Altered degradation of epidermal growth factor in a diphtheria toxin‐resistant clone of KB cells
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Abstract We have investigated the cellular fate of epidermal growth factor (EGF) in KB cells and a variant, KB‐R2A, that was isolated based on its resistance to diphtheria toxin and subsequently was shown to be resistant to infection by RNA viruses (Moehring and Moehring, 1972, Infect. Immunity. 6: 487–492). Both cell lines bind 125 I‐EGF and internalize the cell‐bound hormone at the same rate. However, when the degradation of internalized 125 I‐EGF was measured by the release of low molecular weight (mw) hydrolysis products into the medium, the toxin‐resistant KB‐R2A cells degraded the hormone at a drastically reduced rate; 50% and 3% of the cell‐bound 125 I‐EGF was degraded and released by 80 min in the KB and KB‐R2A cells, respectively. To investigate the fate of cell‐associated EGF prior to release into the medium, the radioactivity in extracts of cells labeled with 125 I‐EGF was fractionated by native gel electrophoresis. In KB cells three peaks of radioactivity other than native 125 I‐EGF were resolved. Time course and subcellular fractionation studies showed that the first processed product appeared while the hormone was located in the endocytic vesicles and the appearance of the other two peaks correlated with the arrival of the hormone in the lysosomal compartment. KB‐R2A cells also produced the first intermediate but they produced only very low amounts of the other two peaks. These studies show that endocytic vesicles in both cell lines contain enzymes capable of processing EGF prior to the arrival of the hormone in the lysosomes and show that the KB‐R2A cells have a lesion that prevents the complete degradation of the hormone. We propose that the KB‐R2A cell line has a defective mechanism for the intracellular processing of a number of ligands that are internalized by the process of receptor‐mediated endocytosis and that this defect is located beyond the initial endocytic step.Keywords:
Diphtheria Toxin
Cell fractionation
Diphtheria Toxin
Corynebacterium diphtheriae
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A detailed binding study of 125I-labeled diphtheria toxin to isolated cell surface membrane-enriched fractions is reported. The study was undertaken to determine if toxin-resistant species exhibit a defet in either the binding step or the transport step of the intoxication process. Surface membrane fractions were obtained from liver and mammary glands of toxin-sensitive species, rabbit and giunea pig, and toxin-resistant species, rat mouse. All membrane fractions exhibited reversible binding of 125I-toxin which was competitively inhibited by unlabeled toxin. Toxin receptors from liver co-purified with plasma membranes and the plasma membrane marker 5'-nucleotidase. One-half saturation of all receptors occurred between 5 x 10(-8) and 1.8 x 10(-7) M. Scatchard plots were nonlinear and concave upwards. Total receptor sites ranged from 3.4 to 16 pmol/mg of membrane protein, tissue differences being more pronounced than difference between sensitive and nonsensitive species. Over 95% of the toxin specific binding was inhibited by removal of divalent cation from the medium or by the inclusion of 1 mM ATP, procedures which have been shown to protect sensitive cells from intoxication by diphtheria toxin. We conclude that the rat and mouse have surface membrane receptors for diphtheria toxin and that the toxin insensitivity of these species results from a defect in or a lack of the transport process.
Diphtheria Toxin
Cell surface receptor
ADP-ribosylation
Cell membrane
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This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB(389)TTC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB(389)) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB(389)TTC was approximately 1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB(389)MSH. The cytotoxic effect of DAB(389)TTC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB(389)TTC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB(389) catalytic fragment. These results suggest that a catalytically inactive form of DAB(389)TTC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons.
Diphtheria Toxin
Corynebacterium diphtheriae
Anthrax toxin
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Immunochemical Analysis Shows All Three Domains of Diphtheria Toxin Penetrate across Model Membranes
Diphtheria toxin undergoes membrane insertion and translocation across membranes when exposed to low pH. In this study, the translocation of the toxin has been investigated by the binding of antibodies to two preparations of model membrane-inserted toxin. In one preparation, toxin was added externally to model membrane vesicles and then inserted by exposure to low pH. In the other preparation, toxin was entrapped in the vesicles at neutral pH, and then inserted by decreasing pH. At neutral pH, externally added antibodies could not bind to entrapped toxin, although they could bind to externally added native toxin. However, after low pH exposure, antibodies against all three toxin domains (catalytic (C), transmembrane (T), and receptor-binding (R)) could bind to entrapped toxin, and also to externally added membrane-inserted toxin. The binding to the entrapped toxin shows that all three domains of the toxin translocate to the trans face of the membrane after exposure to low pH. The observation that antibodies bind to both external and entrapped preparations of toxin after low pH exposure shows that toxin inserts in a mixed orientation.A difference in antibody binding to low pH-treated toxin in which the C domain is folded (Lr′ conformation) or unfolded (Lr″ conformation) was also observed. An increase in antibody binding to C and T domains in the Lr″ conformation relative to binding to the Lr′ conformation was found for entrapped toxin, suggesting that more of the C and T domains translocate across the bilayer in the Lr″ conformation.These results suggest all three toxin domains insert in the membrane bilayer and participate in translocation in vitro. The C and R domains lack classical transmembrane hydrophobic sequences. However, they possess sequences that have the potential to form membrane-inserting β-sheets. Diphtheria toxin undergoes membrane insertion and translocation across membranes when exposed to low pH. In this study, the translocation of the toxin has been investigated by the binding of antibodies to two preparations of model membrane-inserted toxin. In one preparation, toxin was added externally to model membrane vesicles and then inserted by exposure to low pH. In the other preparation, toxin was entrapped in the vesicles at neutral pH, and then inserted by decreasing pH. At neutral pH, externally added antibodies could not bind to entrapped toxin, although they could bind to externally added native toxin. However, after low pH exposure, antibodies against all three toxin domains (catalytic (C), transmembrane (T), and receptor-binding (R)) could bind to entrapped toxin, and also to externally added membrane-inserted toxin. The binding to the entrapped toxin shows that all three domains of the toxin translocate to the trans face of the membrane after exposure to low pH. The observation that antibodies bind to both external and entrapped preparations of toxin after low pH exposure shows that toxin inserts in a mixed orientation. A difference in antibody binding to low pH-treated toxin in which the C domain is folded (Lr′ conformation) or unfolded (Lr″ conformation) was also observed. An increase in antibody binding to C and T domains in the Lr″ conformation relative to binding to the Lr′ conformation was found for entrapped toxin, suggesting that more of the C and T domains translocate across the bilayer in the Lr″ conformation. These results suggest all three toxin domains insert in the membrane bilayer and participate in translocation in vitro. The C and R domains lack classical transmembrane hydrophobic sequences. However, they possess sequences that have the potential to form membrane-inserting β-sheets.
Diphtheria Toxin
Anthrax toxin
Cholera toxin
Corynebacterium diphtheriae
Single-domain antibody
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Diphtheria toxin-binding glycoproteins of high molecular weight (greater than 100,000) were identified on the surface of lymph node and thymus cells from hamsters, a diphtheria toxin-sensitive species. These diphtheria toxin-binding glycoproteins also interacted with CRM197 protein, which possesses toxin-blocking activity, but not with diphtheria toxoid, fragment A of diphtheria toxin, or cholera toxin, all of which lack toxin-blocking activity. These observations are consistent with the hypothesis that the detected diphtheria toxin-binding glycoproteins are involved in intoxication of cells by this toxin and possibly serve as the plasma membrane receptors for diphtheria toxin.
Diphtheria Toxin
ADP-ribosylation
Cholera toxin
Corynebacterium diphtheriae
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Differences in sensitivity to diphtheria toxin of several toxin-sensitive and toxin-resistant human and non-human cell lines were compared. A method is described whereby it is possible to compare the sensitivity of one cell line with another and obtain meaningful quantitative results. Based on the concentration of toxin required to produce 50% inhibition of protein synthesis after 24 h of exposure the ID50 (24) value toxin-resistant cells were found to be 105 to 106 times more resistant to toxin than toxin-sensitive cells. There was little variation in the ID50 (24) values for cells in each of the two groups. The toxin-resistant cells used in this study, naturally resistant as well as selected variants, possess elongation factor 2 which is susceptible to inactivation by toxin. It is suggested that they are capable of activation of toxin but either cannot bind toxin or are unable to transport toxin across the plasma membrane. Protein synthesis is inhibited when these resistant cells are exposed to high concentrations of toxin. Under these conditions it is likely that enough toxin is able to bypass the block in toxin-specific entry and reach the cytosol by a second, less efficient, nonspecific mechanism to catalyze the inactivation of elongation factor 2 and inhibit protein synthesis.
Diphtheria Toxin
ADP-ribosylation
Elongation factor
Anthrax toxin
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The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement.
Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm.
Diphtheria Toxin
Antitoxin
Ammonium chloride
Ricin
Anthrax toxin
ADP-ribosylation
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Diphtheria Toxin
Corynebacterium diphtheriae
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Primary heart cell cultures of embryonic guinea pigs and the neonatal rat were established and incubated with purified diphtheria toxin. The rat heart cell cultures were refractory to the effects of the toxin; protein synthesis proceeded normally as measured by the incorporation of tritiated leucine into cell proteins; beating heart cells continued to contract; and the cell monolayers remained intact after exposure to the toxin for periods as long as 72 hr. These findings are compatible with the species resistance of the rat to diphtheria toxin. The guinea pig heart cell cultures were found to be extremely sensitive to the toxin. Protein synthesis was inhibited by approximately 50% after incubation with small quantities of toxin for 3 hr. Increasing the concentration of the length of exposure to the toxin did not increase this inhibition significantly. In addition, diphtheria toxin exerted a true cytopathic effect on the guinea pig heart cells. Monolayers were destroyed when incubated with the toxin for 2 to 3 days. The results show that the heart cells reflect species resistance or sensitivity to diphtheria toxin in the absence of neural or endocrine influences and suggest further that the toxin exerts a direct toxicity to muscle cells of the heart. It is not yet possible to explain in biochemical terms why the toxin seems to act specifically on cardiac tissues.
Diphtheria Toxin
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Summary Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin‐sensitivity to mouse cells, which are normally toxin‐resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin‐sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.
Diphtheria Toxin
Corynebacterium diphtheriae
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