High Mobility of N-Terminal Parts of A and B Subunits of Ricin

Abstract

¹H-NMR spectra (500 MHz) of ricin and its isolated A and B subunits have been analyzed in this study. It is shown that together with tight packing of the polypeptide chain there also exist flexible highly mobile segments in the structure of the proteins. Examination of the line-widths of resonances and the identification of sharp signals with protons of amino acids indicates that N-terminal peptide segments of A and B subunits are free and undergo rapid segmental motions. Spin-echo sequence (90-τ-180-τ) was used to suppress an intensive unresolved background signal from protons involved in the globular part of ricin, thus permitting selective identification of the unusually sharp signals from mobile side chains.     

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.     

Ricin subunit association. Thermodynamics and the role of the disulfide bond in toxicity

The values of the thermodynamic parameters characterizing the association of the subunits of reduced ricin have been determined from equilibrium studies in the analytical ultracentrifuge. van't Hoff analysis indicates that the Gibbs free energy change for subunit association is predominantly of entropic origin. The positive values for the entropy and enthalpy changes suggest that hydrophobic forces may play a dominant role in the association. The association is characterized by values of Ka of 1.72 X 10(6) M-1 at 22 degrees C and 5.66 X 10(6) M-1 at 37 degrees C. The association was not affected by the presence of 20 mM lactose. Toxicity studies demonstrated that reduced ricin at a concentration where it was 52% associated had a toxicity equal to that of native ricin at that same concentration. At higher concentrations, reduced ricin was even more toxic than native ricin. Diethyl maleate, which reduces intracellular glutathione levels, blocked the toxicity of ricin but not the toxicity of reduced ricin. The disulfide bond linking the A and B subunits appears to play no role in toxicity other than to hold the two subunits together at low concentrations.     

Comparison of cooperative and isolated site binding of T4 gene 32 protein to ssDNA by 1H NMR

Deuteriation of all aromatic protons of gene 32 protein (g32P) from phage T4, followed by selective introduction of specific protons, has allowed the precise identification of the number and magnitude of the chemical shift changes induced in the aromatic protons when g32P binds noncooperatively or cooperatively to nucleotides. Signals from five Tyr residues are shifted by binding of g32P to d(pA)8 or d(pA)40-60; however, the change from noncooperative, d(pA)8, to cooperative, d(pA)40-60, binding causes significant increases in the magnitudes of the shifts for only two of these Tyr signals. These two Tyr residues may interact directly with the nucleotide bases, while the shifts associated with the other three Tyr may be due to conformational changes in g32P upon ssDNA binding. Similar conclusions can be drawn for two of the six Phe residues whose protons undergo shifts upon nucleotide binding. Observation of selected proton signals allows for the first time detection by 1H NMR of changes in the proton signals from two Trp residues upon nucleotide binding. The side chains of two Tyr, one or two Phe, and one Trp are probably directly involved in nucleotide base-protein interactions. As assayed by the signals from the H2 and H8 protons of adenine, the bases of a bound nucleotide are undergoing a fast chemical exchange in the noncooperative mode of binding, but shift to slow exchange upon assuming the cooperative mode of ssDNA interaction. When bound to a polynucleotide, the A domain of g32P (residues 254-301) becomes more mobile, as reflected in sharpening of the 1H NMR signals from the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

An interaction between ricin and calreticulin that may have implications for toxin trafficking

Here we demonstrate that ricin is able to interact with the molecular chaperone calreticulin both in vitro and in vivo. The interaction occurred with ricin holotoxin, but not with free ricin A chain; and it was prevented in the presence of lactose, suggesting that it was mediated by the lectin activity of the ricin B chain. This lectin is galactose-specific, and metabolic labeling with [(3)H]galactose or treating galactose oxidase-modified calreticulin with sodium [(3)H]borohydride indicated that Vero cell calreticulin possesses a terminally galactosylated oligosaccharide. Brefeldin A treatment indicated that the intracellular interaction occurred initially in a post-Golgi stack compartment, possibly the trans-Golgi network, whereas the reductive separation of ricin subunits occurred in an earlier part of the secretory pathway, most probably the endoplasmic reticulum (ER). Intoxicating Vero cells with ricin whose A chain had been modified to include either a tyrosine sulfation site or the sulfation site plus available N-glycosylation sites, in the presence of Na(2)35SO(4), confirmed that calreticulin interacted with endocytosed ricin that had already undergone retrograde transport to both the Golgi and the ER. Although we cannot exclude the possibility that the interaction between ricin and calreticulin is an indirect one, the data presented are consistent with the idea that calreticulin may function as a recycling carrier for retrograde transport of ricin from the Golgi to the ER.     

1H NMR studies on the CuA center of nitrous oxide reductase from Pseudomonas stutzeri.

1H NMR spectra of the CuA center of N2OR from Pseudomonas stutzeri, and a mutant enzyme that contains only CuA, were recorded in both H2O- and D2O-buffered solution at pH 7.5. Several sharp, well-resolved hyperfine-shifted 1H NMR signals were observed in the 60 to -10 ppm chemical shift range. Comparison of the native and mutant N2OR spectra recorded in H2O-buffered solutions indicated that several additional signals are present in the native protein spectrum. These signals are attributed to a dinuclear copperII center. At least two of the observed hyperfine-shifted signals associated with the dinuclear center, those at 23.0 and 13.2 ppm, are lost upon replacement of H2O buffer with D2O buffer. These data indicate that at least two histidine residues are ligands of a dinuclear CuII center. Comparison of the mutant N2OR 1H NMR spectra recorded in H2O and D2O indicates that three signals, c (27.5 ppm), e (23.6 ppm), and i (12.4 ppm), are solvent exchangeable. The two most strongly downfield-shifted signals (c and e) are assigned to the two N epsilon 2H (N-H) protons of the coordinated histidine residues, while the remaining exchangeable signal is assigned to a backbone N-H proton in close proximity to the CuA cluster. Signal e was found to decrease in intensity as the temperature was increased, indicating that proton e resides on a more solvent-exposed histidine residue. One-dimensional nOe studies at pH 7.5 allowed the histidine ring protons to be definitively assigned, while the remaining signals were assigned by comparison to previously reported spectra from CuA centers. The temperature dependence of the observed hyperfine-shifted 1H NMR signals of mutant N2OR were recorded over the temperature range of 276-315 K. Both Curie and anti-Curie temperature dependencies are observed for sets of hyperfine-shifted protons. Signals a and h (cysteine protons) follow anti-Curie behavior (contact shift increases with increasing temperatures), while signals b-g, i, and j (histidine protons) follow Curie behavior (contact shift decreases with increasing temperatures). Fits of the temperature dependence of the observed hyperfine-shifted signals provided the energy separation (Delta EL) between the ground (2B3u) and excited (2B2u) states. The temperature data obtained for all of the observed hyperfine-shifted histidine ligand protons provided a Delta EL value of 62 +/- 35 cm-1. The temperature dependence of the observed cysteine C beta H and C alpha H protons (a and h) were fit in a separate experiment providing a Delta EL value of 585 +/- 125 cm-1. The differences between the Delta EL values determined by 1H NMR spectroscopy and those determined by EPR or MCD likely arise from coupling between relatively low-frequency vibrational states and the ground and excited electronic states.     

Interaction of the toxic plant protein--ricin, with model membranes. A fluorescence method of study]

The fluorescence method has been used to investigate ricin and its isolated subunits interaction with some model membranes. Three liposome types were used as a model of biological membrane: 1) liposomes constructed from lecithin and cholesterol (9:1, M:M) 2) from ganglioside receptors GM1 and 3) from the mixture of GM1, lecithin and cholesterol (1:9:1). Interaction of the protein with liposome evokes changes in the parameters of both intrinsic protein fluorescence and fluorescence of the covalently bound dansyl. Binding constants were calculated from a decrease of the intrinsic fluorescence intensity as well as from the changes in the dansyl rotation anisotropy. Measurements were carried out at neutral and acidic pH. There was good correlation of the results obtained by different methods. It was shown that association constants were different for intact ricin and its subunits. The constants also depend on liposome composition and pH of the solution. The present study has demonstrated that interaction of ricin with liposome is accounted for not only by receptor centers but also by other hydrophobic regions of ricin that are inaccessible in the native toxin and may represent the region of the subunits interaction.     

Physical,Chemical and Physiological Properties of S-CM Subunits of Ricin D

Some physical, chemical and physiological properties of two S-CM subunits (S-CM Ile and S-CM Ala chain) of ricin D were studied. Physical properties of subunits are summerized in Table I. The S-CM subunits consisted of 244 (S-CM Ile chain) and 254 (S-CM Ala chain) amino acid residues. By the specific cleavage of the single intermolecular disulfide bond of ricin D, no remarkable change in conformation of polypeptide chains of ricin D was detected, but the toxicity was markedly decreased. The toxicity of ricin D is due to the quaternary structure of the ricin D molecule.     

Identification of three oligosaccharide binding sites in ricin.

The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.     

NMR studies of multiple conformations in complexes of Lactobacillus casei dihydrofolate reductase with analogues of pyrimethamine

1H and 19F NMR signals from bound ligands have been assigned in one- and two-dimensional NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase with various pyrimethamine analogues (including pyrimethamine [1, 2,4-diamino-5-(4'-chlorophenyl)-6-ethylpyrimidine], fluoropyrimethamine [2, 2,4-diamino-5-(4'-fluorophenyl)-6-ethylpyrimidine], fluoronitropyrimethamine [3, 2,4-diamino-5-(4'-fluoro-3'-nitrophenyl) -6-ethylpyrimidine], and methylbenzoprim [4, 2,4-diamino-5-[4'- (methylbenzylamino)-3'-nitrophenyl]-6-ethylpyrimidine]). The signals were identified mainly by correlating signals from bound and free ligands by using 2D exchange experiments. Analogues (such as 1 and 2) with symmetrically substituted phenyl rings give rise to 1H signals from four nonequivalent aromatic protons, clearly indicating the presence of hindered rotation about the pyrimidine-phenyl bond. Analogues containing asymmetrically substituted aromatic rings (such as 3 and 4) exist as mixtures of two rotational isomers (an enantiomeric pair) because of this hindered rotation and the NMR spectra revealed that both isomers (forms A and B) bind to the enzyme with comparable, though unequal, binding energies. In this case two complete sets of bound proton signals were observed. The phenyl ring protons in each of the two forms experience essentially the same protein environment (same shielding) as that experienced by the corresponding protons in bound pyrimethamine: this confirms that forms A and B correspond to two rotational isomers resulting from approximately 180 degrees rotation about the pyrimidine-phenyl bond, with the 2,4-diaminopyrimidine ring being bound similarly in both forms. The relative orientations of the two forms have been determined from NOE through-space connections between protons on the ligand and protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit stoichiometry of the CNG channel of rod photoreceptors

Cyclic nucleotide-gated (CNG) channels play a central role in the conversion of sensory stimuli into electrical signals. CNG channels form heterooligomeric complexes built of A and B subunits. Here, we study the subunit stoichiometry of the native rod CNG channel by chemical crosslinking. The apparent molecular weight (M(w)) of each crosslink product was determined by SDS-PAGE, and its composition was analyzed by Western blotting using antibodies specific for the A1 or B1 subunit. The number of crosslink products and their M(w) as well as the immunological identification of A1 and B1 subunits in the crosslink products led us to conclude that the native rod CNG channel is a tetramer composed of three A1 and one B1 subunit. This is an example of violation of symmetry in tetrameric channels.     

Proton nuclear magnetic resonance study of human plasma alpha-2-macroglobulin

A proton nuclear magnetic resonance (NMR) study is reported of human alpha-2-macroglobulin (alpha-2-M). It was observed that alpha-2-M, which consists of four identical subunits and has a molecular weight of 720,000, gives several sharp resonances. After cleavage of the "bait" region peptide with trypsin and subsequent removal of the peptide under a high salt condition, most of the sharp resonances disappeared, indicating that the sharp resonances observed in the native alpha-2-M originate from the amino acid residues in the bait region. Resonances due to the aromatic protons of the Tyr residue, which exists in the bait region, have been assigned on the basis of chemical shift. It was observed that the C3- and C5-H proton resonances for the Tyr residue are especially narrow, indicating that the side chain of the Tyr residue in the bait region is in a highly mobile state. Photochemically induced dynamic nuclear polarization experiments clearly show that the Tyr residue is actually exposed to the solvent. It was possible to identify resonances due to several His residues that are exposed to solvent. Other resonances, which probably originate from Arg residues in the bait region, were also observable in the conventional NMR spectra. On the basis of the present NMR data, we conclude that the bait region of the native alpha-2-M is highly flexible and exposed to solvent. On treatment of alpha-2-M with methylamine, no significant change has been detected in the NMR spectra observed in both the conventional and CIDNP mode.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective detection of rapid motions in spectrin by NMR

Human erythrocyte spectrin molecules exhibit relatively sharp (30-50 Hz) proton NMR signals in the aliphatic region. A standard solvent presaturation pulse sequence that also partially suppresses the broad envelope from protons with rigid structures in spectrin and selectively enhances the sharp resonances has been used to characterize the behavior of these resonances. The overall resonance pattern strongly resembles that of the denatured spectrin. The observed spectra are also quite similar to the line-broadened spectrum from a mixture of amino acids that corresponds to the composition of the spectrin molecule. These data indicate the existence of regions exhibiting rapid internal motions within the intact spectrin molecule, and suggest that the amino acid composition of the residues giving rise to the sharp resonances is quite similar to that of the full spectrin molecule.     

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.     

The effect of amphotericin B on lectin-induced aggregation of cell surface receptors

The mobility of concanavalin A (ConA) and ricin receptors from NS20 neuroblastoma and C₆ glioma cells was studied using an electrophoretic technique. Cells attached to a solid support were exposed to an electrical field (12V cm⁻¹) at room temperature. The distribution of lectin receptors on the cell surface was revealed by fluorescent conjugates of lectins and microscopic observation of the fixed cells. This technique allowed the estimation of the mobilities of lectin receptors either in free or liganded form, depending on the time at which the cells are labeled with lectins (either after or before electrophoresis). In line with previous observations [1] it is shown that in their free form ConA and ricin receptors are mobile all over the cell surface. Ligand binding induced an apparent receptor immobilization. Immobilization of ricin receptors from C₆ glioma cells could be induced either by the multivalent or the monovalent form of the lectin indicating that cross-linking of receptors by the ligand did not play a predominant role in the process of receptor immobilization. Amphotericin B but not ionophores like valinomycin or gramicidin blocked ligand-induced receptor immobilization. It is concluded from this observation that the effect of amphotericin B is not related to its ionophoretic properties but more likely to its capacity to interact with membrane cholesterol. When cells were incubated at 37 °C extensive patching of lectin receptors could be observed. This process was also inhibited by amphotericin B. A model is proposed to account for a role of cholesterol in ligand-induced receptor immobilization and patching.     

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B‐chain

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome‐associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5–90 min) and was later (15–90 min) partially translocated to cytosol as A‐ and B‐chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell‐free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time‐dependent translocation of ricin across the endosomal membrane occurred in cell‐free endosomes. Endosome‐located thioredoxin reductase‐1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase‐9 and caspase‐3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B‐chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B‐chain with mitochondria is involved in ricin‐induced apoptosis.     

Structural Analysis of Toxin-Neutralizing,Single-Domain Antibodies that Bridge Ricin’s A-B Subunit Interface

Ricin toxin kills mammalian cells with notorious efficiency. The toxin’s B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin’s A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin’s individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (V_HHs) that span the RTA-RTB interface, including four Tier 1 V_HHs with IC₅₀ values <1 nM. Crystal structures of each V_HH bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA’s F-G loop (mode 1), RTB’s subdomain 2γ (mode 2) or both (mode 3). V_HHs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2γ Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 V_HHs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin’s cytotoxic pathway.     

Defined sites of interaction between subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the stator structure of the vacuolar H+-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.     

Selection and characterization of peptide memitopes binding to ricin

A combinatorial random peptide display library expressed in E. coli was employed to identify short, linear peptide sequences that showed affinity for ricin and could be used as reagents for detection and identification of ricin. One peptide, P3, from a collection of four short peptides showed specific binding to ricin. The kinetic analysis of this peptide binding to the ricin showed lower equilibrium binding constants for the peptide P3 than monoclonal antibody. This is attributed due to both slower association and faster dissociation rates for the peptide P3. The random ricin peptide P3 binds to ricin with a K_D of 1 M versus the antibody's K_D of 14 nM. This particular peptide memitope P3 against ricin showed specific binding to ricin without any significant cross-reactivity against other proteins such as bovine serum albumin (BSA), lysozyme and natural bacterial toxins such as Staphylococcal enterotoxins A and B. The results provided proof-of-principal that peptide memitopes are another choice of reagents due to ease in production to be used for the detection of highly toxic bio-threat or biowarfare agents such as ricin.     

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.