GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.

We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.     

CD9 amino acids critical for upregulation of diphtheria toxin binding.

CD9 associates with a diphtheria toxin receptor (DTR) that is identical to the membrane-anchored form of heparin-binding EGF-like growth factor. We determined the region of CD9 important for upregulation activity. Human and monkey CD9 upregulates DT binding activity of DTR, while mouse CD9 has no upregulation activity. Transfection of chimeric constructs comprising monkey and mouse CD9s showed that the human sequence between Ala156 and Asp183 is essential for the upregulation activity. Studies of mutants, replacing a single amino acid within the region between Ala156 and Asp183 of monkey CD9 with the corresponding amino acid residue in mouse CD9, revealed that substitution of Gly158 is critical for the reduction of the upregulation activity and secondly for the substitution of Val159 and Thr175. These three amino acid residues were deduced to be located on the head domain of the second extracellular loop, suggesting that interactions of CD9 with DTR or DT at the domain containing these three amino acids were important for the upregulation of DT binding.     

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes -helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery

After reaching early endosomes by receptor-mediated endocytosis, diphtheria toxin (DT) molecules have two possible fates. A large pool enters the degradative pathway whereas a few molecules become cytotoxic by translocating their catalytic fragment A (DTA) into the cytosol. Impairment of DT degradation by microtubule depolymerization does not block DT cytotoxicity. Therefore, DTA membrane translocation into the cytosol occurs from an endocytic compartment located upstream of late endosomes. Comparisons between early endosomes and endocytic carrier vesicles in a cell-free translocation assay have demonstrated that early endosomes are the earliest endocytic compartment from which DTA translocates. DTA translocation is ATP-dependent, requires early endosomal acidification, and is increased by the addition of cytosol. Cytosol-dependent DTA translocation is GTPγS-insensitive but is blocked by anti-βCOP antibodies.     

A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration

A new system for lineage ablation is based on transgenic expression of a diphtheria toxin receptor (DTR) in mouse cells and application of diphtheria toxin (DT). To streamline this approach, we generated Cre-inducible DTR transgenic mice (iDTR) in which Cre-mediated excision of a STOP cassette renders cells sensitive to DT. We tested the iDTR strain by crossing to the T cell- and B cell-specific CD4-Cre and CD19-Cre strains, respectively, and observed efficient ablation of T and B cells after exposure to DT. In MOGi-Cre/iDTR double transgenic mice expressing Cre recombinase in oligodendrocytes, we observed myelin loss after intraperitoneal DT injections. Thus, DT crosses the blood-brain barrier and promotes cell ablation in the central nervous system. Notably, we show that the developing DT-specific antibody response is weak and not neutralizing, and thus does not impede the efficacy of DT. Our results validate the use of iDTR mice as a tool for cell ablation in vivo.     

Refined structure of monomeric diphtheria toxin at 2.3 A resolution.

The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 A resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3'-5' uridine 3' monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the "open" monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000-0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main-chain conformations of 8 residues acting as a hinge to "open" or "close" the receptor-binding (R) domain, and (2) a possible receptor-docking site, a beta-hairpin loop protruding from the R domain containing residues that bind the cell-surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5-step structure-based mechanism of intoxication: (1) proteolysis of a disulfide-linked surface loop (residues 186-201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a beta-hairpin loop protruding from the R domain to the DT receptor, leading to receptor-mediated endocytosis; (3) low pH-triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP-ribosylation of elongation factor 2.     

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.     

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.     

A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.     

Oligomerization of membrane-bound diphtheria toxin (CRM197) facilitates a transition to the open form and deep insertion

Diphtheria toxin (DT) contains separate domains for receptor-specific binding, translocation, and enzymatic activity. After binding to cells, DT is taken up into endosome-like acidic compartments where the translocation domain inserts into the endosomal membrane and releases the catalytic domain into the cytosol. The process by which the catalytic domain is translocated across the endosomal membrane is known to involve pH-induced conformational changes; however, the molecular mechanisms are not yet understood, in large part due to the challenge of probing the conformation of the membrane-bound protein. In this work neutron reflection provided detailed conformational information for membrane-bound DT (CRM197) in situ. The data revealed that the bound toxin oligomerizes with increasing DT concentration and that the oligomeric form (and only the oligomeric form) undergoes a large extension into solution with decreasing pH that coincides with deep insertion of residues into the membrane. We interpret the large extension as a transition to the open form. These results thus indicate that as a function of bulk DT concentration, adsorbed DT passes from an inactive state with a monomeric dimension normal to the plane of the membrane to an active state with a dimeric dimension normal to the plane of the membrane.     

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.     

Domain analysis of the tetraspanins: studies of CD9/CD63 chimeric molecules on subcellular localization and upregulation activity for diphtheria toxin binding

CD9 and CD63 belong to a tetramembrane-spanning glycoprotein family called tetraspanin, and are involved in a wide variety of cellular processes, but the structure-function relationship of this family of proteins has yet to be clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activity in spite of the fact that this protein also associates with DTR/proHB-EGF on the cell surface. CD9 molecules localize on the cell surface, while those of CD63 localize predominantly at lysosomes and intracellular compartments. We made CD9/CD63 chimeric molecules and then studied their intracellular localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effect on the expression of the chimeric proteins at the cell surface, while mutants lacking the lysosome sorting motif delivered more efficiently on the cell surface, indicating that the lysosome sorting motif contributes to the inhibitory effect of the C-terminal region. However, the N-terminal half of this family of proteins containing the 1st to 3rd transmembrane domains also seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essential, while the remaining regions influenced the upregulation activity by changing the efficiency of cell surface expression. From these results we discussed the structure-function relationship of this family of proteins.     

Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B fragment. Effect on membrane translocation and on formation of cation channels.

Diphtheria toxin B fragment is capable of forming cation-selective channels in the plasma membrane. Such channels may be involved in the translocation of the toxin A fragment to the cytosol. Seven negatively charged amino acids in the B fragment were replaced one by one by lysines, followed by studies of cytotoxicity and channel-forming ability of the different mutants. The mutant D392K showed a strong reduction in binding to cell surface receptors. Of the six mutants that showed wild-type binding affinity, the two mutants D295K and D318K were very inefficient in forming channels. These two mutants had the lowest ability to mediate A fragment translocation. The mutant E362K was able both to induce cation channel formation and to mediate A fragment translocation at a higher pH value than the wild-type B fragment. The results support the notion that formation of cation channels is of importance for the translocation of the A fragment across the plasma membrane, and they indicate that the pH requirement for translocation of the A fragment to the cytosol is partly determined by the B fragment.     

The effect of receptor rapid-internalization signals on diphtheria toxin endocytosis and cell sensitivity

Diphtheria toxin enters toxin-sensitive mammalian cells by receptor-mediated endocytosis employing the heparin-binding EGF-like growth factor precursor as its receptor. We reported previously (Almond and Eidels, 1994) that cytoplasmic domain mutants of the toxin receptor and cells expressing wild-type receptor internalize toxin slowly, the rate being approximately that of normal turnover of the plasma membrane. To determine whether it was possible to increase toxin sensitivity by increasing the rate of toxin internalization, we constructed diphtheria toxin cytoplasmic domain mutant cell lines containing rapid-internalization signals from either the low density lipoprotein receptor or from the lysosomal acid phosphatase precursor. Although cells transfected with mutant receptor genes internalized toxin at a faster rate than those expressing the wild-type receptor, they showed a decrease in toxin sensitivity. This decreased sensitivity may be accounted for by an observed decrease in the number of toxin-binding sites and by an increased rate of toxin internalization and degradation. These results suggest that the rate of toxin internalization may not be the rate-limiting step in the cytotoxic process.     

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre‐loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site‐directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine‐rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild‐type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.     

Temporal separation of protein toxin translocation from processing events

Intoxication of Vero cells by ricin, modeccin, diphtheria toxin (DT), and Pseudomonas exotoxin A requires: 1) binding to cell surface receptors; 2) transport to the cytoplasm; and 3) enzymatic inactivation of a component of the protein synthetic machinery. The kinetic profiles of all four toxins consist of a lag followed by the apparent first-order decrease in protein synthesis. Autoradiographic analysis of DT-intoxicated cell populations has demonstrated that two subpopulations of cells exist during the period of decreasing protein synthesis: one population synthesizing at control levels and the other synthesizing little or no protein (Hudson, T. H., and Neville, D. M., Jr. (1985) J. Biol. Chem. 260, 2675-2680). The present study correlates the autoradiographic data with the rates of protein synthesis decline in cells intoxicated with modeccin, ricin, Pseudomonas exotoxin A, as well DT. In all cases, the first time point which exhibits a decrease in protein synthetic activity also exhibits two subpopulations of cells, one synthesizing protein at control rates and the other synthesizing little or no protein. As the intoxication progresses, cells leave the control population by the rapid cessation of all protein synthesis. These experiments demonstrate that transport of all four toxins to the cytosol is the rate-limiting step during the pseudo first-order decline in protein synthesis. Furthermore, the final step in the transport process (translocation) must result in the release to the cytoplasm of a quantity of toxin sufficient to rapidly inactivate all protein synthesis in that cell. The probability of a translocation event occurring in any cell of the population is established during the lag and remains constant throughout the first-order decrease in protein synthesis. The requirement for acidification during the intoxication by DT, Pseudomonas exotoxin A, or modeccin is restricted to the lag period. Acidification is therefore necessary to establish the probability of translocation, but it is not directly involved in the actual translocation of these toxins. The pseudo first-order passage of DT intoxications through antitoxin and NH4Cl- or monensin-sensitive stages are shown to have the same cellular basis as the pseudo first-order decrease in protein synthesis. A kinetic model is presented which defines the DT intoxication process from one of its earliest events (endocytosis) to its penultimate event (translocation of toxin to the cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin

A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N-terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N-terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti-ETA and anti-DT antisera. Furthermore, the chimaeric protein displayed ADP-ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.     

Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes.

Translocation is a necessary and rate-limiting step for diphtheria toxin (DT) cytotoxicity. We have reconstituted DT translocation in a cell-free system using endosomes purified from lymphocytes and have demonstrated this using two different probe/cell systems, which provided identical results: 125I-DT/human CEM cells and 125I-transferrin-DT/mouse BW cells. The cell-free DT translocation process was found to be dependent on the presence of the pH gradient endosome (pH 5.3)/cytosol (pH 7). Among the pH equilibrating agents, nigericin (5 microM) was found to be the most effective, inhibiting DT translocation by 88%. An optimum pH value of 7 on the cytosolic side of the membrane (pH gradient approximately 1.7) was determined. ATP per se is not required for DT translocation. 125I-DT translocation was 3-fold more active from late than from early endosomes, probably because of their slightly more acidic pH. Only the A chain of the toxin was found to escape from either 125I-DT/CEM or 125I-transferrin-DT/BW endosomes. Translocation of control endosome labels (125I-transferrin and 125I-horseradish peroxidase) was never observed. We also show that DT receptors present on resistant (mouse) cells block the translocation of the toxin and are responsible for the resistance of these cells to DT.     

Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin.

Acidic conditions within the endosomal lumen induce the T domain of receptor-bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A + D352A and fourfold for E349Q + D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.