Structure of recombinant ricin A chain at 2.3 A.

The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N-glycosidase. RTA must be separated from B chain to attack rRNA. The X-ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild-type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site-directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136-1145).     

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.     

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)     

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.     

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.     

High-resolution NMR study of a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein, ricin.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Its substrate domain forms a double helical stem and a 17-base loop that includes the sequence GAGA, the second adenosine of which corresponds to A4324. Recently, studies of mutant RNAs have shown that the four-nucleotide loop, GAGA, can function as a substrate for ricin. To investigate the structure that is recognized by ricin, we studied the properties of a short synthetic substrate, the dodecaribonucleotide r-CUCAGAGAUGAG, which forms a RNA hairpin structure with a GABA loop and a stem of four base pairs. The results of NMR spectroscopy allowed us to construct the solution structure of this oligonucleotide by restrained molecular-dynamic calculations. We found that the stem region exists as an A-form duplex. 5G and 8A in the loop region form an unusual G:A base pair, and the phosphodiester backbone has a turn between 5G and 6A. This turn seems to help ricin to gain access to 6A which is the only site of depurination in the entire structure. The overall structure of the GAGA loop is similar to those of the GAAA and GCAA loops that have been described but that are not recognized by ricin. Therefore, in addition to the adenosine at the depurination site, the neighboring guanosine on the 3' side (7G) may also play a role in the recognition mechanism together with 5G and 8A.     

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.     

Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation

Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met-tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation-factor-2-catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine-containing peptides bound to 80S monosomes in ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, programmed with globin mRNA, revealed a predominance of Met-Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin-A-chain-modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.     

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.     

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.     

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.     

Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity.

Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contact residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99% of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99% of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.     

In vitro selection of RNA molecules that inhibit the activity of ricin A-chain

The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K(d) = 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC(50) of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors.     

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.     

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.     

RIP and RALyase cleave the sarcin/ricin domain, a critical domain for ribosome function, during senescence of wheat coleoptiles

Type-I ribosome-inactivating protein (RIP), which is found in many plants, catalyzes depurination of a specific adenine in the sarcin/ricin domain (SRD) of the large rRNA causing loss of ribosomal activity. Previously, we found a RNA apurinic site-specific lyase (RALyase) that catalytically cleaved the phosphodiester bond at the RIP-dependent depurination site by β-elimination reaction. Here we show that both the RIP activity and RIP-RALyase-mediated cleavage of SRD in the cytoplasmic ribosome were induced at the late stage of senescence of wheat coleoptiles. Following this process, tissue death was observed. Furthermore, transgenic tobacco plants expressing glucocorticoid-induced RIP developed senescence-like phenotype. Our results suggest that ribosome inactivation due to the cleavage of SRD by the inducible RIP and constitutively expressed RALyase may be a unique plant system that mediates programmed cell death at the late senescent stage.