DIFFERENCES IN CYTOTOXICITY OF NATIVE AND ENGINEERED RIPS CAN BE USEDTO ASSESS THEIR ABILITY TO REACH THE CYTOPLASM

Ricin is a heterodimeric cytotoxin composed of RTB, a galactose bindin g lectin, and RTA, an enzymatic N-glycosidase. The toxin is endocytose d, and after intracellular routing, RTA is translocated to the cytopla sm where it inactivates ribosomes resulting in a loss of host cell pro tein synthesis and cell death. We show for the first time that the cyt otoxicity against cultured T cells by several RTA mutants is directly proportional to the enzyme activity of RTA, suggesting this is a relia ble system to measure translocation effects. Large discrepancies betwe en cytotoxicity and enzyme action for a given pair of toxins are there fore attributable to differences in cell, binding, uptake, or membrane translocation. Fluid phase uptake and cytotoxicity of isolated RTA ar e essentially identical to that of the single chain toxin PAP. This im portant finding suggests that RTA, and the A chain of class 2 RIPs in general, has not evolved special translocation signals to complement t he increased target cell binding facilitated by RTB. Experiments with the lectin RCA and with ebulin suggest those toxins have diminished cy totoxicity probably mediated by comparative deficiencies in B chain bi nding. Addition of a KDEL sequence to RTA increases fluid phase uptake , consistent with the notion that transport to the ER is important for cytotoxicity. Fusion of MBP or GST to the amino terminus of RTA has l ittle effect on enzyme action or cytotoxicity. This result is not alte red by protease inhibitors, suggesting the fusion proteins are probabl y not cleaved prior to translocation of the toxic A chain and implying that the toxins can carry large passenger proteins into the cytoplasm , an observation with interesting potential for analytical and therape utic chemistry. (C) 1998 Academic Press.