Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II.

A hybrid protein of ricin and the enzymatically active fragment A of diphtheria toxin (toxin A) has been synthesized and purified. The diphtheria toxin A fragment of the hybrid protein is shown to enter the cytosol compartment of HeLa cells, its presence assayed by the fall of intracellular elongation factor II (EF-2) and the rise of ADP-ribosylated EF-2. Hybrid entrance to HeLa cells is blocked by lactose which blocks receptor-mediated entry of ricin but not by NH4Cl which blocks the transport of diphtheria toxin. It is concluded that the diphtheria toxin fragment A moiety of the hybrid enters the cell cytosol via the ricin receptor-mediated transport system. The kinetics of intracellular ADP-ribosylation of EF-2 by diphtheria toxin have also been studied. Ribosylation is preceded by a toxin dose-dependent lag period. The data suggest that the time constant responsible for the lag period is in the transport step. Models consistent with these data are discussed.     

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.     

Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins

Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors.     

A single affinity column step method for the purification of ricin toxin from castor beans (Ricinus communis)

A rapid method for purifying ricin toxin from castor beans is presented which uses a single affinity column step to obtain pure toxin from a crude extract of castor beans. A galactosyl-Sepharose affinity matrix was used to bind ricin toxin and its associated agglutinin, which both bind specifically to galactose, from a crude extract. The selective elution of ricin toxin and agglutinin was then achieved by eluting the affinity column with a galactose gradient, which sequentially elutes the two proteins due to a difference in binding avidity to the matrix.     

Selective destruction of nerve growth factor receptor-bearing cells in vitro using a hybrid toxin composed of ricin A chain and a monoclonal antibody against the nerve growth factor receptor

A hybrid toxin composed of ricin A chain and a monoclonal antibody directed against the rat nerve growth factor (NGF) receptor (192-IgG) was prepared using the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)-propionate and purified by affinity chromatography. Characterization studies showed that the hybrid, 192-s-s-A, displaced bound 125I-labeled 192-IgG from rat superior cervical ganglion (SCG) membranes with an IC50 3-5 times lower than that of unconjugated 192-IgG. When incubated with cultured rat SCG neurons, 192-s-s-A inhibited protein synthesis in a concentration-dependent fashion. The effect of 192-s-s-A on these neurons was reversed by coincubation with an excess of 192-IgG. The IC50 of 192-s-s-A on protein synthesis in rat SCG neurons was 4 nM. Intact ricin and ricin A chain inhibited protein synthesis in these neurons with IC50 values of 5 pM and 500 nM, respectively. The 192-s-s-A hybrid had no effect on mouse SCG neurons or a human melanoma cell line known to have NGF receptors. This is consistent with the finding that 192-IgG recognizes only the rat NGF receptor. Also, 192-s-s-A did not inhibit protein synthesis in primary cultures of rat skeletal muscle or Vero cells, which do not have cell surface receptors for NGF. 192-s-s-A was able to inhibit protein synthesis in PC12 cells but the potency was 10-100 times less in these cells compared to rat SCG neurons. Ricin and A chain were also 10-100 times less potent in PC12 cells than neurons. Rat SCG neurons exposed to 192-s-s-A lost their refractile appearance under phase-contrast optics, showed granular degeneration of neurites, and died. Thus the decreased protein synthesis caused by the hybrid toxin correlated with the morphological destruction of the neurons. 192-s-s-A represents a potentially powerful tool by which to selectively destroy NGF receptor-bearing cells in vitro. The hybrid toxin may prove useful as an in vivo toxin.     

Studies on the galactose-binding site of ricin and the hybrid toxin man6P-ricin

N-acetylimidazole (NAI) was used to O-acetylate the plant seed toxin ricin. O-acetylation of one to two tyrosine residues per molecule of ricin inhibited ricin binding to Sepharose 4B and decreased toxicity by 90% in a protein synthesis inhibition assay in HeLa cells. Lactose, known to block the binding site on the ricin B subunit, protected ricin from NAI modification of binding or toxicity. Thus NAI, under these conditions, can be a lactose site-specific inhibitor. The lactose site-specific modification of the hybrid toxin, Man6P-ricin, performed under the same conditions, exhibited the same 90% inhibition of Man6P receptor-mediated toxicity as the galactose-containing receptor-mediated toxicity of either Man6P-ricin or ricin. Thus the ricin B chain lactose-binding site appears to be essential for the high potency of Man6P-ricin via the new cell type-specific Man6P receptor. Treatment of fibroblasts with neuraminidase exposes galactose residues, thus increasing the sensitivity to ricin eight fold. The Man6P receptor-mediated toxicity of Man6P-ricin is not affected by this treatment, although the galactose-inhibited route is potentiated eight fold. The Man6P-ricin hybrid appears to require the ricin B chain galactose-binding site to enter the cytosol after initially binding to the Man6P receptor. These data provide some insights into the proper design of hybrid toxins. We discuss a number of possible models for hybrid toxin entry.     

Conformation-dependent antigenic determinants in the toxic lectin ricin.

The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.     

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.     

Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

The gene for the A chain of ricin toxin was fused to a beta-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.     

Characterization of a transferrin-diphtheria toxin conjugate

We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis.     

Identification and characterization of a monoclonal antibody recognizing a galactose-binding domain of the toxin ricin

A monoclonal antibody raised against purified ricin B chain, 75/3B12, blocked ricin toxicity 30- to 100-fold in vitro. The 75/3B12 IgG and F(ab')2 blocked ricin binding to cell surface galactose-containing receptors. The 75/3B12 Fab bound ricin D with a Ka of 10(7) M-1, and this binding was blocked by asialofetuin, lactose, and N-acetylgalactosamine--molecules which interact with the ricin galactose-binding site--but not by fetuin, sucrose, or glucose. The 75/3B12 Fab contained no detectable carbohydrate and, according to several lines of evidence, did not bind ricin via Ig carbohydrate determinants. The monoclonal antibody appears to recognize a galactose-binding site on ricin D via the variable region of the antibody. The 75/3B12 Fab bound ricin E only 1/50 as well as ricin D and bound the Ricinus agglutinin only 1/80 as well as ricin D. The antibody specificity indicates that structural differences exist in the galactose-binding sites of the Ricinus communis lectins. Abrin and other lectins which bind galactose or N-acetylgalactosamine were not significantly bound by the monoclonal antibody. In vitro, the antibody blocked the nontarget toxicity of immunotoxins similarly to lactose. However, in vivo, unlike lactose, the 75/3B12 antibody protected mice from ricin toxicity.     

Structure and toxicity of pure ricinus agglutinin

Highly purified ricinus agglutinin was found to inhibit protein synthesis in HeLa cells. This effect could be prevented by the addition of the specific antiricinus agglutinin serum, whereas specific anti-ricin serum did not protect the cells, demonstrating that the toxic effect of ricinus agglutinin is not due to contamination with ricin.After reduction of ricinus agglutinin with 2-mercaptoethanol in the presence of 0.5 M galactose the constituent peptide chains were separated by chromatography on a DE-52 column. The B′-chain passed through the column, whereas the A′-chain bound and was eluted with a salt gradient. The B′-chain was further purified by chromatography on a CM-52 column.The shortest chain, the A′-chain, was found to inhibit cell-free protein synthesis, whereas the B′-chain did not have this ability. On the other hand, the B′-chain was able to induce agglutination of erythrocytes when tested together with anti-ricinus agglutinin serum indicating that the B′-chains bind to the cells.Ouchterlony immunodiffusion tests with crude anti-ricin and anti-ricinus agglutinin sera revealed that the two constituent chains of ricinus agglutinin are immunologically partial identical and that they also show reaction of partial identity with both chains of the toxic lectin ricin.The data indicate that a similar structure-function relationship exists in ricinus agglutinin as in ricin. The reason for the much lower toxicity of ricinus agglutinin than of ricin in living animals is discussed.     

Amino sugars in the glycoprotein toxin from Bacillus thuringiensis subsp. israelensis.

The carbohydrate content of purified Bacillus thuriniensis subsp. israelensis crystal toxin was determined by six biochemical tests, column chromatography on an amino acid analyzer, and the binding of 11 fluorescent lectins. The crystals contained approximately 1.0% neutral sugars and 1.7% amino sugars. The amino sugars consisted of 70% glucosamine and 30% galactosamine. No N-acetylneuraminic acid (sialic acid) was detected. The presence of amino sugars was confirmed by the strong binding of fluorescent wheat germ agglutinin and the weak binding of fluorescent soybean agglutinin. These lectins recognize N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, respectively. The lectin-binding sites appeared evenly distributed among the protein subunits of the crystal. The sugars were covalently attached to the crystal toxin because wheat germ agglutinin still bound alkali-solubilized toxin which had been boiled in sodium dodecyl sulfate, separate by polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. This study demonstrates the covalent attachment of amino sugars and indicates that the B. thuringiensis subsp. israelensis protein toxins should be viewed as glycoprotein toxins. The crystals used in the present study were purified on sodium bromide density gradients. Studies employing crystals purified on Renografin density gradients can give artificially high values for the anthrone test for neutral sugars.     

Endosomes and toxin translocation

In this review we discuss data obtained by our group regarding the entry of toxins, especially ricin, diphtheria toxin (DT) and Pseudomonas exotoxin A (PE) into animal cells. We studied the translocation process of these toxins using endosomes purified from lymphocytes. This process is rate-limiting for toxicity and enables these toxins to reach the cytosol where they will inactivate the protein synthesis system and kill the cell. We could show that each of these toxins uses a different strategy to cross the endosome membrane. Whereas ricin transmembrane transport only relies on cytosolic ATP hydrolysis, PE first requires exposure to the low endosomal pH (pH-6), presumably to insert into the endosome membrane, before being translocated via a process which also requires cytosolic ATP hydrolysis. DT translocation is directly triggered and energized by the endosome-cytosol pH gradient. Using conjugates with dihydrofolate reductase we could indirectly show that ricin and PE require unfolding for translocation. A deletion approach enabled to produce a more cytotoxic PE mutant by increasing its translocation activity.     

Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains.

A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor (EF-2). The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes. There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1-0.2 muM for all three A-chains. The activity of the A-chains in the intact cell is discussed.     

Effect of pH on the conformation and stability of the plant toxin ricin

Intrinsic protein fluorescence of native plant toxin and its isolated subunits were studied. The effect of pH was studied on: conformation of ricin and its A- and R-chains; affinity to galactose of ricin and its binding B-subunit. At two pH 5.0 and 7.0, the structural stability of toxin and subunits was estimated according to denaturational action of guanidine chloride. It was demonstrated that position of maximum and the spectrum shape of fluorescence of native toxin and catalytical A-subunit insignificantly depends on pH in the range of 3-8, whereas sufficient changes of the separameters for the ricin B-chain reveal structural transition at pH 4-5. The affinity of galactose of ricin and its isolated B-chain depends on pH, the maximal binding is observed at pH 7. The structural stability of ricin and isolated chains significantly differs at pH 7.5 and 5.0, thus the structure stability of ricin and A-chain increases, and that of B-chain decreases at pH 5.0.     

Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites. To establish the importance of these regions of homology, we used site-directed mutagenesis to alter the A-subunit sequences of two members of the Shiga toxin family. Substitution of an aspartic acid for glutamic acid 166 of the Slt-IIA subunit decreased the capacity of the polypeptides to inhibit protein synthesis at least 100-fold in a cell-free translation system. However, this mutation did not prevent the expression of immunoreactive, full-length Slt-IIA. In addition, SLT-II holotoxin containing the mutated A subunit was 1,000-fold less toxic to Vero cells. Finally, site-directed mutagenesis was used to delete sequences encoding amino acids 202 through 213 of the Shiga toxin A subunit. Although this deletion did not prevent holotoxin assembly, it abolished cytotoxic activity.     

Characterization of a cDNA encoding ricin E,a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.     

The mechanism of action of barley toxin: a type 1 ribosome-inactivating protein with RNA N-glycosidase activity

In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.     

Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.