evidence and a research window for the mechanism of intestinal injury induced by ricin, also set up a new platform to clarify the specific toxic effect of ricin.