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PubMed1979

Effects of pseudomonas toxin A, diphtheria toxin, and cholera toxin on electrical characteristics of turtle bladder.

Rapidly developing changes in the short-circuiting current (Isc), conductance (G), and potential (PD) of turtle bladders in Na-rich or Na-free media are seen after the mucosal addition, at 10 nM, of each of three toxins that contain ADP-ribosylation activity: Pseudomonas aeruginosa toxin A, diphtheria toxin, and cholera toxin. Toxin A irreversibility decreased the Isc, PD, and G of bladders in Na-rich media and the Isc and PD of bladders in Na-free media. Diphtheria or cholera toxin reversibly increased Isc and PD (not G), but only in Na-free media. The effects of toxin A in the turtle bladder, like those in other host cell systems, were eliminated by preexposure of this toxin to heat, specific antitoxin, or dithiothreitol and urea. Because exposure to this last condition increases the ADP-ribosylation activity of toxin A, it is suggested that the proenzyme is the required transport-inhibiting form of toxin A. The effects of all three toxins occurred rapidly, possibly before any of the possible intracellular ADP-ribosylation reactions are initiated. Whereas a recognition binding of toxin of toxin to receptors on the apical membrane completely accounts for the reversible effects of diphtheria or cholera toxin, this and additional toxin-membrane interactions (e.g., translocation) are needed to account for the irreversible effects of toxin A.

Animals↗
PubMed1994

Effect of lovastatin on the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in brefeldin A-sensitive and -resistant cell lines.

Lovastatin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits the biosynthesis of cholesterol and the prenylation of proteins. In this study, we have found that lovastatin inhibited the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in Vero and a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40) to different extents. Among these toxins tested, the cytotoxicity of modeccin was most strongly inhibited by lovastatin in a dose-dependent manner. The protective effect of lovastatin was completely reversed by the addition of mevalonic acid, while the addition of cholesterol had no effect on the cytotoxicity of modeccin in lovastatin-treated cells. These results suggest that prenylated proteins are involved in the intoxication process of modeccin. The addition of cycloheximide to the growth medium also reversed the protective effect of lovastatin, suggesting a requirement of de novo protein synthesis for the protection by lovastatin against toxins. In contrast to Vero and BER-40 cells, no significant effect of lovastatin was observed in naturally BFA-resistant cell lines, PtK1 and MDCK cells, even though similar morphological changes and disassembly of the actin microfilaments were induced by lovastatin in these cell lines as observed in Vero and BER-40 cells. Lovastatin did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells. Our results suggest that prenylated cellular proteins are involved in intracellular trafficking or processing of protein toxins, especially modeccin. In PtK1 and MDCK cells, such intracellular vesicle trafficking of protein toxins may be regulated by lovastatin-resistant mechanisms.

ADP Ribose Transferases↗
PubMed1993

Cerulenin inhibits the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in brefeldin A-resistant cell lines.

We have found that cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis and fatty acylation of proteins, strongly inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40). The protective effect of cerulenin against ricin was also observed in two other BFA-resistant cell lines, Madin-Darby canine kidney, and PtK1 cells. In contrast to BER-40 cells, no significant effect of cerulenin was observed in Vero cells. Cerulenin did not affect the binding of ricin to the cell-surface receptors, but reduced significantly the internalization of ricin in BER-40 cells; no effect of cerulenin on the binding or internalization of ricin was observed in Vero, PtK1, and Madin-Darby canine kidney cells. Endocytic uptake of fluid-phase markers such as horseradish peroxidase and lucifer yellow was inhibited by cerulenin in BER-40 cells, but the endocytosis of transferrin via the coated pit/coated vesicle pathway was slightly increased. Cerulenin inhibited the degradation and excretion of ricin in BER-40 cells, and this effect of cerulenin was not observed in Vero cells. Furthermore, cerulenin inhibited the bulk protein secretion in a dose-dependent manner, with BER-40 cells being more susceptible than Vero cells. These results suggest that in addition to its effect on endocytosis, cerulenin interferes with the intracellular trafficking or processing of toxin molecules, and the vesicle transport system in BER-40 cells appears to be cerulenin-sensitive. Since addition of fatty acids and cholesterol did not reverse the effects of cerulenin, the protective effect of cerulenin against protein toxins is not due to an inhibition of de novo fatty acids and cholesterol biosynthesis.

ADP Ribose Transferases↗
PubMed1995

Heparin-like molecules on the cell surface potentiate binding of diphtheria toxin to the diphtheria toxin receptor/membrane-anchored heparin-binding epidermal growth factor-like growth factor.

Diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF), has a high affinity for heparin. We studied the effect of heparin-like molecules on the binding of diphtheria toxin (DT) to DTR/proHB-EGF. Mutant Chinese hamster ovary (CHO) cells deficient in heparan sulfate (HS) proteoglycans were about 15 times less sensitive to DT than wild type CHO-K1 cells. When free heparan sulfate or heparin was added to the culture medium, DT sensitivity of the mutant cells was fully restored. Studies of binding of 125I-labeled DT to HS-deficient CHO cells transfected with human DTR/proHB-EGF cDNA indicated that the increased sensitivity to DT after addition of heparin is due to increased binding of DT to cells. Vero cells display a relatively large amount of heparan sulfate residues compared to CHO-K1 cells or L cells. Enhancement of DT binding by the addition of heparin was also observed with CHO-K1 cells and L cells that had been transfected with human DTR/proHB-EGF cDNA, but the degree of enhancement was less than that observed with the HS-deficient CHO cells. Addition of heparin did not affect DT binding or DT sensitivity of Vero cells. Heparin-dependent binding was observed when intact Vero cells were treated with heparitinase or when the cell membrane was solubilized with a neutral detergent. Scatchard plot analysis for the binding of DT to a recombinant HB-EGF in vitro and to L cells expressing human DTR/proHB-EGF revealed that heparin increases the affinity of DTR/proHB-EGF for DT but does not change the number of binding sites. Although DRAP27/CD9 is known to enhance DT binding to DTR/proHB-EGF, the results indicate that heparin and DRAP27/CD9 increase DT binding by independent mechanisms. Thus, heparin-like molecules, probably in the form of heparan sulfate proteoglycan on the cell surface, are a third factor required for maximal DT binding activity of cells.

Animals↗
PubMed1994

Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells.

To investigate the ability of anthrax toxin lethal factor (LF) to translocate foreign proteins into the cytosol of eukaryotic cells and to characterize the structural requirements of this process, fusion proteins containing a portion of LF and the catalytic domains of either diphtheria toxin or Shiga toxin were constructed. Previous work showed that residues 1 to 254 of anthrax toxin lethal factor (LF1-254) are sufficient for binding to the protective antigen component of the toxin and that portions of Pseudomonas exotoxin A fused to LF1-254 are efficiently translocated to the cytosol of eukaryotic cells (N. Arora and S. H. Leppla, J. Biol. Chem. 268:3334-3341, 1993). In this study, it was found that fusion proteins containing the ADP-ribosylation domain of diphtheria toxin fused at either the amino end or the carboxyl end of LF1-254 are highly toxic to Chinese hamster ovary (CHO) cells, indicating that translocation does not strictly require that the amino terminus of LF be free. A fusion protein containing the ribosome-inactivating A1 subunit of Shiga toxin fused to the carboxyl terminus of LF1-254 was also highly toxic for CHO cells. All fusion proteins were toxic only when administered with the anthrax toxin protective antigen component. The data show that the combination of protective antigen and LF fusion proteins can efficiently import polypeptides from diverse bacterial sources to the cytosol of eukaryotic cells and that LF fusion proteins may have the passenger polypeptides fused at either the amino terminus or the carboxyl terminus of LF1-254. These LF fusion proteins could potentially be used as components of a therapeutic agent when the destruction of certain types of cells is desired (e.g., in treating cancer).

Amino Acid Sequence↗
PubMed1993

Cytotoxic activities of recombinant immunotoxins composed of Pseudomonas toxin or diphtheria toxin toward lymphocytes from patients with adult T-cell leukemia.

We have previously shown that the recombinant single-chain immunotoxin anti-Tac(Fv)-PE40, composed of the variable domains of the anti-Tac monoclonal antibody in a single-chain form joined to a derivative of Pseudomonas exotoxin (PE), is cytotoxic toward malignant cells from adult T-cell leukemia (ATL) patients. Using this assay, we have now compared the activity of anti-Tac(Fv)-PE40 with that of an improved version, anti-Tac(Fv)-PE40KDEL which contains an altered carboxyl terminus, and also with two chimeric toxins made with diphtheria toxin (DT). One of these is a fusion of amino acids 1-388 of DT with anti-Tac(Fv) and is termed DT388-anti-Tac(Fv). The other, DT388-IL2, contains interleukin 2 (IL2) at the carboxyl terminus of the same DT derivative. We incubated these toxins with malignant ATL peripheral blood mononuclear cells (PBMCs) for 1-3 days and then measured [3H]leucine incorporation. We found that anti-Tac(Fv)-PE40KDEL was the most cytotoxic agent and was followed in decreasing order of activity by anti-Tac(Fv)-PE40, DT388-anti-Tac(Fv), and finally DT388-IL2. Trypan blue staining showed that inhibition of protein synthesis correlated with cell death. Time course studies showed that the recombinant toxins containing anti-Tac(Fv) were cytotoxic even if exposed to the cells for only one hour. After intravenous injection into mice, the half-life of anti-Tac(Fv)-PE40 or anti-Tac(Fv)-PE40KDEL was 30 minutes. Normal PBMCs were resistant to all four toxins. Recombinant immunotoxins made with anti-Tac merit further study as potential reagents in the treatment of ATL.

Adult↗
PubMed2000

Cell surface monkey CD9 antigen is a coreceptor that increases diphtheria toxin sensitivity and diphtheria toxin receptor affinity.

Monkey (Mk) CD9 antigen has been shown previously to increase the diphtheria toxin (DT) sensitivity of cells when co-expressed with Mk proHB-EGF (DT receptor). We have elucidated here the mechanism whereby Mk CD9 influences Mk proHB-EGF and present evidence that Mk CD9 is a coreceptor for DT. We observed that Mk CD9 not only increased the DT sensitivity but also increased the DT receptor affinity of cells. Furthermore, the higher the Mk CD9/Mk proHB-EGF ratio, the higher the affinity. In contrast, mouse (Ms) CD9 did not increase the toxin sensitivity or receptor affinity of cells when co-expressed with Mk proHB-EGF. Using Mk/Ms chimeric CD9 molecules, we determined that the second extracellular domain of Mk CD9 is responsible for both increased sensitivity and receptor affinity. This domain of Mk CD9 also interacts with Mk proHB-EGF in a yeast two-hybrid system. Our findings thus suggest that Mk CD9 has a direct physical interaction with Mk proHB-EGF to form a DT receptor complex and that this contact may change the conformation of the receptor to increase DT binding affinity and consequently increase toxin sensitivity. We thus propose that Mk CD9 is a coreceptor for DT.

Amino Acid Sequence↗
PubMed1979

Codominant translational mutants of Chinese hamster ovary cells selected with diphtheria toxin.

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a "universal hybridizer" (10).

Animals↗
PubMed2006

Expression and purification of a trivalent pertussis toxin-diphtheria toxin-tetanus toxin fusion protein in Escherichia coli.

Pertussis toxoid, diphtheria toxoid, and tetanus toxoid are key components of diphtheria-tetanus-acellular pertussis vaccines. The efficacy of the vaccines is well documented, however, the vaccines are expensive partly because the antigens are derived from three different bacteria. In this study, a fusion protein (PDT) composed of the immunoprotective S1 fragment of pertussis toxin, the full-length non-toxic diphtheria toxin, and fragment C of tetanus toxin was constructed via genetic means. The correct fusion was verified by restriction endonuclease analysis and Western immunoblotting. Escherichia coli carrying the recombinant plasmid (pCoPDT) produced a 161kDa protein that was recognized by antibodies specific to the three toxins. The expression of the PDT protein was inducible by isopropyl-beta-d-thio-galactoside but the total amount of protein produced was relatively low. Attempts to improve the protein yield by expression in an E. coli strain (Rosetta-gami 2) that could alleviate rare-codon usage bias and by supplementation of the growth media with amino acids deemed to be a limiting factor in translation were not successful. The PDT protein remained in the insoluble fraction when the recombinant E. coli was grown at 37 degrees C but the protein became soluble when the bacteria were grown at 22 degrees C. The PDT protein was isolated via affinity chromatography on a NiCAM column. The protein was associated with five other proteins via disulfide bonds and non-covalent interactions. Following treatment with beta-mercaptoethanol, the PDT fusion was purified to homogeneity by preparative polyacrylamide gel electrophoresis with a yield of 45 microg/L of culture. Antisera generated against the purified PDT protein recognized the native toxins indicating that some, if not all, of the native epitopes were conserved.

Animals↗
PubMed1982

Preparation and properties of chimeric toxins prepared from the constituent polypeptides of diphtheria toxin and ricin. Evidence for entry of ricin A-chain via the diphtheria toxin pathway.

A highly toxic conjugate of ricin A-chain and diphtheria toxin fragment B was prepared by disulfide exchange reaction. A similar conjugate between diphtheria toxin fragment A and ricin B-chain was nontoxic. Like native diphtheria toxin, the conjugate ricin A/diphtheria toxin B was much more toxic to Vero than to HeLa cells. Ricin was equally toxic to these cell lines. Lactose, which inhibits ricin binding, did not protect against the conjugate. Cells resistant to ricin, partly due to a reduced number of ricin-binding sites, were fully sensitive to the conjugate, indicating that the conjugate binds to diphtheria toxin receptors. The conjugate was fully toxic to two Vero cell mutants, resistant to diphtheria toxin because the elongation factor 2 could not be ADP-ribosylated by the diphtheria toxin A-fragment. Therefore, the inhibition of protein synthesis by the conjugate must be caused by the ricin A-chain. Ammonium chloride which prevents entry of diphtheria toxin, but not of ricin, also protected against the conjugate. Like diphtheria toxin, the conjugate was most toxic at low pH, whereas ricin is most active at pH above neutrality and inactive at low pH. The results indicate that the conjugate ricin A/diphtheria toxin B binds to diphtheria toxin receptors and inhibits cellular protein synthesis due to the action of ricin A-chain which appears to enter the cell by the diphtheria toxin pathway.

Ammonium Chloride↗
PubMed1982

Immunological cross-reactivity in the absence of DNA homology between Pseudomonas toxin A and diphtheria toxin.

The immunodominant determinant of Pseudomonas toxin A was shown to cross-react with a normally inaccessible determinant in fragment A of diphtheria toxin. Trypsin-treated diphtheria toxin and fragment A of diphtheria toxin inhibited binding of toxin A antibody to whole toxin A, whereas whole diphtheria toxin did not inhibit this reaction. However, even at the lowest stringency no hybridization was detected between diphtheria tox probe and Pseudomonas aeruginosa DNA.

ADP Ribose Transferases↗
PubMed1998

Toxin binding site of the diphtheria toxin receptor: loss and gain of diphtheria toxin binding of monkey and mouse heparin-binding, epidermal growth factor-like growth factor precursors by reciprocal site-directed mutagenesis.

The transmembrane precursor of the monkey (Mk) heparin-binding, epidermal growth factor-like growth factor (proHB-EGF) functions as a diphtheria toxin (DT) receptor, whereas the mouse (Ms) precursor does not. Previously, using chimeric Ms/Mk precursors, we have shown that DT resistance of cells bearing Ms proHB-EGF may be accounted for by several amino acid substitutions between residues 122 and 148 within the EGF-like domain and that Glu-141 is an important amino acid residue for DT binding. In this study, reciprocal site-directed mutagenesis was performed on the major non-conserved residues in the region of 122-148, alone or in combination, between Mk and Ms precursors to identify more precisely which amino acid residues are important for DT binding. Two approaches were used. The first, more traditional approach was to destroy DT sensitivity and binding of Mk proHB-EGF by substitution(s) with the corresponding Ms residue(s). From the single mutations, the greatest loss of DT sensitivity was observed with Mk/Glu-141His (approximately 4000-fold) and the next greatest with Mk/Ile-133Lys (approximately fourfold). The double mutations Mk/Leu-127Phe/Glu-141His, Mk/Ile-133Lys/Glu-141His and Mk/His-135Leu/Glu-141His resulted in complete toxin resistance (> 100000-fold). The second approach, both novel and complementary, was to gain DT binding and sensitivity of Ms proHB-EGF by substitution(s) with the corresponding Mk residue(s). Surprisingly, the single mutation Ms/His-141Glu resulted in the gain of moderate DT sensitivity (> 260-fold). The double mutation Ms/Lys-133Ile/His-141Glu and the triple mutation Ms/Lys-133Ile/Leu-135His/His-141Glu resulted in a progressive gain in toxin sensitivity (> 4700-fold and >16000-fold respectively) and affinity. This triple mutant cell line is essentially as sensitive (IC50 = 3.1 ng ml(-1)) as the highly toxin-sensitive monkey Vero cell line (IC50 = 4 ng ml(-1)), indicating that these three Mk residues enable the Ms proHB-EGF to act as a fully functional DT receptor. Taken together, these results indicate that Glu-141 plays the most critical role in DT binding and sensitivity and that two additional amino acid residues, Ile-133 and His-135, also play significant roles.

Amino Acid Sequence↗
PubMed2001

Extension of juxtamembrane domain of diphtheria toxin receptor arrests translocation of diphtheria toxin fragment A into cytosol.

Diphtheria toxin (DT) binds to the EGF-like domain of the DT receptor (DTR), followed by internalization and translocation of the enzymatically active fragment A into the cytosol. The juxtamembrane domain (JM) of the DTR is the linker domain connecting the transmembrane and EGF-like domains. We constructed mutants of DTRs with altered JMs and studied their abilities for DT intoxication. Although DTR mutants with extended JMs showed normal DT binding activity, the cells expressing the mutants showed both reduced translocation of DT fragment A into the cytosol and reduced sensitivity to DT, when compared with cells expressing wild-type DTR. These results indicate that the JM contributes to DT intoxication by providing a space appropriate for the interaction of DT with the cell membrane. The present study also indicates that consideration of epitopes of an immunotoxins would be an important factor in the design of potent immunotoxins.

Animals↗