Topology of diphtheria toxin B fragment inserted in lipid vesicies

Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells In response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresls, transferred to a membrane and their N-terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194–223, 266–375 and 429–528. The secondary structures of the peptides inserted in the membrane, determined by Fourier-transformed infrared spectroscopy, were shown to be mostly α-helices and β-sheets (44% and 53%, respectively). On the basis of these data and the recently published X-ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.     

Specific chemical cleavage of diphtheria toxin with hydroxylamine. Purification and characterization of the modified proteins

Specific chemical cleavage of diphtheria toxin with hydroxylamine was performed to remove peptides of 10 and 7 kDa from the carboxyl terminus. The resulting modified proteins of 51 and 48 kDa (HA51DT and HA48DT, respectively) were purified and characterized with respect to structural and biological properties. The 51-kDa toxin binds to ATP-agarose, as does intact diphtheria toxin, while HA48DT does not bind to the nucleotide matrix. Neither modified toxin binds to the membranes of diptheria toxin-sensitive cells, and, consequently, neither is toxic. However, when covalently linked to a membrane binding moiety, both HA51DT and HA48DT are toxic. Cell-killing ability during a short exposure time indicated that concanavalin A (Con A) derivatives of diphtheria toxin and HA51DT are equally toxic, ConA HA48DT being somewhat less toxic, while the conjugate of ConA to A-chain kills a small number of cells only at inordinately high concentration (1 microM). We have thus separated the cell membrane binding function of diphtheria toxin from its membrane permeation function by removing specific small peptides from the carboxyl terminus. These modified toxins may have applications in the preparation of highly potent hybrid toxins.     

Interactions of diphtheria toxin with lipid vesicles: determinants of ion channel formation

Lipid vesicles have been utilized to study the interactions of diphtheria toxin (DT) with membranes. The assay for DT ion channel formation was fluorescence-detected membrane potential depolarization of vesicles in which valinomycin-induced potassium diffusion gradients had been generated. The following requirements for ion channel formation have been identified: (1) acid pH (less than 5); (2) trans-negative membrane potentials (35-fold increase in channel-forming activity from -6 mV to -59 mV); and (3) negatively charged phospholipid headgroups (about 100-fold more activity using vesicles formed from asolectin compared to soybean phosphatidylcholine). Concentration dependence plots of toxin activity showed a linear response with logarithmic slopes of nearly one for each lipid composition. These results show a close parallel to those obtained previously with planar lipid bilayers and thus provide guidelines for conditions which facilitate functional insertion of the toxin into vesicles.     

Lipid interaction of diphtheria toxin and mutants. A study with phospholipid and protein monolayers

To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface-pressure-increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm). DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm). The low-pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.     

Intracellular localization and degradation of diphtheria toxin

The internalization of surface-bound diphtheria toxin (DT) in BS-C-1 cells correlated with its appearance in intracellular endosomal vesicles; essentially no toxin appeared within secondary lysosomal vesicles. In contrast, internalized epidermal growth factor (EGF) was localized within both endosomal and lysosomal vesicles. Upon preincubation of cells with leupeptin, a lysosomal protease inhibitor, a threefold increase in the accumulation of EGF into lysosomes was observed. Under identical conditions, essentially all of the diphtheria toxin remained within endosomes (less than 2% of the intracellular diphtheria toxin accumulated in the lysosomal fraction), indicating that the inability to detect diphtheria toxin in lysosomes was not due to its rapid turnover within this vesicle. Following internalization of EGF or DT, up to 40% of the ligand appeared in the medium as TCA-soluble radioactivity. EGF degradation was partially leupeptin-sensitive and markedly NH4Cl-sensitive, indicating lysosomal degradation. In contrast, DT A-fragment degradation was resistant to these inhibitors, while B-fragment showed only partial sensitivity. These data suggest that the bulk of endocytosed diphtheria toxin is localized within endosomes and degraded by a pathway essentially independent of lysosomes.     

Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo

A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target. Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits. Assessment of the subsequent post-endosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus. In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4). Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH. Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu. Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and/or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes.     

Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.

A monkey cDNA (pDTS) encoding a diphtheria toxin (DT) sensitivity determinant was isolated by expression cloning in mouse L-M cells. Mouse cells are naturally resistant to DT, because they lack functional cell surface receptors for the toxin. Unlike wild-type L-M cells, pDTS-transfected mouse cells are extremely toxin sensitive and specifically bind radioiodinated DT. Intoxication of the transfected cells requires receptor-mediated endocytosis of the bound toxin. The cDNA is predicted to encode an integral membrane protein that is identical to the precursor of a heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor that DT exploits as a receptor.     

Characterization of diphtheria toxin's catalytic domain interaction with lipid membranes

In response to a low environmental pH and with the help of the B fragment (DTB) the catalytic domain of diphtheria toxin (DTA) crosses the endosomal membrane to inhibit protein synthesis. In this study, we investigated the interaction of DTA with lipid membranes by biochemical and biophysical approaches. Data obtained from proteinase K and trypsin digestion experiments of membrane-inserted DTA suggested that residues 134-157 may adopt a transmembrane orientation and residues 77-100 could be membrane-associated, adopting either a surface or a transmembrane orientation. Fourier transform infrared spectroscopy analysis (FTIR) was used to characterize the secondary and tertiary structure of DTA along its pathway, from the native secreted form at pH 7.2 to the refolded structure at neutral pH after interaction with and desorption from a lipid membrane. We found that the association of DTA with lipid membranes at low pH was characterized by an increase of beta-sheet structures and that the refolded structure at neutral pH after interaction with the membrane was identical to the native structure at the same pH. We also investigated the desorption of DTA from the membrane at neutral pH as a function of temperature. Although a complete desorption was observed at 37 degrees C, no desorption took place at 4 degrees C. A model of translocation involving the possibility that DTA might insert one or several transient transmembrane domains during translocation is discussed.     

A superactive hormonotoxin prepared with truncated diphtheria toxin

A chemically truncated form of diphtheria toxin, DT51, which lacks the cell-binding site but retains the membrane-translocating function, was covalently linked to luteinizing hormone (LH) and compared to similar conjugates containing diphtheria toxin (DT) or diphtheria toxin A-chain (DTA). The DT51 hormonotoxin killed cells possessing an LH receptor at concentrations similar to that of DT hormonotoxin and orders of magnitude lower than DTA hormonotoxin. The DTA hormonotoxin exhibited an LD-50 similar to that of previously reported hormonotoxins which employed DTA, ricin A-chain, or gelonin as toxic moieties.     

Characterization of diphtheria toxin's catalytic domain interaction with lipid membranes

In response to a low environmental pH and with the help of the B fragment (DTB) the catalytic domain of diphtheria toxin (DTA) crosses the endosomal membrane to inhibit protein synthesis. In this study, we investigated the interaction of DTA with lipid membranes by biochemical and biophysical approaches. Data obtained from proteinase K and trypsin digestion experiments of membrane-inserted DTA suggested that residues 134-157 may adopt a transmembrane orientation and residues 77-100 could be membrane-associated, adopting either a surface or a transmembrane orientation. Fourier transform infrared spectroscopy analysis (FTIR) was used to characterize the secondary and tertiary structure of DTA along its pathway, from the native secreted form at pH 7.2 to the refolded structure at neutral pH after interaction with and desorption from a lipid membrane. We found that the association of DTA with lipid membranes at low pH was characterized by an increase of β-sheet structures and that the refolded structure at neutral pH after interaction with the membrane was identical to the native structure at the same pH. We also investigated the desorption of DTA from the membrane at neutral pH as a function of temperature. Although a complete desorption was observed at 37 °C, no desorption took place at 4 °C. A model of translocation involving the possibility that DTA might insert one or several transient transmembrane domains during translocation is discussed.     

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.     

Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane binding. A site-directed spin labeling study

The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of delta-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an alpha-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes.     

Characterization of high-order diphtheria toxin oligomers

In 3D domain swapping, a domain of a protein breaks its noncovalent bonds with the protein core and its place is taken by the identical domain of another molecule, creating a strongly bound dimer or higher order oligomer. For some proteins, including diphtheria toxin, 3D domain swapping may affect protein function. To explore the molecular basis of 3D domain swapping in a well-characterized protein system, domain-swapped oligomers of diphtheria toxin were produced by freezing and thawing under a variety conditions, including in various salts and buffers, and at various temperatures. Reaction yields were followed by high-performance size-exclusion chromatography. The traditional low pH pulse produced by freeze-thawing in mixed sodium phosphate buffer induces the oligomerization of DT, but the addition of alkali chloride salts was found to increase the yield in the order of Li(+) > Na(+) > K(+). Unexpectedly, oligomers also formed when DT was frozen and thawed in the presence of 1 M LiCl alone. Slower freezing and thawing of the mixture led to the production of more and larger oligomers. DT oligomers were also produced by exposure to acidic buffers, and were found by electron microscopy to adopt both linear and cyclized forms in a wide distribution of sizes. Upon the basis of these results, the model for the production of DT oligomers by freezing and thawing was expanded to include a salt-mediated pathway. We present a mechanism for the formation of high-order DT oligomers by acidification that takes into account domain swapping and hydrophobic interactions.     

Computer modelling of the transmembrane channel formed by a CNBr peptide of diphtheria toxin B fragment

Abstract Diphtheria toxin (DT) forms transmembrane, voltage-dependent channels in a planar lipid bilayer. Channels with similar characteristics were obtained with CB1, a cyanogen bromide peptide of diphtheria toxin B fragment (DTB) (res 340–459). Tryptophan 398 is in interaction with the hydrophobic core of the lipid bilayer. Using the Eisenberg method in association with the Shiffer-Edmunson wheel representation, we have identified two amphipathic α-helices within CB1 (res 346–364 and 389–406) that could be involved in the interaction with lipids. Bearing this information in mind, we are providing a model for the structure of the CB1 channel.     

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.     

Amino acid substitution in alpha-helix 7 of Cry1Ac delta-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner.

Insecticidal proteins or delta-endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests. The toxins are formed of three structural domains. The N-terminal domain is a bundle of eight alpha-helices and is implicated in pore formation in insect midgut epithelial membranes. All the delta-endotoxins share a common hydrophobic motif of eight amino acids in alpha-helix 7. A similar motif is also present in fragment B of diphtheria toxin (DT). Site-directed mutagenesis of Cry1Ac delta-endotoxin of B. thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B. The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild-type toxin. Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild-type toxin.     

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.     

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.     

Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance

The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membranes depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly.

We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.