The influence of anti-(ricin toxin A chain) monoclonal antibodies on the pharmacokinetics of ricin toxin A chain and recombinant ricin A chain in mice

Summary Two monoclonal antibodies against ricin toxin A chain (RTA) have been examined for their effects on the blood survival and biodistribution of RTA and recombinant ricin A chain in mice. When admixed with the toxins at 1:1 molar ratios prior to intravenous injection, the antibodies prolonged blood survival and whole-body retention of both species of RTA, and this was due essentially to reduced renal clearance of the toxins. Immune complexes were identified by gel filtration chromatography and immune precipitation with anti-IgG antiserum in mixtures prior to injection and in the serum of mice injected with the mixtures. An irrelevant monoclonal antibody showed no complex formation, and no effect on biodistribution. These studies have shown that immune complexes formed between monoclonal antibodies and protein antigens of molecular mass up to at least 30 kDa survive in the circulation, rather than being cleared by the reticuloendothelial system. Such antibodies could be used to modulate the biodistribution of toxic molecules such as ribosome-inhibiting proteins like RTA. This might be exploited therapeutically, for example in the construction of bispecific antibodies against ribosomal inhibiting proteins and tumour-associated antigens.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia

The therapeutic efficacy of whole ricin, or recombinant ricin A chain, coupled to a monoclonal antibody that reacts with the idiotype of the surface IgM expressed on guinea pig L2C lymphoblasts, was assessed. In vitro studies were done to characterize the immunotoxins (IT) and to demonstrate their specificity before use in vivo. The concentration of whole ricin IT (M6-Ricin) that inhibited protein synthesis by 50% (IC50) in L2C cells was 1.4 X 10(-9) M, in a 5-hr assay, in the presence of lactose to block non-antibody-directed toxicity. M6-Ricin did not inhibit protein synthesis in two control guinea pig cell lines that did not express the idiotype, nor did a whole ricin IT prepared with an isotype-matched monoclonal antibody of irrelevant specificity inhibit protein synthesis in L2C cells. Two recombinant ricin A chain IT, which differed from one another by a factor of 2 to 3 in the number of A chains conjugated per antibody molecule, were less effective in vitro than M6-Ricin (IC50 of greater than 5 X 10(-8) M). For in vivo experiments, the IT were given by the i.p. route 24 hr after the i.p. inoculation of 1 X 10(5) L2C cells. The highest doses of M6-Ricin and M6-Ricin A chain IT tested, 30 micrograms/kg and 3000 micrograms/kg, respectively, were within fourfold to fivefold of their maximum tolerated doses; no deaths or ill effects due to ricin toxicity were noted. These doses increased the median survival time of L2C-bearing guinea pigs to 31 to 34 days, compared with 15 days for untreated animals. This magnitude of increase in survival indicates that 99.999% (5 logs) of injected tumor cells were eliminated, thus accounting for the 12% long-term survival rate obtained. Median survival times for guinea pigs treated with 30 micrograms/kg of the A chain IT were 18 and 21 days for the two conjugates tested, and the median survival for guinea pigs treated with 3000 micrograms/kg of unconjugated antibody was 18 days. Our data demonstrate that recombinant A chain IT are active in vivo and that the B chain of ricin can potentiate IT activity in vivo. Although the potency differs by 100-fold, the therapeutic index of the intact ricin IT is similar to that of the ricin A chain IT.     

Homology between ricin and Ricinus communie agglutinin: Amino terminal sequence analysis and protein synthesis inhibition studies

The existence of three forms of ricin and two forms of the Ricinus communis agglutinin (RCA) was established using cation exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The preparation of the RCA we obtained was 60–75 times more potent than ricin in the agglutination of erythrocytes, but was about 4% as effective as an inhibitor of cell-free protein synthesis. When reduced with 2-mercaptoethanol, the RCA was activated 3000-fold as an inhibitor of cell-free protein synthesis, whereas ricin was activated about 600-fold by the same treatment. A mixture of the RCA A chains was about one-fifth as effective as the ricin A chain in the inhibition of cell-free protein synthesis. The purified polypeptide subunits of the castor bean lectins were subjected to automated Edman degradation. The sequence for 17 of the first 19 residues of the agglutinin A chain was determined. The first seven residues of the ricin A chain were determined and they are identical with those of the RCA A chain. Nineteen turns of Edman degradation on the RCA B chain resulted in the identification of 18 amino acids. The sequence determined for the first 17 residues of the ricin B chain was identical with that of the RCA B chain. It is likely that the identity of the ricin/RCA A and B chain sequences extends further along the polypeptide chains than the sequences we have determined. The similar structural and catalytic potentials of the RCA and ricin suggest that they bear a precursor-product relationship.     

A comparative study of ricin and diphtheria toxin-antibody-conjugate kinetics on protein synthesis inactivation

Kinetic data on toxin and antibody-toxin-conjugate inactivation of protein synthesis have been used to assess the variables which affect the transport of these toxins into the cytosol compartment. First-order inactivation rate constants of protein synthesis (ki) are compared under conditions of known receptor occupancy. The effect of inclusion of toxin B chains, both homologous and heterologous, in antibody-toxin conjugates is observed, and factors which affect toxin lag periods are studied. The results show that the inclusion of B chains in conjugates increases ki values 3-10-fold, but only if the B chain is homologous with the A chain. In spite of the augmentation of antibody-toxin-conjugate ki values by homologous toxin B chain, these ki values are only 1/20 those observed with unmodified toxins on sensitive cells. A further difference noted between toxins and antibody-toxin conjugates is the presence of a dose-dependent lag when toxins, but not antibody-toxin conjugates, effect sensitive cell types. This lag period for ricin can be shortened by alkalinizing the cell medium. The kinetic data can be fit by assuming a processing step interposed between the binding of ricin to surface receptors and the interaction of the A chain with ribosomes which is first-order in toxin concentration and pH-dependent. The time constant of this event is reflected in the dose-dependent lag period. It is proposed that antibody-toxin conjugates do not participate in this processing event and therefore fail to achieve the high entry levels exhibited by unmodified toxins.     

Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line.

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

Reduction of ricin and other plant toxins by thiol:protein disulfide oxidoreductases

The inhibitory effect of ricin, abrin, and modeccin on protein synthesis by a rabbit reticulocyte lysate is enhanced after preincubation of the toxins with GSH in the presence of a thiol:protein disulfide oxidoreductase purified from bovine liver. The same toxins, as well as the toxin from Viscum album, are reduced also by another thiol:protein disulfide oxidoreductase purified from rat liver cytosol.     

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B‐chain

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome‐associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5–90 min) and was later (15–90 min) partially translocated to cytosol as A‐ and B‐chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell‐free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time‐dependent translocation of ricin across the endosomal membrane occurred in cell‐free endosomes. Endosome‐located thioredoxin reductase‐1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase‐9 and caspase‐3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B‐chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B‐chain with mitochondria is involved in ricin‐induced apoptosis.     

Saporin induces multiple death pathways in lymphoma cells with different intensity and timing as compared to ricin

Ribosome-inactivating protein (RIP)-containing immunotoxins are currently used in clinical trials as anti-tumour drugs, in particular against haematological malignancies. In cell killing-based therapies it is important to identify the death pathways induced by the cytotoxic agent. The purpose of this work was to compare the pathways of cell death induced by the RIP saporin with those carried out by ricin in the L540 human Hodgkin's lymphoma-derived cell line. Protein synthesis inhibition, activation of caspases, DNA fragmentation and loss of viability have been evaluated. The two toxins triggered a similar DNA fragmentation and cell death, at concentrations giving the same level of cell protein synthesis inhibition, although the inhibitory effect of ricin on protein synthesis was more rapid than that of saporin. Moreover, the intrinsic apoptotic pathway was equally activated by both toxins, whilst ricin activated the extrinsic caspase pathway and the effector caspase-3/7 more efficiently than saporin. The complete inhibition of caspases by Z-VAD was only partially effective in cell rescue which appeared to be time limited. Necrostatin-1, a new inhibitor of non-apoptotic death, rescued cells from death by RIPs, although the effect was also partial and temporary. Despite the high RIP doses used no necrosis was detectable by Annexin V/Propidium Iodide (PI) test. These results suggest that more than one death mechanism was elicited by both ricin and saporin, however, with different timing and strength. The perspective of modulating cell death of neoplastic lymphocytes through different pathways could add new opportunities to reduce side effects and develop combined synergic immuno-chemotherapy.     

Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs-a type 1 RIP (dianthin) and a type 2 RIP (ricin)-that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.     

The ribosomal stalk is required for ribosome binding,depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α‐sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild‐type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild‐type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.     

Bioactivities of ricin retained and its immunoreactivity to anti-ricin polyclonal antibodies alleviated through pegylation

Ricin has long been employed to construct immunotoxins, whose efficacy was often undermined by immunogenicity. Pegylation (modification of proteins with polyethylene glycol, PEG) was one of those recently developed approaches to circumvent immunogenicity of legions of drugs. Herein, pegylation of ricin was found to have barely changed the RNA N-glycosidase activity and protein synthesis inhibiting activity of ricin, but remarkably altered the cytotoxicity of ricin on hepatoma cell line (BEL7404) or the immunoreactivity with polyclonal anti-ricin antibodies. This result suggested that the attached PEG or monomethyloxyl polyethylene glycol (mPEG) groups did not hinder ricin from hydrolyzing ribosomal RNA, but indeed covered some areas on the surface of ricin molecule, including those involved in the interaction with cellular receptors and epitopes recognized by polyclonal antibodies. Pegylation, masking certain epitopes of ricin, might contribute to alleviate the immunogenicity of the toxin. Approach in this work, if applied to thereby constructed immunotoxins, would help improve the prospective efficacy of these toxins.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells

In attempts to assess how many molecules of the toxic lectins abrin, ricin and modeccin are needed in the cytosol to kill HeLa cells the effect of these toxins on protein synthesis and plating efficiency was studied. The incubation time of the cells after a 1 h exposure to the toxins influenced strongly the extent of inhibition of protein synthesis. The full toxic effect was expressed about 20 h of incubation after the exposure. On further incubation, protein synthesis again increased at a rate comparable to that in the control cells. After exposure to increasing concentrations of toxins the inhibition of cellular protein synthesis measured after 20 h showed excellent agreement with the inhibition of plating efficiency, indicating that the inhibition of protein synthesis can be used as a measure of cell killing. The inhibition of protein synthesis by toxins was found to follow first order kinetics, indicating that the cells are killed by an all- or none-effect. Autoradiographic studies indicated that after exposure to intermediate toxin concentrations protein synthesis was completely abolished in some cells, whereas it appeared to proceed at a normal rate in the remaining cells. The results provide evidence that penetration of one molecule of abrin, ricin or modeccin into cytosol is lethal to HeLa cells and that the efficiency of toxin entry into the cytoplasm is very low compared to the rate of bulk toxin uptake.     

High-level expression and simplified purification of recombinant ricin A chain.

Ricin toxin is a glycoprotein which catalytically inactivates eukaryotic ribosomes by depurination of a single adenosine residue from the 28S ribosomal RNA. The enzymatic activity is present in the A chain of the toxin molecule, whereas the B chain contains two binding sites for galactose. Since it is highly potent in inhibiting protein synthesis, the A chain is used to prepare cytotoxic conjugates effective against tumor cells. Such chimeric proteins are highly selective and have a wide range of clinical applications. Extensive preclinical studies on these conjugates require large amounts of purified A chain. Native ricin A chain is heterogeneous, since plants produce a number of isoforms of ricin toxin. Purified, native preparations often contain two types of ricin A chain which differ in the extent of glycosylation. By cloning and expressing the gene of A chain, one could obtain homogeneous toxin molecules devoid of carbohydrates. In addition, structural changes in the toxin polypeptide could be introduced by in vitro mutagenesis, which can improve the pharmacological properties and antitumor activity. Earlier methods of expression strategies using Escherichia coli have yielded only moderate levels of expression. In the present study, the coding region of ricin A chain was cloned into pET3b, a high-level expression vector under the control of the T7 promoter. Recombinant ricin A chain produced by this construct has an additional 14 amino acid residues at the NH2 terminus. Subsequently, a NdeI site was created at the 5' end of the gene by oligonucleotide-directed mutagenesis. The modified fragment was then introduced into pET3b vector to produce toxin polypeptide identical to the native sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of eucaryotic ribosomes by the toxic plant proteins abrin and ricin

The effect of abrin and ricin on protein synthesizing systems from different sources was studied. The protein synthesis in a cell-free system from rabbit reticulocytes and from rat liver was strongly inhibited by the toxins, whereas a system from E. coli was not affected. Separate treatments of ribosomes and postribosomal supernatant from a rabbit reticulocyte lysate showed that the site of action of the toxins is on the ribosomes. The inactivation of the rabbit reticulocyte lysate by the toxins was a function of the incubation time and temperature. Protein synthesis was not necessary for the toxins to exert their effect. The data indicate that abrin and ricin act only on the eucaryotic type of ribosomes, and that they exert their effect by enzymatic action.     

Role of lipids in the retrograde pathway of ricin intoxication

The plant toxin ricin binds to both glycosphingolipids and glycoproteins with terminal galactose and is transported to the Golgi apparatus in a cholesterol-dependent manner. To explore the question of whether glycosphingolipid binding of ricin or glycosphingolipid synthesis is essential for transport of ricin from the plasma membrane to the Golgi apparatus, retrogradely to the endoplasmic reticulum or for translocation of the toxin to the cytosol, we have investigated the effect of ricin and the intracellular transport of this toxin in a glycosphingolipid-deficient mouse melanoma cell line (GM95), in the same cell line transfected with ceramide glucosyltransferase to restore glycosphingolipid synthesis (GM95-CGlcT-KKVK) and in the parental cell line (MEB4). Ricin transport to the Golgi apparatus was monitored by quantifying sulfation of a modified ricin molecule, and toxicity was studied by measuring protein synthesis. The data reveal that ricin is transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum and translocated to the cytosol equally well and apparently at the same rate in cells with and without glycosphingolipids. Importantly cholesterol depletion reduced endosome to Golgi transport of ricin even in cells without glycosphingolipids, demonstrating that cholesterol is required for Golgi transport of ricin bound to glycoproteins. The rate of retrograde transport of ricin was increased strongly by monensin and the lag time for intoxication was reduced both in cells with and in those without glycosphingolipids. In conclusion, neither glycosphingolipid synthesis nor binding of ricin to glycosphingolipids is essential for cholesterol-dependent retrograde transport of ricin. Binding of ricin to glycoproteins is sufficient for all transport steps required for ricin intoxication.     

Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin,abrin, and ricin

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.     

The diphthamide modification on elongation factor-2 renders mammalian cells resistant to ricin

Diphthamide is a post-translational derivative of histidine in protein synthesis elongation factor-2 (eEF-2) that is present in all eukaryotes with no known normal physiological role. Five proteins Dph1–Dph5 are required for the biosynthesis of diphthamide. Chinese hamster ovary (CHO) cells mutated in the biosynthetic genes lack diphthamide and are resistant to bacterial toxins such as diphtheria toxin. We found that diphthamide-deficient cultured cells were threefold more sensitive than their parental cells towards ricin, a r ibosome- i nactivating p rotein (RIP). RIPs bind to ribosomes at the same site as eEF-2 and cleave the large ribosomal RNA, inhibiting translation and causing cell death. We hypothesized that one role of diphthamide may be to protect ribosomes, and therefore all eukaryotic life forms, from RIPs, which are widely distributed in nature. A protective role of diphthamide against ricin was further demonstrated by complementation where dph mutant CHO cells transfected with the corresponding DPH gene acquired increased resistance to ricin in comparison with the control-transfected cells, and resembled the parental CHO cells in their response to the toxin. These data show that the presence of diphthamide in eEF-2 provides protection against ricin and suggest the hypothesis that diphthamide may have evolved to provide protection against RIPs.