Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition.

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.     

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.     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

Identification of a novel functional domain of ricin responsible for its potent toxicity

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved α-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an α-helix comprising the residues 99-106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the α-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the α-helix were rationally designed. Our data clearly demonstrated that the flexibility of the α-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved α-helix might be considered as a potential target for the prevention and treatment of RIP poisoning.     

Preparation and characterization of recombinant proricin containing an alternative protease-sensitive linker sequence.

The aim of this study was to determine the feasibility of utilizing a factor Xa-specific cleavage site within a recombinant protein containing the ricin A chain (RTA) sequence. Release of RTA is believed to be an essential step during the intracellular phase of ricin intoxication. Failure to incorporate such cleavage sites in fusions containing RTA results in a loss of toxin action (O'Hare, M., et al. (1990) FEBS Lett. 273,200. Kim, J., and Weaver, R.F. (1988) Gene 68,315). In this report we describe the introduction of a factor Xa-specific site in the linker of proricin, which we use here as a model substrate. Upon purification of the recombinant mutant proricin after expression in Xenopus oocytes, we demonstrate that the protease does have access to the engineered recognition sequence (albeit at low efficiency) and that the presence of the latter does not interfere with disulfide bond formation or the lectin activity of the ricin B chain moiety. Upon cleavage and reduction, the RTA polypeptide displays ribosome-inactivating ability, indicating that the presence of the modified linker at its C-terminus does not interfere with its catalytic activity. The general applicability of using such a cleavage site in recombinant fusions with RTA is discussed.     

High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein.

Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain.     

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.     

Non-specific depurination activity of saporin-S6, a ribosome-inactivating protein, under acidic conditions

Among five ribosome-inactivating proteins tested only saporin-S6 could efficiently release the adenine from adenosine 20 of the synthetic oligoribonucleotide (SRD RNA) mimic of the sarcin/ricin domain of rat 28S rRNA with a Km of 9 microM and a kcat of approximately 0.4 min(-1) at pH 7.6. The optimal pH for the depurination activity of saporin-S6 is 5.0. However, saporin-S6 lost its site-specificity of depurination on SRD RNA around the optimal pH. The non-specific depurination activity of saporin-S6 was dependent on the enzyme concentration and pH conditions. These results are valuable to understand the diversity and the depurination mechanism of ribosome-inactivating proteins.     

A relatively low level of ribosome depurination by mutant forms of ricin toxin A chain can trigger protein synthesis inhibition,cell signaling and apoptosis in mammalian cells

The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination.     

N‐glycosylation Does Not Affect the Catalytic Activity of Ricin A Chain but Stimulates Cytotoxicity by Promoting Its Transport Out of the Endoplasmic Reticulum

Ricin A chain (RTA) depurinates the α‐sarcin/ricin loop after it undergoes retrograde trafficking to the cytosol. The structural features of RTA involved in intracellular transport are not known. To explore this, we fused enhanced green fluorescent protein (EGFP) to precursor (preRTA‐EGFP), containing a 35‐residue leader, and mature RTA (matRTA‐EGFP). Both were enzymatically active and toxic in Saccharomyces cerevisiae. PreRTA‐EGFP was localized in the endoplasmic reticulum (ER) initially and was subsequently transported to the vacuole, whereas matRTA‐EGFP remained in the cytosol, indicating that ER localization is a prerequisite for vacuole transport. When the two glycosylation sites in RTA were mutated, the mature form was fully active and toxic, suggesting that the mutations do not affect catalytic activity. However, nonglycosylated preRTA‐EGFP had reduced toxicity, depurination and delayed vacuole transport, indicating that N‐glycosylation affects transport of RTA out of the ER. Point mutations in the C‐terminal hydrophobic region restricted RTA to the ER and eliminated toxicity and depurination, indicating that this sequence is critical for ER exit. These results demonstrate that N‐glycosylation and the C‐terminal hydrophobic region stimulate the toxicity of RTA by promoting ER export. The timing of depurination coincided with the timing of vacuole transport, suggesting that RTA may enter the cytosol during vacuole transport.     

Pokeweed antiviral protein depurinates the sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site

Ribosome-inactivating proteins, such as the pokeweed antiviral protein (PAP), inhibit translation by depurinating the conserved sarcin/ricin loop of the large ribosomal RNA. Depurinated ribosomes are unable to bind elongation factor 2, and, thus, the translocation step of the elongation cycle is inhibited. Though the consequences of depurination are well characterized, the ribosome conformation required for depurination to take place has not been described. In this report, we correlate biochemical and genetic data to conclude that pokeweed antiviral protein depurinates the sarcin/ricin loop when the A-site of the ribosomal peptidyl-transferase center is unoccupied. We show that prior incubation of ribosomes with puromycin, an analog of the 3'-terminus of aminoacyl-tRNA, inhibits both binding and depurination by PAP in a concentration-dependent manner. Expression of PAP in the yeast strain mak8-1 results in little depurination unless the cells are lysed, a process that would promote loss of aminoacyl-tRNA from the ribosome. The mak8-1 strain is known to exhibit a higher affinity for aminoacyl-tRNA compared with wild-type cells, and therefore, its ribosomes are more resistant to PAP in vivo. These data contribute to the mechanism of action of pokeweed antiviral protein; specifically, they have uncovered the ribosomal conformation required for depurination that leads to subsequent translation inhibition.     

The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity

The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the sarcin-ricin-loop (SRL) on the 60S ribosomal subunit is a major source of its toxicity. The model proposes that SRL depurination leads to protein synthesis inhibition, evoking ribotoxic stress with concomitant induction of numerous metabolic pathways, which lead to cell death. However, the direct relationship between the depurination and its impact on the translational machinery in vivo has never been satisfactorily explained. In this work, we approached a long-standing question about the influence of SRL depurination on the functioning of the translational machinery in vivo. We have shown that an already low level of depurinated ribosomes exert an effect on cell metabolism, indicating that minute modification within the ribosomal pool is sufficient to elicit a toxic effect. Importantly, depurination does not affect notably any particular step of translation, and translational slowdown caused by ricin is not a direct consequence of depurination and cannot be considered as the sole source of cell death. Instead, SRL depurination in a small fraction of ribosomes blocks cell cycle progression with no effect on cell viability. In this work, we have provided a comprehensive picture of the impact of SRL depurination on the translational apparatus in vivo. We propose that ribosomes with depurinated SRL represent a small imprinted ribosomal pool, which generates a specific signal for the cell to halt the cell cycle.     

Pokeweed antiviral protein isoforms PAP-I, PAP-II, and PAP-III depurinate RNA of human immunodeficiency virus (HIV)-1.

Pokeweed antiviral protein (PAP) is a naturally occurring broad-spectrum antiviral agent with potent anti-human immunodeficiency virus (HIV)-1 activity by an as yet undeciphered molecular mechanism. In the present study, we sought to determine if PAP is capable of recognizing and depurinating viral RNA. Depurination of viral RNA was monitored by directly measuring the amount of the adenine base released from the viral RNA species using quantitative high-performance liquid chromatography. Our findings presented herein provide direct evidence that three different PAP isoforms from Phytolacca americana (PAP-I from spring leaves, PAP-II from early summer leaves, and PAP-III from late summer leaves) cause concentration-dependent depurination of genomic RNA (63 to 400 pmols of adenine released per micrograms of RNA) purified from human immunodeficiency virus type-I (HIV-I), plant virus (tobacco mosaic virus (TMV), and bacteriophage (MS 2). In contrast to the three PAP isoforms, ricin A chain (RTA) failed to cause detectable depurination of viral RNA even at 5 microM, although it was as effective as PAP in inhibiting protein synthesis in cell-free translation assays. PAP-I, PAP-II, and PAP-III (but not RTA) inhibited the replication of HIV-1 in human peripheral blood mononuclear cells with IC(50) values of 17 nM, 25 nM, and 16 nM, respectively. These findings indicate that the highly conserved active site residues responsible for the depurination of rRNA by PAP or RTA are not sufficient for the recognition and depurination of viral RNA. Our study prompts the hypothesis that the potent antiviral activity of PAP may in part be due to its unique ability to extensively depurinate viral RNA, including HIV-1 RNA.     

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.     

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.     

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.     

Evidence for retro-translocation of pokeweed antiviral protein from endoplasmic reticulum into cytosol and separation of its activity on ribosomes from its activity on capped RNA

Pokeweed antiviral protein (PAP) is a single-chain ribosome inactivating protein (RIP) that binds to ribosomes and depurinates the highly conserved alpha-sarcin/ricin loop (SRL) of the large subunit rRNA. Catalytic depurination of a specific adenine has been proposed to result in translation arrest and cytotoxicity. Here, we show that both precursor and mature forms of PAP are localized in the endoplasmic reticulum (ER) in yeast. The mature form is retro-translocated from the ER into the cytosol where it escapes degradation unlike the other substrates of the retro-translocation pathway. A mutation of a highly conserved asparagine residue at position 70 (N70A) delays ribosome depurination and the onset of translation arrest. The ribosomes are eventually depurinated, yet cytotoxicity and loss of viability are markedly absent. Analysis of the variant protein, N70A, does not reveal any decrease in the rate of synthesis, subcellular localization, or the rate of transport into the cytosol. N70A destabilizes its own mRNA, binds to cap, and blocks cap dependent translation, as previously reported for the wild-type PAP. However, it cannot depurinate ribosomes in a translation-independent manner. These results demonstrate that N70 near the active-site pocket is required for depurination of cytosolic ribosomes but not for cap binding or mRNA destabilization, indicating that the activity of PAP on capped RNA can be uncoupled from its activity on rRNA. These findings suggest that the altered active site of PAP might accommodate a narrower range of substrates, thus reducing ribotoxicity while maintaining potential therapeutic benefits.     

The position of the proricin vacuolar targeting signal is functionally important

Ricin is synthesised as an ER-targeted precursor containing an enzymatic A chain and a galactose-binding B chain separated by a 12-amino acid linker propeptide. This internal propeptide is known to contain a sequence-specific vacuolar sorting signal whose functionality depends on the presence of an isoleucine residue. Conversion of this isoleucine to glycine completely abolished vacuolar targeting of proricin and led to its secretion. However, when this mutated signal was positioned at the C-terminus of a normally secreted reporter, vacuolar targeting of a significant fraction still occurred. Likewise, when the corrupted linker was C-terminally exposed within its natural context following the mature ricin A chain, and then co-expressed with ricin B chain, toxin heterodimers were still partially transported to tobacco cell vacuoles. By contrast, when placed at the N-terminus of the secreted reporter, or at the N-terminus of ricin B chain for co-expression with ricin A chain, the propeptide behaved most strikingly as a sequence-specific vacuolar targeting signal that, when mutated, resulted in complete secretion of the proteins. It would appear that the position of the linker peptide influences the specificity of its vacuolar targeting function.