Abstract
An Escherichia coli expression system was used to produce recombinant ricin A chain (RTA) and RTA modified either by the addition of a carboxyl-terminal endoplasmic reticulum retrieval sequence Lys-Asp-Glu-Leu (RTAKDEL) or a nonfunctional analogue Lys-Asp-Glu-Ala (RTAKDEA). These RTA molecules can enter mammalian cells by fluid phase endocytosis. RTAKDEL was significantly more cytotoxic than either RTA or RTAKDEA to both Vero cells and HeLa cells (250- and 10-fold, respectively), despite the fact that all these RTA molecules had comparable enzymatic activities. This difference did not result from KDEL-mediated binding of RTAKDEL to the cell surface. Enhanced cytotoxicity could be correlated with an increased level of ribosome inactivation, measured as the RTA-catalyzed depurination of 28 S ribosomal RNA. These results indicate that the added KDEL sequence facilitated RTA entry into the cytosol. We propose that interaction with the intracellular KDEL receptor promotes retrograde transport of the toxin to the endoplasmic reticulum, where translocation of RTA into the cytosol occurs.
Highlights
Kingdom Science and Engineering Research Council Biotechnology Directorate
Ricin containingmutantRTB no longer capable of binding galactose [12] was not cytotoxic to macrophages even though the toxin, which is itself N-glycosylated, was able to enter thecells via the macrophage mannose receptor [13].It is possible that ricin binds intracellularly to an endogenous, galactosylated KDEL-containing protein, such as the resident rat liver ER luminal protein calreticulin [14],which is retrieved by the KDEL receptor from the trans Golgi cisternae or the transGolgi network (TGN)
Plasmids encoding RTA, RTAKDEL,and RTAKDEA were expressed in E. coli, and therecombinant proteins were purified to homogeneity by two rounds of CM-Sepharose chromatography and quantified as described previously [16]
Summary
Kingdom Science and Engineering Research Council Biotechnology Directorate. The costs of publication of this article were defrayed in part by the payment of page charges. A speculative interpretation of these data is that PE interacts with the KDEL receptor during cell entry, possibly to facilitate the later stages of its journey from the cell surface to the ER lumen. If this is the case, how might ricin reach the ER lumen since neither RTA nor RTB containsa carboxyl-terminal KDEL or related sequence? In order to examine further the potential involvement of the KDEL receptor in the retrograde transport of toxins, we prepared mutant ricin in which the tetrapeptide KDEL (or a related sequence that does not function as an ER retention signal, KDEA [15]) was added to the carboxyl terminus of RTA. The results, which are presented here, clearly confirm that the addition of KDEL to RTA markedly enhances itscytotoxicity in comparison to either RTA or RTAKDEA
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