RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes

The modification reaction of 28 S rRNA in eukaryotic ribosomes by ricin A-chain was characterized. To examine whether ricin A-chain release any bases from 28 S rRNA, rat liver ribosomes were incubated with a catalytic amount of the toxin, and a fraction containing free bases and nucleosides was prepared from the postribosomal fraction of the reaction mixture by means of ion-exchange column chromatography. Thin-layer chromatographic analysis of this fraction revealed a release of 1 mol of adenine from 1 mol of ribosome. When the ribosomes or naked total RNAs were treated with ricin A-chain in the presence of [32P] phosphate, little incorporation of the radioactivity into 28 S rRNA was observed, indicating that the release is not mediated by phosphorolysis. Thus, considering together with the previous result (Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912), the results in the present experiments demonstrated that ricin A-chain inactivates the ribosomes by cleaving the N-glycosidic bond of A4324 of 28 S rRNA in a hydrolytic fashion.     

The mechanism of action of barley toxin: a type 1 ribosome-inactivating protein with RNA N-glycosidase activity

In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.     

Ribosome-bound elongation factor 2 escapes phosphorylation by Ca2+/calmodulin-dependent protein kinase III.

The activity of eukaryotic elongation factor 2 is regulated by phosphorylation catalysed by a highly specific Ca2+/calmodulin-dependent protein kinase. Phosphorylated EF2 binds to ribosomes with decreased affinity. The present evidence indicates that EF2 prebound to ribosomes is protected from phosphorylation, just as earlier evidence indicated that ribosome-bound EF2 is protected from ADP-ribosylation catalysed by diphtheria toxin. Ribosome-inactivating proteins ricin and gelonin, by interfering with the EF2-ribosome interaction, allow full phosphorylation of EF2.     

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

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)     

Inhibition by ricin of protein synthesis in vitro. Ribosomes as the target of the toxin

1. Ricin (a toxic protein from the seeds of Ricinus communis) is a powerful inhibitor of the poly(U)-directed incorporation of phenylalanine into polypeptides catalysed by isolated rat liver ribosomes and elongation factors 1 and 2 (EF 1 and EF 2). The inhibition can be largely overcome by increasing the concentration of ribosomes. 2. The toxin does not affect the binding of phenylalanyl-tRNA to ribosomes catalysed by EF 1, nor does it inhibit the puromycin reaction used as a test for peptide-bond formation catalysed by ribosomes. 3. Ricin inhibits the ribosome-linked GTP hydrolysis catalysed by EF 2. 4. Ribosomes treated with ricin and washed through sucrose gradients containing 0.6m-NH(4)Cl are functionally inactive in those assay systems that are sensitive to the presence of added toxin. 5. It is suggested that ricin brings about an irreversible modification of ribosomes which impairs their ability to interact with EF 2. Since ricin inhibits at a molar concentration much lower than that of ribosomes it probably acts catalytically. No added cofactor is necessary for the inhibitory action of the toxin.     

Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains.

A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor (EF-2). The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes. There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1-0.2 muM for all three A-chains. The activity of the A-chains in the intact cell is discussed.     

Identification of the ricin lipase site and implication in cytotoxicity

Ricin is a heterodimeric plant toxin and the prototype of type II ribosome-inactivating proteins. Its B-chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the membrane of intracellular compartments to reach the cytosol where its N-glycosidase activity inactivates ribosomes, thereby arresting protein synthesis. We here show that ricin possesses a functional lipase active site at the interface between the two subunits. It involves residues from both chains. Mutation to alanine of catalytic serine 221 on the A-chain abolished ricin lipase activity. Moreover, this mutation slowed down the A-chain translocation rate and inhibited toxicity by 35%. Lipase activity is therefore required for efficient ricin A-chain translocation and cytotoxicity. This conclusion was further supported by structural examination of type II ribosome-inactivating proteins that showed that this lipase site is present in toxic (ricin and abrin) but is altered in nontoxic (ebulin 1 and mistletoe lectin I) members of this family.     

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.     

Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes

Ricin, Shiga toxin, and Shiga-like toxin II (SLT-II, Vero toxin 2) exhibit an RNA N-glycosidase activity which specifically removes a single base near the 3' end of 28 S rRNA in isolated rat liver ribosomes and deproteinized 28 S rRNA (Endo Y., Mitsui, K., Motizuki, M., & Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912; Endo Y. & Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130, Endo, Y., Tsurugi, K., Yutsudo, T., Takeda, Y., Ogasawara, K. & Igarashi, K. (1988) Eur. J. Biochem. 171, 45-50). These workers identified the single base removed, A-4324, by examining a 28 S rRNA degradation product which was generated by contaminating ribonucleases associated with the ribosomes. To determine whether this N-glycosidase activity applies in living cells, we microinjected ricin into Xenopus oocytes. We also microinjected Shiga toxin and a variant of Shiga-like toxin II (SLT-IIv). All three toxins specifically removed A-3732, located 378 nucleotides from the 3' end of 28 S rRNA. This base is analogous to the site observed in rat 28 S rRNA for ricin, Shiga toxin, and SLT-II. Purified, glycosylated, ricin A chain contains this RNA N-glycosidase activity in oocytes. We also demonstrated that the nonglycosylated A subunit of recombinant ricin exhibits this RNA N-glycosidase activity when injected into Xenopus oocytes. Ricin, Shiga toxin, and SLT-IIv also caused a rapid decline in oocyte protein synthesis for nonsecretory proteins.     

Depurination of yeast 26S ribosomal RNA by recombinant ricin A chain

The nature of the modification of yeast ribosomes by the recombinant form of the ricin A chain has been examined. Evidence is presented that the 26S rRNA molecule is depurinated at a specific site and that the activity is inhibited by antibody raised to ricin A chain. It thus appears that the recombinant form of this toxin retains the depurination activity of the native molecule. These results are consistent with the model that the site of depurination is in a highly conserved sequence forming a loop on the surface of the ribosome, a domain involved in elongation factor-dependent binding of aminoacyl-tRNA.     

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.     

The preparation of deglycosylated ricin by recombination of glycosidase-treated A- and B-chains: effects of deglycosylation on toxicity and in vivo distribution

Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target.     

Ribosome-mediated folding of partially unfolded ricin A-chain

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.     

Ricin and alpha-sarcin alter the conformation of 60S ribosomal subunits at neighboring but different sites

The effects of ricin and alpha-sarcin separately or in combination on the conformation of rat liver ribosomes were investigated by measuring the relative accessibility of individual ribosomal proteins to N-ethylmaleimide after 80S ribosomes were treated with these toxins. By using a double-labelling technique in which ribosomes were incubated with the toxins and then treated with 3H-labelled or 14C-labelled N-ethylmaleimide, it was found that labelling of protein L14 was specifically reduced by treatment with ricin, and that of proteins L3 and L4 by treatment with alpha-sarcin, suggesting that the toxins alter the conformation of ribosomes in the vicinity of these proteins. When ribosomes were treated with both ricin and alpha-sarcin, the extent of labelling of protein L3 was reduced compared to that observed after treatment with alpha-sarcin alone. These results are discussed in relation to previous observations showing that these three proteins are neighbours in the 60S ribosomal subunit and probably play important roles in protein biosynthesis, and in the actions of ricin and alpha-sarcin on 28S rRNA.     

Effect of ribosome treatment on sensitivity to ricin A chain

Rat liver ribosomes treated with catalytic amounts (30 ng/ml) of ricin A chain are inhibited about 80% when assayed immediately. However, the same ribosomes assayed after separation from A chain by centrifugation have partially recovered their activity in the translation of polyuridylic acid. The extent of recovery is dependent on magnesium ion concentration. Even though the activity of A chain-treated ribosomes is increased after centrifugation, they are not sensitive to further treatment with ricin A chain. Except for impure ribosomes, isolated by centrifugation of crude homogenate, the overall sensitivity of ribosomes after different treatments was the same.     

Inhibition by ricin of protein synthesis in vitro: 60S ribosomal subunit as the target of the toxin (Short Communication)

Poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes is strongly inhibited by ricin. Experiments involving hybridization between subunits derived from normal and ricin-treated ribosomes demonstrate that the 60S subunit is the site of action of the toxin. The toxin inactivates the 60S subunit independently of the presence of the 40S subunit.     

RNAi screening of Drosophila (Sophophora) melanogaster S2 cells for ricin sensitivity and resistance

The ribosome-inhibiting toxin ricin binds exposed β1→4 linked galactosyls on multiple glycolipids and glycoproteins on the cell surface of most eukaryotic cells. After endocytosis, internal cell trafficking is promiscuous, with only a small proportion of ricin proceeding down a productive (cytotoxic) trafficking route to the endoplasmic reticulum (ER). Here, the catalytic ricin A chain traverses the membrane to inactivate the cytosolic ribosomes, which can be monitored by measuring reduction in protein biosynthetic capacity or cell viability. Although some markers have been discovered for the productive pathway, many molecular details are lacking. To identify a more comprehensive set of requirements for ricin intoxication, the authors have developed an RNAi screen in Drosophila S2 cells, screening in parallel the effects of individual RNAi treatments alone and when combined with a ricin challenge. Initial screening of 806 gene knockdowns has revealed a number of candidates for both productive and nonproductive ricin trafficking, including proteins required for transport to the Golgi, plus potential toxin interactors within the ER and cytosol.