Wales R, Roberts L M, Lord J M
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
J Biol Chem. 1993 Nov 15;268(32):23986-90.
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.
利用大肠杆菌表达系统生产重组蓖麻毒素A链（RTA）以及通过添加羧基末端内质网回收序列赖氨酸-天冬氨酸-谷氨酸-亮氨酸（RTAKDEL）或无功能类似物赖氨酸-天冬氨酸-谷氨酸-丙氨酸（RTAKDEA）修饰的RTA。这些RTA分子可通过液相内吞作用进入哺乳动物细胞。尽管所有这些RTA分子具有相当的酶活性，但RTAKDEL对Vero细胞和HeLa细胞的细胞毒性分别比RTA或RTAKDEA显著高250倍和10倍。这种差异并非源于KDEL介导的RTAKDEL与细胞表面的结合。增强的细胞毒性可能与核糖体失活水平的增加相关，核糖体失活水平通过RTA催化的28 S核糖体RNA脱嘌呤来衡量。这些结果表明，添加的KDEL序列促进了RTA进入细胞质。我们推测，与细胞内KDEL受体的相互作用促进了毒素向内质网的逆向转运，在内质网中RTA发生向细胞质的转运。
