A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity

The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.     

Interaction of diphtheria toxin B subunit with sensitive and insensitive mammalian cells

The recombinant fluorescent derivative of diphtheria toxin (EGFP-SbB) obtained by the replacement of toxin A subunit by enhanced green fluorescent protein (EGFP) has been used for visualization of the interaction of diphtheria toxin (DT) with sensitive and insensitive cells. It was shown that EGFP-SbB could interact with cell surface of both toxin-sensitive monkey cells (Vero cell line) and toxin-resistant mouse cells (3T3 cell line). The affinity of this protein for receptors of Vero cells was three times higher as compared with 3T3 cells. It was demonstrated that fluorescent derivate was able to interact with receptors of both cell lines and to internalize into these cells. Internalization of EGFP-SbB into the cells was inhibited by endocytosis inhibitor phenyl arsine oxide. We suppose that diverse sensitivity to DT of monkey and mouse cells can be explained not only by differences in their receptor affinity for DT but also by the processes that occur after internalization of the toxin into the cells.     

Quantal entry of diphtheria toxin to the cytosol

The rate-limiting step in diphtheria toxin (DT) intoxication of Vero cells has been determined utilizing cycloheximide as an inhibitor of the intoxication process. Cycloheximide is shown to inhibit the toxin catalyzed ADP-ribosylation of elongation factor 2 (EF-2). The inhibition is blocked by puromycin thus establishing the ribosome as the location of cycloheximide protection. Washing cells free of cycloheximide rapidly reverses the protective effect. The initial rates of protein synthesis inhibition observed after removal of cycloheximide from DT-intoxicated cells are 5 to 12-fold greater than rates observed in unprotected cells and are shown to reflect ADP-ribosylation of EF-2 by cytosolic DT. Ten to thirty minutes after cycloheximide removal, the rate of protein synthesis inhibition abruptly changes to values identical to those of unprotected cells. Both the initial rates and extent of the initial rapid inactivation are directly related to toxin concentration and time of incubation with DT in the presence of cycloheximide. We concluded that: the rate-limiting step in protein synthesis inhibition by DT is not the ADP-ribosylation of EF-2 by cytosolic toxin but rather the earlier entry step of DT into the cytosol. DT enters the cytosol as a bolus of sufficient size to rapidly inactivate all EF-2 in that cell. It is inferred from 1 and 2 that the first order inactivation rate exhibited by DT is the result of the probability of the release of a bolus of toxin to the cytosol of any cell in the population per unit time. Autoradiographic analysis of intoxicated cell populations support this two-population state model. The size of a single bolus or quantum of DT is calculated from data over the range of 10(-11) to 10(-9) M DT and is found to remain constant. We suggest that the cytosolic entry mechanism of DT results from a unique ability of the internalized toxin molecules to destabilize the vesicular membrane resulting in a random release of a bolus of toxin into the cytosol. Because the bolus size remains constant over a 50-fold change in receptor occupancy the possibility is raised that DT undergoes a post-receptor packaging process, package size remaining a constant and package number increasing with receptor occupancy.     

Requirement of specific receptors for efficient translocation of diphtheria toxin A fragment across the plasma membrane

The role of specific receptors in the translocation of diphtheria toxin A fragment to the cytosol and for the insertion of the B fragment into the cell membrane was studied. To induce nonspecific binding to cells, toxin was either added at low pH, or biotinylated toxin was added at neutral pH to cells that had been treated with avidin. In both cases large amounts of diphtheria toxin became associated with the cells, but there was no increase in the toxic effect. There was also no increase in the amount of A fragment that was translocated to the cytosol, as estimated from protection against externally added Pronase E. In cells where specific binding was abolished by treatment with 12-O-tetradecanoyl-phorbol 13-acetate, trypsin, or 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, unspecific binding did not induce intoxication or protection against protease. This was also the case in untreated L cells, which showed no specific binding of the toxin. When Vero cells with diphtheria toxin bound to specific receptors were exposed to low pH, the cells were permeabilized to K+, whereas this was not the case when the toxin was bound nonspecifically at low pH or via avidin-biotin. The data indicate that the cell-surface receptor for diphtheria toxin facilitates both insertion of the B fragment into the cell membrane and translocation of the A fragment to the cytosol.     

Energy requirements for diphtheria toxin translocation are coupled to the maintenance of a plasma membrane potential and a proton gradient

Translocation of diphtheria toxin (DT) or ricin to the cytosol is the rate-limiting step responsible for (pseudo) first-order decline in protein synthesis observed in intoxicated cell populations. The requirements for energy utilization in the translocation of both toxins are examined by perturbing the intoxication during this period of protein synthesis decline. Translocation of either toxin is blocked at 4 degrees C and requires energy. Ricin translocation is tightly coupled to ATP hydrolysis with no involvement of membrane potential. Cell depolarization slows the rate of DT translocation but does not block completely. Elimination of transmembrane pH gradients alone does not affect DT translocation; however, in combination with depolarization, translocation is blocked virtually completely. Energy requirements for DT intoxication are mediated by establishing a plasma membrane potential and a pH gradient across some cellular membrane. It is proposed that a postendocytotic vesicle containing processed DT fuses with the plasma membrane. Either component of the proton motive force across the plasma membrane then drives DT translocation. Ricin apparently utilizes a different energy coupling mechanism at a different intracellular site, thus demonstrating toxin specificity in the translocation mechanism.     

ADP-ribosylation of a small size GTP-binding protein in bovine neutrophils by the C3 exoenzyme of Clostridium botulinum and effect on the cell motility.

A 24-kDa G protein, ADP-ribosylable by exoenzyme C3 from Clostridium botulinum and therefore related to the rho family, was found to be abundantly present in human and bovine neutrophils, and preferentially located in cytosol. In human myeloid HL60 cells, the amount of C3 substrate increased during differentiation of the HL60 cells into granulocytes. The effect of exoenzyme C3 on different functions of bovine neutrophils, namely generation of O-2, degranulation and chemotaxis, has been tested, using electropermeabilized cells. Exoenzyme C3 hardly affected the respiratory burst and the degranulation. In contrast, it efficiently inhibited the spontaneous and chemoattractant-induced motility of the cells and disorganized the actin microfilament assembly.     

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.     

The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.     

Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells

Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (approximately 65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.     

Entry of diphtheria toxin linked to concanavalin A into primate and murine cells

Diphtheria toxin linked by a disulfide bridge to concanavalin A was highly toxic to HeLa S3 and Vero cells, as well as to murine L cells. The cells could be protected with alpha-methyl mannoside, indicating that the conjugate binds mainly through its concanavalin A moiety. Treatment of Vero cells with phospholipase C, TPA (12-O-tetradecanoylphorbol-13-acetate), and vanadate, which strongly reduce the ability of the cells to bind free diphtheria toxin, had little protective effect against the conjugate, whereas SITS (L-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid), which inhibits diphtheria toxin binding, as well as the subsequent entry, protected Vero cells, but not L cells. Both types of cells are protected against the conjugate by NH4Cl and monensin, indicating that an acidified compartment is necessary for entry into the cytosol. Exposure of cells, bound with surface conjugate, to low pH induced entry of the toxin into Vero cells, but not into L Cells. Phospholipase C, TPA, and vanadate did not protect L cells against the conjugate. It is concluded that toxin in the conjugate enters L cells by a route which involves low pH, but which is not identical to that in Vero cells.     

The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells.

Clostridium botulinum C3 is a recently discovered exoenzyme that ADP-ribosylates a eukaryotic GTP-binding protein of the ras superfamily. We show now that the bacterially-expressed product of the human rhoC gene is ADP-ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP-ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.     

Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes

The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.     

The role of the diphtheria toxin receptor in cytosol translocation

The role of the receptor in the transport of diphtheria toxin (DT) to the cytosol was examined. A point-mutant form of DT, CRM 107 (CRM represents cross-reacting material), that has an 8,000-fold lower affinity for the DT receptor than native toxin was conjugated to transferrin and monoclonal antibodies specific for the cell-surface receptors T3 and Thy1. Conjugating the binding site-inactivated CRM 107 to new binding moieties reconstituted full toxicity, indistinguishable from native DT linked to the same ligand, indicating that the entry activity of the DT B chain can be fully separated from the receptor binding function. Like DT, the toxin conjugates exhibited a dose-dependent lag period before first-order inactivation of protein synthesis. Inactivation of the binding site of the toxin portion of the conjugate was found to have no effect on the kinetics of protein synthesis inactivation. The receptor used by the toxin determined the length of the lag period relative to the killing rate. Comparing the potency of CRM 107 conjugates with native DT, standardized for receptor occupancy, shows that new receptors can be as or more efficient than the DT receptor in transporting DT to the cytosol. The transferrin-CRM 107 conjugate, unlike native DT, was highly toxic to murine cells. All the data presented are consistent with a model that the DT receptor, other than initiating rapid internalization of the toxin to low pH compartments, is unnecessary for transport of the toxin to the cytosol and that membrane translocation activity is expressed by the DT B subunit independent of the receptor-binding site.     

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.     

Entry of diphtheria toxin-protein A chimeras into cells

Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.     

Enhancement of diphtheria toxin-induced apoptosis in Vero cells by combination treatment with brefeldin A and okadaic acid

In the present study, we compared the abilities of ricin and diphtheria toxin to induce apoptosis in Vero cells. The cytolysis and DNA fragmentation by ricin paralleled its protein synthesis inhibitory activity. However, unlike ricin, diphtheria toxin could induce neither cytolysis nor DNA fragmentation in Vero cells up to very high concentration, in spite of the fact that Vero cells were even more sensitive to protein synthesis inhibition by diphtheria toxin than ricin. Interestingly, coexistence of brefeldin A (BFA) and okadaic acid (OA) significantly enhanced diphtheria toxin-mediated cytolysis and DNA fragmentation without affecting the activity of protein synthesis inhibition. Ammonium chloride almost completely abolished the ability of diphtheria toxin to induce apoptosis in the presence of BFA and OA as well as the protein synthesis inhibitory activity. The mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2), failed to induce apoptosis in Vero cells even in the presence of BFA and OA. Thus, translocation of diphtheria toxin into the cytosol and subsequent enzymatic inactivation of EF-2 may be necessary steps to induce apoptosis. Taken together our results suggest that protein synthesis inhibition by toxins is not sufficient to induce apoptosis, and underlying mechanisms of apoptosis induction may be distinct between ricin and diphtheria toxin. Since a morphological change in the Golgi complex was observed in Vero cells treated with BFA and OA, modulation of the Golgi complex by these reagents may be partly responsible for enhanced apoptosis induction by diphtheria toxin.     

Evidence that membrane phospholipids and protein are required for binding of diphtheria toxin in Vero cells

Treatment with phospholipase C strongly protected monkey kidney (Vero) cells against diphtheria toxin and reduced the ability of the cells to bind 125I-labelled toxin. Treatment with phospholipase D and with trypsin also protected the cells, although to a lesser extent. Phospholipase A2 had no protective effect. Phospholipase C also protected fetal hamster kidney cells against the toxin. After removal of the enzymes, as well as after treatment of the cells with 4-acetamide 4'-isothiocyanostilbene 2,2'-disulfonic acid, diphtheria toxin binding capability was restored slowly, apparently by a process requiring protein synthesis, since cycloheximide blocked the restoration. The data indicate that both phospholipids and protein are involved in the binding sites for diphtheria toxin.     

Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin

We have studied the cytotoxicity of ricin in cells treated with brefeldin A (BFA), which dramatically disrupts the structure of the Golgi apparatus causing Golgi content and membrane to redistribute to the ER. BFA inhibits the cytotoxicity of ricin in Chinese hamster ovary, normal rat kidney, and Vero cells and abolishes the enhancement of ricin cytotoxicity by NH4Cl, nigericin, swainsonine, and tunicamycin or by a mutation in endosomal acidification. BFA protects cells from the cytotoxicities of modeccin and Pseudomonas toxin, but has no effect on the intoxication by diphtheria toxin. Pretreatment of BFA does not protect cells from ricin treatment in the absence of BFA. Our results suggest that ricin, modeccin, and Pseudomonas toxin share a common pathway of intracellular transport from endosomes to the Golgi region where they are released into the cytosol. In contrast, the lack of protection of Vero cells from diphtheria toxin by BFA indicates that diphtheria toxin is released from acidified endosomes without involving the Golgi region.     

Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B

Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by (86) Rb(+) release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.     

Characterization of the diphtheria toxin receptor-binding domain

The carboxyl-terminal region of diphtheria toxin (DT) has been analysed in order to determine regions of receptor recognition. Biochemical cleavage of the toxin with hydroxylamine (HA) was used to generate the peptides HA9DT (residues 454–535), HA6DT (residues 482–535), and HA3DT (residues 454–461). Characterization of HA6DT demonstrated that the final 54 amino acids of DT are sufficient to constitute the receptor-binding domain of the toxin. Within HA9DT, the region encompassing HA3DT and containing the highly cationic polyphosphate-binding site did not contribute to the binding ability of HA6DT. Consistent with this observation, HA3DT itself did not compete for binding of radiolabelled DT to Vero cells. A 30-amino acid synthetic peptide composed of residues 506–535 did not block receptor binding of DT, indicating that residues toward the amino-terminus of HA6DT, or the entire HA6DT region, are required for receptor recognition.