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.     

Direct molecular cloning and expression of two distinct abrin A-chains

The protein toxin abrin, which possesses an N-glycosylase activity toward eukaryotic 28 S rRNA, may have a potential in the deliberate eradication of certain cells. Here we report, by polymerase chain reaction technique, the isolation of genomic DNA sequences encoding two distinct abrin A-chains. A third sequence which encoded a part of a third type of A-chain was also isolated. The deduced amino acid sequences of the two full-length A-chains were about 84% similar. Addition of mRNA encoding the full-length A-chains to reticulocyte lysate strongly inhibited protein synthesis in the lysates, and a corresponding glycosylase activity on rRNA was observed. Addition of the same mRNA to toxin-resistant wheat germ extracts led to synthesis of the expected 30-kDa protein which could be precipitated with antibodies specific for abrin.     

Differential effects of abrin on normal and tumor cells

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK- and S-180) were studied. Measurements of cell growth and colony formation showed that MEF and S-180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK- cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S-180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125I]abrin binding studies: NIH 3T3 and LMTK- cells had significantly fewer abrin binding sites than MEF and S-180 cells. The fate of the [125I]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time-dependent degradation was observed, degradation being more rapid in NIH 3T3 and S-180 cells than in LMTK- and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.     

相思豆毒素研究及应用

相思豆毒素类似于蓖麻毒素,是一种植物蛋白毒素,具有极强的细胞毒性作用,特别是对某些恶性肿瘤细胞的毒性更强,这使它成为用于杀伤肿瘤细胞的候选毒素之一。本就其性质、基因克隆、结构与功能,以及在肿瘤导向治疗方面的应用作一简要的综述。     

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.     

Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells

In attempts to assess how many molecules of the toxic lectins abrin, ricin and modeccin are needed in the cytosol to kill HeLa cells the effect of these toxins on protein synthesis and plating efficiency was studied. The incubation time of the cells after a 1 h exposure to the toxins influenced strongly the extent of inhibition of protein synthesis. The full toxic effect was expressed about 20 h of incubation after the exposure. On further incubation, protein synthesis again increased at a rate comparable to that in the control cells. After exposure to increasing concentrations of toxins the inhibition of cellular protein synthesis measured after 20 h showed excellent agreement with the inhibition of plating efficiency, indicating that the inhibition of protein synthesis can be used as a measure of cell killing. The inhibition of protein synthesis by toxins was found to follow first order kinetics, indicating that the cells are killed by an all- or none-effect. Autoradiographic studies indicated that after exposure to intermediate toxin concentrations protein synthesis was completely abolished in some cells, whereas it appeared to proceed at a normal rate in the remaining cells. The results provide evidence that penetration of one molecule of abrin, ricin or modeccin into cytosol is lethal to HeLa cells and that the efficiency of toxin entry into the cytoplasm is very low compared to the rate of bulk toxin uptake.     

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean)

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I > Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.     

Temporal behavior of abrin in the intoxication of chinese hamster cells (line CHO)

Abrin, a potent cytotoxin, was utilized as a probe to elucidate the mechanism by which external proteins are delivered to the cytoplasm of mammalian cells. Abrin bound rapidly to the surface receptors of the Chinese hamster cells (line CHO) and appeared to be internalized immediately without any significant lag. The maximum level of abrin internalization was achieved within eight minutes, based on both biochemical and electron microscopic autoradiographic studies with [¹²⁵|] abrin. About 10% of the silver grains of internalized [¹²⁵|] abrin were associated with vesicular structures, irrespective of the incubation time. Inhibition of protein synthesis began 30 minutes postincubation, and this latent period was not dependent on extracellular toxin concentration. SDS-polyacrylamide gel electrophoresis of the internalized [¹²⁵|] abrin indicated that internalized abrin molecules remained intact even after two hours of incubation.     

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.     

Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin,abrin, and ricin

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.     

Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin

After binding, the protein toxins ricin, abrin, and modeccin are endocytosed and processed through the cell's vesicular system in a poorly understood fashion, prior to translocation to the cytosol. The role of the Golgi apparatus in toxin processing was studied using brefeldin-A (BFA), a fungal metabolite which blocks Golgi function. At concentrations that inhibit secretion of interleukin-2 (IL-2), BFA blocks ricin, modeccin, and abrin intoxication of a lymphocyte derived cell line (Jurkat). Paradoxically, BFA enhances the toxicity of two ricin A-chain immunotoxins targeted against distinct cell surface determinants. BFA concentrations which are optimal for immunotoxin enhancement are below those needed to affect ricin intoxication or IL-2 secretion. BFA blockade of ricin does not involve effects on ricin endocytosis, toxin translocation to the cytosol, or the enzymatic activity of toxin A-chain. In contrast, BFA has no effect on immunotoxin processing but does enhance the immunotoxin translocation step. It is concluded that: 1) intact Golgi function is required for holotoxin processing. 2) Intact Golgi function is not required for holotoxin translocation. 3) Golgi function is tightly linked to immunotoxin translocation. 4) BFA has effects on vesicular routing in addition to the block of Golgi function in secretion which has been reported.     

Concanavalin A-induced inhibition of abrin and ricin activity parallels with a decrease of the number of toxin-binding uptake-elution cycles

Treatment of either Friend leukemia cells (FLC) or HeLa cells with concanavalin A (conA) causes a significant reduction of abrin and ricin activity. In order to elucidate the mechanism of this phenomenon, the toxin uptake and release were studied. ConA-treated cells show an increase of toxin bulk uptake, but a marked decrease of toxin entry into the cytosol. Analysis of toxin elution into the medium has demonstrated that conA induces a reduction of the toxin excretion process from the cell membrane. No significant degradation of either internalized or released toxin has been detected either in conA-treated or in untreated cells. Moreover, the toxin released into the medium is still biologically active. Treatment of cultures with lactose, in conditions which inhibit de novo binding of eluted toxin, determines a protective effect on the ricin and abrin activity; this phenomenon resembles the conA-induced protective effect. Our results suggest that conA decreases the number of toxin binding uptake-elution cycles, causing thereby a lower probability of toxin entry into the cytoplasm as compared to control cells.     

Chimeric protein: abrin B chain-trypsin inhibitor conjugate as a new antitumor agent

Abrin B chain and trypsin inhibitor isolated from Acacia confusa (ACTI) were covalently linked to form a chimeric protein (ANB-ACTI) with N-succinimidyl-3-(-2-pyridyldithio)propionate. The chimeric protein had 31% of trypsin inhibitory activity of ACTI and 7% of hemagglutinating activity of abrin B chain, but no inhibition on protein biosynthesis. ANB-ACTI had strong inhibitory effects on the growth of sarcoma 180 cells and Hela cell culture while the mixture of an equivalent amount of free abrin B chain and ACTI did not. The results suggests that abrin B chain of chimeric protein may act as a vector to carry ACTI into the tumor cells. ACTI into the tumor cells. ACTI in the chimeric protein potentiates its antitumor activity as well as its resistance to tryptic digestion.     

Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.     

Structure-function analysis and insights into the reduced toxicity of Abrus precatorius agglutinin I in relation to abrin

Abrin and agglutinin-I from the seeds of Abrus precatorius are type II ribosome-inactivating proteins that inhibit protein synthesis in eukaryotic cells. The two toxins share a high degree of sequence similarity; however, agglutinin-I is weaker in its activity. We compared the kinetics of protein synthesis inhibition by abrin and agglutinin-I in two different cell lines and found that approximately 200-2000-fold higher concentration of agglutinin-I is needed for the same degree of inhibition. Like abrin, agglutinin-I also induced apoptosis in the cells by triggering the intrinsic mitochondrial pathway, although at higher concentrations as compared with abrin. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison with that of the reported structure of abrin. The overall protein folding of agglutinin-I is similar to that of abrin-a with a single disulfide bond holding the toxic A subunit and the lectin-like B-subunit together, constituting a heterodimer. However, there are significant differences in the secondary structural elements, mostly in the A chain. The substitution of Asn-200 in abrin-a with Pro-199 in agglutinin-I seems to be a major cause for the decreased toxicity of agglutinin-I. This perhaps is not a consequence of any kink formation by a proline residue in the helical segment, as reported by others earlier, but due to fewer interactions that proline can possibly have with the bound substrate.     

Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells.

The effect of temperature on the uptake of abrin and ricin and on the subsequent excretion and degradation of the toxins was measured. Uptake was assessed either by monitoring the amount of cell-bound ¹²⁵I-labelled toxin that could not be released with lactose or by measuring the time required for transport of the toxins into a state where they were protected against neutralizing antibodies. The presence of toxin in this state was monitored by measuring inhibition of protein synthesis after a subsequent prolonged incubation period. In the case of abrin, straight lines were found in both cases when the data were plotted according to Arrhenius. The activation energies estimated was 18–21 kcal/mol (75–88 kJ/mol) in the case of uptake of [¹²⁵I]abrin and 15–19 kcal/mol (63–79 kJ/mol) when the indirect method was used.After internalization of [¹²⁵I]abrin and ricin a fraction of the radioactive material is released to the medium. Most of this material can be precipitated by trichloroacetic acid (TCA). There is a rapid release during the first 30 min and then over the next few hours the release occurs at a constant, but lower rate. The release of ricin was not affected by addition of colchicine, cytochalasin B (CB), ammonium chloride, sodium azide or bovine serum albumin, whereas the degradation of ricin was reduced by the above mentioned compounds (except albumin). The release of ricin was strongly temperature-dependent with a sharp transition at about 20 °C. The activation energies for the release above and below 20 °C were found to be 2.5 and 31 kcal/mol (10.5 and 172 kJ/mol), respectively.     

Abrin Immunotoxin: Targeted Cytotoxicity and Intracellular Trafficking Pathway

Background

Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy.

Methods

Protein synthesis assay using ³[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins.

Results

We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells.

Conclusions

This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.     

Studies on the toxicity and binding kinetics of abrin in normal and Epstein-Barr virus-transformed lymphocyte culture-I. Experimental results - 4

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein-Barr virus (EBV)-transformed lymphocytes have been previously investigated. Using data on EBV-transformed lymphocyte cell density as a function of both time and dose of abrin, the authors introduced the concept of self- and cross-coupling metabolic variables as a means of understanding how abrin affected DNA and protein uptake. In this paper, the self-coupling constant is studied in more detail and the relationship between DNA and protein synthesis is further expanded. We find that there is a significant linear relationship between DNA and protein synthesis in normal lymphocyte culture as measured by abrin interaction in the culture. We further find that there is a much stronger relationship between these variables in EBV-transformed lymphocyte culture. This relationship is further examined, and possible analytic equations are expressed.     

Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 μg/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl- -glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl- -glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4−7 × 10⁻⁸/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.     

Isolation, purification, and some properties of a lectin and abrin from Abrus precatorius linn.

An alternative and simple procedure has been described for the simultaneous separation of the lectin and abrin from the seeds of Abrus precatorius and their purification free from each other. Both the lectin and abrin have been crystallized, the latter as salt-free crystals. One variety of abrin which was nonhemagglutinating and did not bind d-galactose was obtained. The lectin found homogeneous by immunodiffusion, immunoelectrophoresis and sedimentation had a molecular weight of 132,000 which underwent pH-dependent reversible association-dissociation at pH 7 and 2, dissociating into non-covalently bound subunits of approximately 64,000 molecular weight. The protein was stable in the pH range 2–10. The abrin molecule did not undergo any change at low pH values. The C- and NH₂-terminal groups of the lectin were found to be Ala-Leu (or Leu-Ala) and valine, respectively. Crystalline lectin showed the presence of three isolectins in isoelectric focusing.