Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells.
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.
Wales
R
R
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Roberts
L M
LM
Lord
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D004705:Endocytosis; D004721:Endoplasmic Reticulum; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*; D021382:Protein Sorting Signals; D018000:Receptors, Peptide / Q000378:metabolism; D012276:Ricin / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D013696:Temperature; D014709:Vero Cells
C064459:KDEL receptor; D009842:Oligopeptides; D021382:Protein Sorting Signals; D018000:Receptors, Peptide; C073762:lysyl-aspartyl-glutamyl-leucine; D012276:Ricin