Study On Lysosomes Degradation Of Ricin A Chain

[Purposes]Ricin toxin (RT) is a plant toxin isolated from the seeds of Ricinus Communis. It is one of the most powerful plant toxins discovered. The toxin protein consists of two chains linked by a disulfide bond. Ricin A chain (RTA) is an active chain and has specific N-glycosidase activity, which excises a specific adenine residue (A4324 in rat) from a highly conserved loop of 26 or 28S rRNA in 60S ribosomal subunits. This cleavage of adenine can lead to the disruption of ribosomal function, thereby, inhibits the protein synthesis and then causes the death of cells. Ricin B chain (RTB) can specifically recognize receptors on the cell membrane, which have a terminal galactose. RTB is responsible for the binding to the cell receptors and may facilitate the translocation of RTA. Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular compartments.Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular organdies. In this study, a lysosomes retention signal KFERQ was fused to the C-terminus of ricin (RTA) by polymerase chain reaction. The recombinant RTA and RTA-KFERQ were expressed in Escherichia coli using plasmid pKK223.3 under the control of a tac promoter. The recombinant proteins were purified by affinity chromatography on a Blue-Sepharose 6B column. The cytotoxicities of RTA and the fusion toxin RTA-KFERQ were measured by the MTT assay in HEPG2, Hela, and A549 cells following fluid-phase endocytosis.[Methods]1. Expression and purification of recombinant RTAsThe expression plasmid pKK223.3-RTA was introduced into E. coli JM 109 by CaCb-medicated method. The cells were grown until an optical density at 600 nm reached 0.6. Expression was induced in the presence of IPTG to a final concentration of 1 mM and incubation with shaking at 30°C for 3 h. In order to evaluate the effect of temperature on expression of rRTAs, we have investigated the distribution of the recombinant proteins expressed at different temperatures. To identify an optimal length of the incubation, the bacteria were induced at 30°C for 1 h, 3 h and 5 h respectively. The distribution of the recombinant proteins was analyzed on 12% SDS-PAGE. After induced by 0.1 M IPTG, the pellet was resuspended in phosphate-buffered saline (PBS)/5 mM EDTA, and sonicated three times for 20 s at maximal output. Debris was removed by centrifugation at 15000 rpm for 15 min at 4℃. The supernatants were dialyzed against 50 mM phosphate buffer (PB) (pH 6.5), and recombinant RTAs (rRTAs) were affinitively purified on a Blue-Sepharose 6B column by elution with 0.65 M sodium chloride. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to check the purity. The fractions containing the rRTAs were collected, and the protein concentration was measured by Bradford method.2. Cytotoxicity AssayHEPG2, Hela and A549 cells were planted out in 96-well plates at a density of 1.2 x 10~4 cells per well in the serum-free RPMI-1640 medium. Various concentrations of rRTA and rRTA-KFERQ were added into the wells and incubated for 8 h to allow internalization of toxins by fluid-phase endocytosis. The cells were then incubated in RPMI-1640 medium containing 10% fetal calf serum for another 48 h. Cell viability was measured by MTT assay. In this assay, trtrazolium salt (MTT) is converted to a blue formazan product by enzymes active only in living cells. The amount of formazan produced can be quantitated using a microtitre plate reader. In order to get precise data, each concentration was set for 5 wells.[Results]1. Expression of recombinant RTA and RTA-KFERQExpression of rRTA and rRTA-KFERQ was induced with 0.1 mM IPTG at 30℃ for 3 h. SDS-PAGE showed that the target proteins with approximate molecular weight, 31 kD could be detected in the lysates of the bacteria. The rRTA induced at 30℃ for 3h was soluble and fully active. In comparison to rRTA, a major part of rRTA-KFERQ was expressed in a soluble style at 30℃ for 3h, whereas that expressed...