Cytotoxic ribosome-inactivating lectins from plants

A class of heterodimeric plant proteins consisting of a carbohydrate-binding B-chain and an enzymatic A-chain which act on ribosomes to inhibit protein synthesis are amongst the most toxic substances known. The best known example of such a toxic lectin is ricin, produced by the seeds of the castor oil plant, Ricinnus communis. For ricin to reach its substrate in the cytosol, it must be endocytosed, transported through the endomembrane system to reach the compartment from which it is translocated into the cytosol, and there avoid degradation making it possible for a few molecules to inactivate a large proportion of the ribosomes and hence kill the cell. Cell entry by ricin involves the following steps: (i) binding to cell-surface glycolipids and glycoproteins bearing beta-1,4-linked galactose residues through the lectin activity of the B-chain (RTB); (ii) uptake by endocytosis and entry into early endosomes; (iii) transfer by vesicular transport to the trans-Golgi network; (iv) retrograde vesicular transport through the Golgi complex and into the endoplasmic reticulum (ER); (v) reduction of the disulfide bond connecting the A- and B-chains; (vi) a partial unfolding of the A-chain (RTA) to enable it to translocate across the ER membrane via the Sec61p translocon using the pathway normally followed by misfolded ER proteins for targeting to the ER-associated degradation (ERAD) machinery; (vi) refolding in the cytosol into a protease-resistant, enzymatically active structure; (vii) interaction with the sarcin-ricin domain (SRD) of the large ribosome subunit RNA followed by cleavage of a single N-glycosidic bond in the RNA to generate a depurinated, inactive ribosome. In addition to the highly specific action on ribosomes, ricin and related ribosome-inactivating proteins (RIPs) have a less specific action in vitro on DNA and RNA substrates releasing multiple adenine, and in some instances, guanine residues. This polynucleotide:adenosine glycosidase activity has been implicated in the general antiviral, and specifically, the anti HIV-1 activity of several single-chain RIPs which are homologous to the A-chains of the heterodimeric lectins. However, in the absence of clear cause and effect evidence in vivo, such claims should be regarded with caution.     

Dependence of ricin toxicity on translocation of the toxin A-chain from the endoplasmic reticulum to the cytosol

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61alpha precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.     

Free Ricin A Chain Reaches an Early Compartment of the Secretory Pathway before It Enters the Cytosol

During the intoxication of mammalian cells by ricin, the catalytically active A chain must cross the membrane of an intracellular compartment in order to reach its ribosomal substrates in the cytosol. The actual site of ricin A chain translocation is unclear, and conflicting views hold that it enters the cytosol from endosomes or from an early compartment of the secretory pathway, possibly the lumen of the endoplasmic reticulum. Here we show that treating cells with brefeldin A, or transiently overexpressing mutant GTPases known to inhibit biochemical complexes mediating anterograde and retrograde transport between the endoplasmic reticulum and the Golgi complex, protected cells from intoxication by free ricin A chain. These data indicate that ricin A chain, either free or as part of intact ricin, reaches an early compartment of the secretory pathway before translocation into the cytosol occurs.     

The N-terminal ricin propeptide influences the fate of ricin A-chain in tobacco protoplasts

The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A- and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed.     

The tRNA N2,N2-dimethylguanosine-26 methyltransferase encoded by gene trm1 increases efficiency of suppression of an ochre codon in Schizosaccharomyces pombe

The plant toxin ricin has proven valuable as a membrane marker in studies of endocytosis as well as studies of different intracellular transport steps. The toxin, which consists of two polypeptide chains, binds by one chain (the B-chain) to both glycolipids and glycoproteins with terminal galactose at the cell surface. The other chain (the A-chain) enters the cytosol and inhibits protein synthesis enzymatically. After binding the toxin is endocytosed by different mechanisms, and it is transported via endosomes to the Golgi apparatus and the endoplasmic reticulum before translocation of the A-chain to the cytosol. The different transport steps have been analyzed by studying trafficking of ricin as well as modified ricin molecules.     

内质网区蓖麻毒素的逆向转运

蓖麻毒素是植物来源的核糖体失活蛋白。蓖麻毒素必须通过细胞的内膜系统到达内质网,然后转位至胞质,才能作用于胞质内的核糖体。在内质网中毒素的两条链分离,具有催化活性的A链被内质网上的蛋白质识别,并被转位到胞质内催化核糖体失活。现对内质网在参与蓖麻毒素胞内转运过程中的作用进行综述。     

Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer

Ricin is a heterodimeric protein toxin in which a catalytic polypeptide (the A-chain or RTA) is linked by a disulfide bond to a cell-binding polypeptide (the B-chain or RTB). During cell entry, ricin undergoes retrograde vesicular transport to reach the endoplasmic reticulum (ER) lumen, from where RTA translocates into the cytosol, probably by masquerading as a substrate for the ER-associated protein degradation (ERAD) pathway. In partitioning studies in Triton X-114 solution, RTA is predominantly found in the detergent phase, whereas ricin holotoxin, native RTB, and several single-chain ribosome-inactivating proteins (RIPs) are in the aqueous phase. Fluorescence spectroscopy and far-UV circular dichroism (CD) demonstrated significant structural changes in RTA as a result of its interaction with liposomes containing negatively charged phospholipid (POPG). These lipid-induced structural changes markedly increased the trypsin sensitivity of RTA and, on the basis of the protein fluorescence determinations, abolished its ability to bind to adenine, the product resulting from RTA-catalyzed depurination of 28S ribosomal RNA. RTA also released trapped calcein from POPG vesicles, indicating that it destabilized the lipid bilayer. We speculate that membrane-induced partial unfolding of RTA during cell entry may facilitate its recognition as an ERAD substrate.     

Activation of cell stress response pathways by Shiga toxins

Shiga toxin-producing bacteria cause widespread outbreaks of bloody diarrhoea that may progress to life-threatening systemic complications. Shiga toxins (Stxs), the main virulence factors expressed by the pathogens, are ribosome-inactivating proteins which inhibit protein synthesis by removing an adenine residue from 28S rRNA. Recently, Stxs were shown to activate multiple stress-associated signalling pathways in mammalian cells. The ribotoxic stress response is activated following the depurination reaction localized to the α-sarcin/ricin loop of eukaryotic ribosomes. The unfolded protein response (UPR) may be initiated by toxin unfolding within the endoplasmic reticulum, and maintained by production of truncated, misfolded proteins following intoxication. Activation of the ribotoxic stress response leads to signalling through MAPK cascades, which appears to be critical for activation of innate immunity and regulation of apoptosis. Precise mechanisms linking ribosomal damage with MAPK activation require clarification but may involve recognition of ribosomal conformational changes and binding of protein kinases to ribosomes, which activate MAP3Ks and MAP2Ks. Stxs appear capable of activating all ER membrane localized UPR sensors. Prolonged signalling through the UPR induces apoptosis in some cell types. The characterization of stress responses activated by Stxs may identify targets for the development of interventional therapies to block cell damage and disease progression.     

The enemy within: ricin and plant cells

Ricin, a ribosome-inactivating protein from the seeds of the castor oil plant (Ricinus communis L.) is one of the most potent cell poisons known. It is able to bind and enter most mammalian cells where it exploits their fully reversible secretory pathway to reach the endoplasmic reticulum. Ricin is then able to exit the endoplasmic reticulum to access the cytosol where it inhibits protein synthesis, thus killing the cells. Castor bean ribosomes are sensitive to ricin, but the plant has developed strategies to protect its own cells from suicide. The intracellular routing of ricin has been traditionally studied by exogenously adding toxin to mammalian cells and by following its path through the cell. However, the extreme potency of this protein has prevented the final membrane transport step from being studied in detail. Now, the expression of ricin in heterologous plant cells is providing helpful in elucidating details of both toxin biosynthesis and vacuolar targeting, and in studying membrane translocation of the catalytic subunit from the endoplasmic reticulum to the cytosol.Keywords: Ricin, ribosome-inactivating protein, castor oil plant, seeds, inhibitor, membrane transport.      

The RNA N-glycosidase activity of ricin A-chain

The RNA N-glycosidase activity of ricin A-chain has been characterized. When rat liver ribosomes were used as substrates, the A-chain cleaved the N-glycosidic bond at A-4324 in 28S rRNA. An apparent Michaelis constant (Km) for the reaction was determined to be 2.6 microM and the turnover number (Kcat) was 1777 min-1. When naked rRNA was the substrate, the A-chain cleaved the same bond in 28S rRNA but at a greatly reduced rate. The Km value was 5.8 microM. The results suggest that the A-chain has a similar affinity for 28S rRNA in both ribosomes and the naked states. When the deproteinized Escherichia coli rRNA was the substrates, ricin A-chain cleaved a N-glycosidic bond at A-2600 in 23S rRNA which corresponds to the ricin-site in 28S rRNA of rat liver ribosomes, while the A-chain has little activity on 23S rRNA in the ribosomes. The results suggest that ricin A-chain acts directly on RNA by recognizing a certain structure in the molecules. Using the secondary structure models for each species of rRNA, we have deduced a loop and stem structure having GAGA in the loop to be a minimum requirement for the substrate of ricin A-chain.     

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.     

Homodimerization of pokeweed antiviral protein as a mechanism to limit depurination of pokeweed ribosomes

Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from depurination during the synthesis of these enzymes. For example, pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.     

EDEM is involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol

The plant toxin ricin is transported retrogradely from the cell surface to the endoplasmic reticulum (ER) from where the enzymatically active part is retrotranslocated to the cytosol, presumably by the same mechanism as used by misfolded proteins. The ER degradation enhancing alpha-mannosidase I-like protein, EDEM, is responsible for directing aberrant proteins for ER-associated protein degradation. In this study, we have investigated whether EDEM is involved in ricin retrotranslocation. Overexpression of EDEM strongly protects against ricin. However, when the interaction between EDEM and misfolded proteins is inhibited by kifunensin, EDEM promotes retrotranslocation of ricin from the ER to the cytosol. Furthermore, puromycin, which inhibits synthesis and thereby transport of proteins into the ER, counteracted the protection seen in EDEM-transfected cells. Coimmunoprecipitation studies revealed that ricin can interact with EDEM and with Sec61alpha, and both kifunensin and puromycin increase these interactions. Importantly, vector-based RNA interference against EDEM, which leads to reduction of the cellular level of EDEM, decreased retrotranslocation of ricin A-chain to the cytosol. In conclusion, our results indicate that EDEM is involved in retrotranslocation of ricin from the ER to the cytosol.     

A class of mutant CHO cells resistant to cholera toxin rapidly degrades the catalytic polypeptide of cholera toxin and exhibits increased endoplasmic reticulum-associated degradation

After binding to the eukaryotic cell surface, cholera toxin undergoes retrograde transport to the endoplasmic reticulum. The catalytic A1 polypeptide of cholera toxin (CTA1) then crosses the endoplasmic reticulum membrane and enters the cytosol in a process that may involve the quality control mechanism known as endoplasmic reticulum-associated degradation. Other toxins such as Pseudomonas exotoxin A and ricin are also thought to exploit endoplasmic reticulum-associated degradation for entry into the cytosol. To test this model, we mutagenized Chinese hamster ovary cells and selected clones that survived a prolonged coincubation with Pseudomonas exotoxin A and ricin. These lethal endoplasmic reticulum-translocating toxins bind different surface receptors and target different cytosolic substrates, so resistance to both would likely result from disruption of a shared trafficking or translocation event. Here we characterize two Pseudomonas exotoxin A/ricin-resistant clones that exhibited increased endoplasmic reticulum-associated degradation. Both clones acquired the following unselected traits: (i) resistance to cholera toxin; (ii) increased degradation of an endoplasmic reticulum-localized CTA1 construct; (iii) increased degradation of an established endoplasmic reticulum-associated degradation substrate, the Z variant of alpha1-antitrypsin (alpha1AT-Z); and (iv) reduced secretion of both alpha1AT-Z and the transport-competent protein alpha1AT-M. Proteosome inhibition partially rescued the alpha1AT-M secretion deficiencies. However, the mutant clones did not exhibit increased proteosomal activity against cytosolic proteins, including a second CTA1 construct that was expressed in the cytosol rather than in the endoplasmic reticulum. These results suggested that accelerated endoplasmic reticulum-associated degradation in the mutant clones produced a cholera toxin/Pseudomonas exotoxin A/ricin-resistant phenotype by increasing the coupling efficiency between toxin translocation and toxin degradation.     

Interaction of Cibacron blue F3GA and polynucleotides with ricin A-chain, 60 S ribosomal subunit-inactivating protein

Cibacron blue F3GA, a sulfonated polyaromatic blue dye, inhibited the ability of ricin A-chain to inactivate ribosomes. Difference-spectroscopic study revealed that the dye bound to the A-chain (Kd = 0.72 microM), producing a difference spectrum with a single maximum at 688 nm and two minima at 585 and 628 nm. Such a significant difference spectrum was not observed in the presence of ricin B-chain or intact ricin, neither of which can inactivate ribosomes. Modification of arginine residues in the A-chain with phenylglyoxal showed a correlation between the loss of inhibitory activity on protein synthesis and the loss of difference absorbance produced by the dye-A-chain interaction. Both losses occurred significantly at an early stage of the modification. Furthermore, the dye protected the A-chain against a loss of its inhibitory activity resulting from the modification of arginine residues. These results suggest that the same arginine residues participate both in the interaction with the dye and in the inactivation of ribosomes. Based on these data, the dye appears to interact with the active site of the A-chain. Addition of several polynucleotides, namely rRNA, tRNA, poly(U) and DNA, to the dye-A-chain complex resulted in a marked displacement of the dye, whereas mono- and dinucleotides had little or no effect on the dye-A-chain interaction. These findings indicate the possible existence of a polynucleotide binding site in the active site of the A-chain. A combination of these and other results suggests that the A-chain recognizes and acts on some part of RNA of the 60 S ribosomal subunit.     

Modulation of toxin stability by 4-phenylbutyric acid and negatively charged phospholipids

AB toxins such as ricin and cholera toxin (CT) consist of an enzymatic A domain and a receptor-binding B domain. After endocytosis of the surface-bound toxin, both ricin and CT are transported by vesicle carriers to the endoplasmic reticulum (ER). The A subunit then dissociates from its holotoxin, unfolds, and crosses the ER membrane to reach its cytosolic target. Since protein unfolding at physiological temperature and neutral pH allows the dissociated A chain to attain a translocation-competent state for export to the cytosol, the underlying regulatory mechanisms of toxin unfolding are of paramount biological interest. Here we report a biophysical analysis of the effects of anionic phospholipid membranes and two chemical chaperones, 4-phenylbutyric acid (PBA) and glycerol, on the thermal stabilities and the toxic potencies of ricin toxin A chain (RTA) and CT A1 chain (CTA1). Phospholipid vesicles that mimic the ER membrane dramatically decreased the thermal stability of RTA but not CTA1. PBA and glycerol both inhibited the thermal disordering of RTA, but only glycerol could reverse the destabilizing effect of anionic phospholipids. In contrast, PBA was able to increase the thermal stability of CTA1 in the presence of anionic phospholipids. PBA inhibits cellular intoxication by CT but not ricin, which is explained by its ability to stabilize CTA1 and its inability to reverse the destabilizing effect of membranes on RTA. Our data highlight the toxin-specific intracellular events underlying ER-to-cytosol translocation of the toxin A chain and identify a potential means to supplement the long-term stabilization of toxin vaccines.     

Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain

Hybrid molecules were prepared from the A- and B-chains of the two toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic to Vero cells than were the parent toxins, native ricin and modeccin. A number of drugs (NH₄Cl, monensin, trifluoperazine, verapamil, ionophore A23187) which protect cells against modeccin, but not against ricin, protected to some extent against the toxic hybrid, but less so than against native modeccin. The possibility is discussed that the modeccin A-chain of the hybrid may enter the cytosol by two routes, one which is highly efficient and identical to that used by native modeccin and another less efficient one which cannot be used by native modeccin.     

A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes

Because it has been proposed that the ribosome–membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T₁) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.     

Dislocation of ricin toxin A chains in human cells utilizes selective cellular factors

Ricin is a potent A-B toxin that is transported from the cell surface to the cytosol, where it inactivates ribosomes, leading to cell death. Ricin enters cells via endocytosis, where only a minute number of ricin molecules reach the endoplasmic reticulum (ER) lumen. Subsequently, the ricin A chain traverses the ER bilayer by a process referred to as dislocation or retrograde translocation to gain access to the cytosol. To study the molecular processes of ricin A chain dislocation, we have established, for the first time, a human cell system in which enzymatically attenuated ricin toxin A chains (RTA(E177D) and RTA(Δ177-181)) are expressed in the cell and directed to the ER. Using this human cell-based system, we found that ricin A chains underwent a rapid dislocation event that was quite distinct from the dislocation of a canonical ER soluble misfolded protein, null Hong Kong variant of α(1)-antitrypsin. Remarkably, ricin A chain dislocation occurred via a membrane-integrated intermediate and utilized the ER protein SEL1L for transport across the ER bilayer to inhibit protein synthesis. The data support a model in which ricin A chain dislocation occurs via a novel strategy of utilizing the hydrophobic nature of the ER membrane and selective ER components to gain access to the cytosol.     

Cation-induced toroidal condensation of DNA studies with Co3+(NH3)6

The unfolding and refolding of Staphylococcus aureus penicillinase have been followed by urea-gradient electrophoresis. Unfolding of the native state proceeds by an all-or-none transition to fully unfolded protein, with no detectable accumulation of partially unfolded states. In contrast, refolding is complex and proceeds by very rapid, reversible formation of a partially folded state, H, which had been detected and characterized previously, as it is the most stable conformation at intermediate denaturant concentrations. At very low urea concentrations, a more compact conformational state was observed as a transient intermediate in refolding. There was little kinetic heterogeneity of the unfolded protein, as is normally observed with proteins containing proline residues.