Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA.

The rRNA N-glycosidase activities of the catalytically active A chains of the heterodimeric ribosome inactivating proteins (RIPs) ricin and abrin, the single-chain RIPs dianthin 30, dianthin 32, and the leaf and seed forms of pokeweed antiviral protein (PAP) were assayed on E. coli ribosomes. All of the single-chain RIPs were active on E. coli ribosomes as judged by the release of a 243 nucleotide fragment from the 3′ end of 23S rRNA following aniline treatment of the RNA. In contrast, E. coli ribosomes were refractory to the A chains of ricin and abrin. The position of the modification of 23S rRNA by dianthin 32 was determined by primer extension and found to be A₂₆₆₀, which lies in a sequence that is highly conserved in all species.     

Depurination of plant ribosomes by pokeweed antiviral protein

Mammalian ribosomes have been shown to be enzymatically modified by ribosomal inactivating protein (RIPs) via specific depurination of rRNA. Here we report that ribosomes isolated from wheat germ contain intact and undepurinated rRNA and are depurinated by pokeweed antiviral protein (PAP). Pokeweed ribosomes isolated under the same conditions are depurinated. Total RNA isolated from pokeweed in the presence of strong denaturants was found to pbe partially depurinated. We conclude that wheat germ ribosomes are resistant to the endogenous RIP, tritin, but are sensitive to PAP and that pokeweed ribosomes can be depurinated by the N-glycosidase activity of endogenous PAP during isolation.     

The C-terminus of pokeweed antiviral protein has distinct roles in transport to the cytosol, ribosome depurination and cytotoxicity

Pokeweed antiviral protein (PAP) produced by pokeweed plants is a single-chain (type I) ribosome-inactivating protein (RIP) that depurinates ribosomes at the alpha-sarcin/ricin loop of the large rRNA, resulting in inhibition of translation. Unlike the type II RIPs, which have an active and a binding moiety, PAP has only the active moiety. The mechanism by which toxins without a binding moiety gain access to cytosolic ribosomes is not known. We set up yeast as a simple and genetically tractable system to investigate how PAP accesses ribosomes and showed that the mature form of PAP is targeted to the cytosol from the endomembrane system in yeast. In the present study, we performed a systematic deletion analysis to identify the signal required for transport of PAP to the cytosol. We demonstrate here that processing of the C-terminal extension and sequences at the C-terminus of the mature protein are critical for its accumulation in the cytosol. Using a series of PAP mutants, we identified the C-terminal signal and demonstrated that it is distinct from the sequences required for ribosome depurination and cytotoxicity. The C-terminal motif showed sequence similarity to type II RIPs that retrotranslocate from the endoplasmic reticulum to the cytosol. These results demonstrate that a conserved sequence at the C-terminus of a type I RIP mediates its transport to the cytosol and suggest that type I and II RIPs may use a common signal to enter the cytosol.     

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.     

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.     

Pokeweed antiviral protein regulates the stability of its own mRNA by a mechanism that requires depurination but can be separated from depurination of the alpha-sarcin/ricin loop of rRNA

Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, alpha-sarcin/ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeast Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to the N-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.     

Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA

Ribosome-inactivating proteins (RIPs) are N-glycosylases that remove a specific adenine from the sarcin/ricin loop of the large rRNA in a manner analogous to N-glycosylases that are involved in DNA repair. Some RIPs have been reported to remove adenines from single-stranded DNA and cleave double-stranded supercoiled DNA. The molecular basis for the activity of RIPs on double-stranded DNA is not known. Pokeweed antiviral protein (PAP), a single-chain RIP from Phytolacca americana, cleaves supercoiled DNA into relaxed and linear forms. Double-stranded DNA treated with PAP contains apurinic/apyrimidinic (AP) sites due to the removal of adenine. Using an active-site mutant of PAP (PAPx) which does not depurinate rRNA, we present evidence that double-stranded DNA treated with PAPx does not contain AP sites and is not cleaved. These results demonstrate for the first time that PAP cleaves supercoiled double-stranded DNA using the same active site that is required for depurination of rRNA.     

Wheat ribosome-inactivating proteins: Seed and leaf forms with different specificities and cofactor requirements

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.Abbreviations CM carboxymethyl - FPLC fast protein liquid chromatography - NEPHGE non-equilibrium pH gradient gel electrophoresis - PAP pokeweed antiviral protein - RIP ribosome-inactivating protein A.J.M. was the recipient of a U.K. Science and Engineering Research Council CASE studentship sponsored by Agricultural Genetics Company Ltd., Cambridge CB4 4GG, UK.     

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.     

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.     

Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP)

Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A⁴³²⁴ in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes.Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.     

A non-toxic pokeweed antiviral protein mutant inhibits pathogen infection via a novel salicylic acid-independent pathway

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from Phytolacca americana, is characterized by its ability to depurinate the sarcin/ricin (S/R) loop of the large rRNA of prokaryotic and eukaryotic ribosomes. In this study, we present evidence that PAP is associated with ribosomes and depurinates tobacco ribosomes in vivo by removing more than one adenine and a guanine. A mutant of pokeweed antiviral protein, PAPn, which has a single amino acid substitution (G75D), did not bind ribosomes efficiently, indicating that Gly-75 in the N-terminal domain is critical for the binding of PAP to ribosomes. PAPn did not depurinate ribosomes and was non-toxic when expressed in transgenic tobacco plants. Unlike wild-type PAP and a C-terminal deletion mutant, transgenic plants expressing PAPn did not have elevated levels of acidic pathogenesis-related (PR) proteins. PAPn, like other forms of PAP, did not trigger production of salicylic acid (SA) in transgenic plants. Expression of the basic PR proteins, the wound-inducible protein kinase and protease inhibitor II, was induced in PAPn-expressing transgenic plants and these plants were resistant to viral and fungal infection. These results demonstrate that PAPn activates a particular SA-independent, stress-associated signal transduction pathway and confers pathogen resistance in the absence of ribosome binding, rRNA depurination and acidic PR protein production.     

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes,and correlation with antifungal activity

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.     

Production of pokeweed antiviral proteins nontoxic to cells by Mutagenesis of PAP-cDNA

Pokeweed antiviral protein (PAP), one of ribosome inactivating proteins (RIPs), has very strong toxicity both to prokaryotic and eukaryotic cells. To produce mutant PAPs nontoxic to cells, the PAP-cDNA was inserted into a yeast-E.coli shuttle vector under the control of galactose promoter, mutagenized using hydroxylamine, and transformed into yeast cells. Transformed yeast cells were selected on the uracil-deficient plate containing glucose or raffinose, and the yeast cells producing mutant PAPs nontoxic to cells were then selected on the galactose plate. Eighteen mutants were obtained by immunoblot analyses of 1,000 transformants: among them, three, ten and five mutants produced unprocessed, mature and truncated PAPs, respectively. Fourteen PAP mutants among them did not inhibit the yeast cell growth, and showed no or less inhibition of protein synthesisin vitro. Six among fourteen mutants were able to protect TMV infection in coinoculation experiment. The mutant PAPs showing an antiviral activity either without or reduced RIP activity contain neither the active site mutation nor C-terminal deletion mutation. These results suggest that both the RIP activity and the antiviral activity will require other amino acid residue(s) besides the active site and that the antiviral acitivity of PAP can be dissociated from its toxicity.     

The 1.4 anstroms structure of dianthin 30 indicates a role of surface potential at the active site of type 1 ribosome inactivating proteins

Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 angstroms, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed alpha-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 angstroms. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands beta7, beta8, and beta9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.     

Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome-linked Protein (VPg)

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.     

The type-1 and type-2 ribosome-inactivating proteins from<Emphasis Type="Italic"> Iris</Emphasis> confer transgenic tobacco plants local but not systemic protection against viruses

The antiviral activity of the type-2 ribosome-inactivating protein (RIP) IRAb from Iris was analyzed by expressing IRAb in tobacco (Nicotiana tabacum L. cv. Samsun NN) plants and challenging the transgenic plants with tobacco mosaic virus (TMV). Although constitutive expression of IRAb resulted in an aberrant phenotype, the plants were fertile. Transgenic tobacco lines expressing IRAb showed a dose-dependent enhanced resistance against TMV infection but the level of protection was markedly lower than in plants expressing IRIP, the type-1 RIP from Iris that closely resembles the A-chain of IRAb. To verify whether IRIP or IRAb can also confer systemic protection against viruses, transgenic RIP-expressing scions were grafted onto control rootstocks and leaves of the rootstocks challenged with tobacco etch virus (TEV). In spite of the strong local antiviral effect of IRIP and IRAb the RIPs could not provide systemic protection against TEV. Hence our results demonstrate that expression of the type-1 and type-2 RIPs from Iris confers tobacco plants local protection against two unrelated viruses. The antiviral activity of both RIPs was not accompanied by an induction of pathogenesis-related proteins. It is suggested that the observed antiviral activity of both Iris RIPs relies on their RNA N-glycohydrolase activity towards TMV RNA and plant rRNA.Abbreviations GUS -Glucuronidase - IRAb Iris agglutinin b - IRIP Iris type-1 RIP - PAG Polynucleotide:adenosine glycosylase - PAP Phytolacca americana antiviral protein - PR Pathogenesis-related - RIP Ribosome-inactivating protein - TCS Trichosanthin - TEV Tobacco etch virus - TMV Tobacco mosaic virus