Reductive activation of type 2 ribosome-inactivating proteins is promoted by transmembrane thioredoxin-related protein

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.     

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.     

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.     

In vitro selection of DNA aptamer against abrin toxin and aptamer-based abrin direct detection

Abrin toxin as the target protein, belongs to class II ribosome-inactivating proteins family, has high toxicity to eukaryotic cells. Here, we firstly report the DNA aptamers, isolated by in vitro selection, recognize abrin toxin with high affinity and specificity, and have the advantage of no cross-reaction with structure-similar protein ricin toxin over antibodies. Then, a highly selective and sensitive aptamer-based abrin assay was established using a molecular light switching reagent [Ru(phen)(2)(dppz)](2+) with a limit of detection of 1 nM and a wide linear range from 1 to 400 nM with the correlation coefficient of 0.993. This assay can be successfully directly performed not only in physiological buffer but also in more complicated biological matrix, such as diluted serum.     

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.     

Cholera toxin B-subunit protects mammalian cells from ricin and abrin toxicity

The glycoproteins ricin and abrin intoxicate cells by inhibiting protein synthesis. Pretreatment of HeLa cells with cholera toxin partially protects them from ricin and abrin activity. The involvement in this phenomenon of the various effects of cholera toxin, namely, redistribution of membrane receptors elicited from protomer B and increasing cyclic AMP concentrations induced by protomer A, were studied. Substances able to enhance cyclic AMP concentrations do not affect ricin and abrin activity, while protomer B alone protects cells. In addition, the effects of several lectins on ricin or abrin toxicity were examined. Almost complete prevention of ricin or abrin activity was obtained using concanavalin A (Con A) and wheat germ agglutinin (WGA). Conversely, neither succinyl Con A nor Ulex europeus agglutinin (UEA) affected the cellular response. Both protomer B of cholera toxin and Con A did not alter the binding of ricin or abrin; they seem to protect cells by altering membrane structure.     

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.     

Homology between ricin and Ricinus communie agglutinin: Amino terminal sequence analysis and protein synthesis inhibition studies

The existence of three forms of ricin and two forms of the Ricinus communis agglutinin (RCA) was established using cation exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The preparation of the RCA we obtained was 60–75 times more potent than ricin in the agglutination of erythrocytes, but was about 4% as effective as an inhibitor of cell-free protein synthesis. When reduced with 2-mercaptoethanol, the RCA was activated 3000-fold as an inhibitor of cell-free protein synthesis, whereas ricin was activated about 600-fold by the same treatment. A mixture of the RCA A chains was about one-fifth as effective as the ricin A chain in the inhibition of cell-free protein synthesis. The purified polypeptide subunits of the castor bean lectins were subjected to automated Edman degradation. The sequence for 17 of the first 19 residues of the agglutinin A chain was determined. The first seven residues of the ricin A chain were determined and they are identical with those of the RCA A chain. Nineteen turns of Edman degradation on the RCA B chain resulted in the identification of 18 amino acids. The sequence determined for the first 17 residues of the ricin B chain was identical with that of the RCA B chain. It is likely that the identity of the ricin/RCA A and B chain sequences extends further along the polypeptide chains than the sequences we have determined. The similar structural and catalytic potentials of the RCA and ricin suggest that they bear a precursor-product relationship.     

Studies on the variants of the protein toxins ricin and abrin.

This study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101-109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.     

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate

Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endocytosis and must then escape the endomembrane system to reach the cytosolic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the present study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitization to saporin/uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preceding cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the catalytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disruption. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments.     

Enhancement of saporin cytotoxicity by Gypsophila saponins—More than stimulation of endocytosis

Saporin is a type I ribosome-inactivating protein with N-glycosidase activity. It removes adenine residues from the 28S ribosomal RNA resulting in inhibition of protein synthesis. Recently we have shown that saporin exerts no cytotoxicity on seven human cell lines. However, the combination of saporin with a special mixture of Gypsophila saponins (Soapwort saponins) from Gypsophila paniculata L. (baby's breath) rendered saporin to a potent cytotoxin comparable to viscumin, a highly toxic type II ribosome-inactivating protein.In this study we investigated whether the enhancement of the saporin-cytotoxicity by Gypsophila saponins is mediated by a saponin-triggered modulation of endocytosis, exocytosis or impaired degradation processes of his-tagged saporin (^hissaporin) in ECV-304 cells. For this purpose ^hissaporin was labelled with tritium and cytotoxicity of the toxin alone and in combination with Gypsophila saponins was scrutinized. The transport and degradation processes of ^hissaporin were not different in Gypsophila saponin-treated and control cells. However, after ultracentrifugation of a post-nuclear supernatant the amount of cytosolic ^hissaporin was significantly higher in saponin-treated cells than in cells, which were only incubated with ^hissaporin. This indicates a saponin mediated endosomal escape of saporin.     

Highly sensitive protein kinase activity assay based on electrochemiluminescence nanoprobes

Herein, we describe a novel electrochemiluminescence (ECL) biosensor for protein kinase activities and inhibition monitoring based on the magnetic beads (MB) technology and signal enhancement of gold nanoparticles (GNP). In this design, ECL nanoprobes were prepared by conjugating GNP with phosphorylated DNA capture probes and tris-(2,2'-bipyridyl) ruthenium (TBR)-cysteamine. Zirconium cations, a specific bridging agent, mediate the linkage between biotin modified phosphorylated peptides and ECL nanoprobes. The complexes were then captured and enriched on the electrode surface by streptavidin-coated MB for ECL reaction. To confirm the feasibility of this biosensor, we employed protein kinase A (PKA) as the model kinase to validate the assay and a satisfactory detection limit of 0.005 U/mL was achieved. The combination of ECL and GNP lays a solid foundation for highly sensitive assay, meanwhile, the coupling of MB surfaces used for separation and capture with unmodified ECL electrode detection results in a greatly simplified and reusable protocol. Thus, our biosensor offers great promise for a highly sensitive and simple assay for protein kinase activity. Furthermore, the inhibition of PKA activity was monitored on the basis of the ECL signals change in response to the concentration of PKA inhibitor.     

Highly sensitive and specific detection of viable Escherichia coli in drinking water

A highly sensitive and specific assay method was developed for the detection of viable Escherichia coli as an indicator organism in water, using nucleic acid sequence-based amplification (NASBA) and electrochemiluminescence (ECL) analysis. Viable E. coli were identified via a 200-nt-long target sequence from mRNA (clpB) coding for a heat shock protein. In the detection assay, a heat shock was applied to the cells prior to disruption to induce the synthesis of clpB mRNA and the mRNA was extracted, purified, and finally amplified using NASBA. The amplified mRNA was quantified with an ECL detection system after hybridization with specific DNA probes. Several disruption methods were investigated to maximize total RNA extracted from viable cells. Optimization was also carried out regarding the design of NASBA primer pairs and detection probes, as well as reaction and detection conditions. Finally, the assay was tested regarding sensitivity and specificity. Analysis of samples revealed that as few as 40 E. coli cells/mL can be detected, with no false positive signals resulting from other microorganisms or nonviable E. coli cells. Also, it was shown that a quantification of E. coli cells was possible with our assay method.     

Correlation of inhibition of ribosomal GTPase and protein synthesis by abrin and ricin.

The rate of inhibition of protein synthesis and ribosome-dependent GTPase activity by abrin and ricin was determined. Ribosomes were treated with toxin A-chains for increasing periods of time and they assayed for their ability to synthesize polyphenylalanine and hydrolyze GTP. The rate of inhibition of GTPase activity of the 60S subunit derived 8S ribonucleoprotein complex was also determined. Although the degree of maximal inhibition of protein synthesis and GTPase activity was different, the rates of inhibition were almost identical. The results support a conclusion that abrin and ricin inhibit protein synthesis by inactivating ribosome-dependent GTPase.     

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.     

Identification of the RNA N-glycosidase activity of ricin in castor bean extracts by an electrochemiluminescence-based assay

A simple electrochemiluminescence-based assay for RNA N-glycosidase activity has been modified to permit its use with authentic extracts of Ricinus communis (castor beans) and Abrus precatorius (jequirity seeds)—the natural sources of ricin and abrin. Modifications include the addition of an RNase inactivator to the reaction mixture, elimination of a signal-enhancing monoclonal antibody, and optimization of the incubation temperature. Concurrent testing with two substrates provides a diagnostic tool enabling castor bean toxins to be differentiated from a larger selection of N-glycosidase toxins than was previously examined.     

Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis.

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.     

Systematic comparison of oligosaccharide specificity of Ricinus communis agglutinin I and Erythrina lectins: a search by frontal affinity chromatography

Ricinus communis agglutinin I (RCA120) is considered a versatile tool for the detection of galactose-containing oligosaccharides. However, possible contamination by the highly toxic isolectin 'ricin' has become a critical issue for RCA120's continued use. From a practical viewpoint, it is necessary to find an effective substitute for RCA120. For this purpose, we examined by means of frontal affinity chromatography over 100 lectins which have similar sugar-binding specificities to that of RCA120. It was found that Erythrina cristagalli lectin (ECL) showed the closest similarity to RCA120. Both lectins prefer Gal beta1-4GlcNAc (type II) to Gal beta1-3GlcNAc (type I) structures, with increased affinity for highly branched N-acetyllactosamine-containing N-glycans. Their binding strength significantly decreased following modification of the 3-OH, 4-OH and 6-OH of the galactose moiety of the disaccharide, as well as the 3-OH of its N-acetylglucosamine residue. Several differences were also observed in the affinity of the two lectins for various other ligands, as well as effects of bisecting GlcNAc and terminal sialylation. Although six other Erythrina-derived lectins have been reported with different amino acid sequences, all showed quite similar profiles to that of ECL, and thus, to RCA120. Erythrina lectins can therefore serve as effective substitutes for RCA120, taking the above differences into consideration.