Effects Of KEEL/KDEL Sequence On Intracellular Transport Action Of Ricin

Many plants produce toxic proteins known as ribosome inactivating proteins (RIPS) whose functions mainly serve as a defensive tool against invasion of other organisms.Ricin is a heterodimeric RIP comprised of A and B chains joined through a disulfide bond. It is toxicicity for most mammalian cells Since ricin A chain (RTA) is an RNA specific N一glycosidase that removes a specific adenine residue in a highly conservative region from among over 4000 nucloside residues present in 28S rRNA,and causes protein synthesis inhibition and cell death. A single molecule of RTA is able to depurinate 1500-2000 ribosomes per minute. Ricin B chain (RTB) is a galactose-specific lectin which binds to the receptors on cell surfaces, and may enhance translocation by forming a pore in the membrane of intracellular organelles.Ricin enters the cells by receptor-mediated endocytosis. In order to reach thesubstrate(ribosomes), must translocate into the cytosol. Translocation from endocytic compartments to the cytosol is the essential and rate-limiting step in the intoxication process of most toxins such as ricin, Diphtheria toxin, Shiga toxin and Pseudomonas exotoxin (PE).A number of these toxins are transported to trans-Golgi network (TGN), and in many cases such transport to the TGN is required for the translocation and cytotoxicity. In deed, 5% of the rcin endocytosed by cells has been shown to reach the TGN. It is known from studies using brefeldin A (BFA) that disruption of TGN can block the cytotoxicity of ricin, suggesting that the intracellular compartment may be an important part of the uptake pathway.The DNA of ricin A chain and KEEL/KDEL sequence was amplified by PCR,and directly subcloned into T vector after confirmed by sequencing to make the unfusion protein。The unfusion gene then was cloned into expression vectoer Pet28a(+) , and the sequence was confirmed by sequencing。Expression was induced by IPTG in E. coli BL21(DE3)。The molecular weight of the recombinant protein was measured by SDS-PAGE and identify the expressed product by Western blot and reacted with sepecific ricin of monoclonal antibody。The recombinant protein can be purified by Blue-Sepharose 6B FF affinity chromato graphy. The cytotoxicity of EGFP and the recombinant RTA(RTA,RTA-KEEL,RTA-KDEL) were evaluated by the MTS assay in different cells following fluid-phase endocytosis. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy(LSCM). This study provided important evidence by a visualized way to prove that RTA and the recombinant RTA does reach the endoplasmic reticulum.From our experiments, we have demonstrated:(1)The plasmid DNA extracted from a single positive colony was digested with HindⅢ,and NcoⅠ. The agarose electrophoresis showed that the fragments were visible. Eventually the DNA sequences of RTA,RTA-KEEL,RTA-KDEL were confirmed by sequencing the desired region;(2)Expression of RTA, RTA-KEEL,RTA-KDEL was induced with IPTG.. After sonication and centrifugation of the bacteria, the supernatants were run on 12%SDS-PAGE. The result that the target proteins with about molecular weight of 31kDa could be detected in the lysates of the bacteria.(3)Blue-Sepharose 6B FFwas to purify. EGFP-RTA. After purification, protein showed a single band on 12%SDS--PAGE. The recombinant proteins produced at 25℃for 4 h were purified from E. coli with yields of approximately10 mg/L.(4)The antigenicity of the recombinant protein RTA,RTA-KEEL,RTA-KDEL analyzed by Western blotting. The results showed the sepecific Ricin of monoclonal antibody can recognize the protein of RTA-KEEL,RTA-KDEL just like the RTA protein.(5)The purified recombinant proteins were tested by MTS assay for cytotoxicity on different cells fluid-phase endocytosis. The results showed that RTA-KEEL,RTA-KDEL had a similar cytotoxicity of RTA indicating that EGFP tag would not influence the intracellular tracking and translocation of RTA;(6) Idifferent cells with EGFP and RTA,RTA-KEEL,RTA-KDEL fusion protein were observed by the laser scanning confocal microscopy.The result indicated that RTA,RTA-KEEL,RTA-KDEL with EGFP labelling was capable of avoiding being degraded inside the intracellular compartments;(7) HEP-G2 cells treated with EGFP or RTA-KEEL with EGFP labelling respectively were observed by LSCM.This result gave direct evidence that EGFP tag would not influence the intracellular trafficking and translocation of RTA and RTA were able to reach the ER of the HEP-G2 cells.