Interaction Of Diphtheria Toxin Research Articles

Interaction of diphtheria toxin (fragment A) with actin

It was shown by gel filtration and viscosity measurements that N-terminal fragment (FA) of diphtheria toxin (DT) can interact with both G- and F-actin (filamentous actin). Elution profiles on Sephadex G-100 indicated the formation of a binary complex of fragment A (FA) with globular actin monomer (G-actin), which was inhibited by gelsolin. Deoxyribonuclease I (DNase I) in turn appeared to interact with this complex. Tritiated FA was found to bind to F-actin stoichiometrically. This binding was inhibited again by gelsolin and G-actin, but not by DNase I. The binding of FA inhibited polymerization of G-actin and induced a time-dependent breakdown of F-actin under polymerization conditions. Inhibition of its ADP-ribosyltransferase activity did not have any effect on the interactions of FA with actin. FA interacted with actin also in the cell. After treatment of human umbilical vein endothelial cells (HUVEC) with biotin-labeled DT, Western blot analysis revealed predominantly the presence of actin in affinity-isolated complexes of the labeled FA. Similarly, FA was found in immunoaffinity-isolated complexes of actin.

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin is not detected, and free antibodies account for 68 to 98% of the original amount. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80-100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalency of serum antibodies, and monovalency of the toxin epitopes. A new model of the toxin-antibody interactions is proposed.

Interaction of diphtheria toxin T domain with molten globule-like proteins and its implications for translocation.

The transmembrane (T) domain of diphtheria toxin has a critical role in the low pH-induced translocation of the catalytic domain (A chain) of the toxin across membranes. Here it is shown that at low pH, addition of proteins in a partly unfolded, molten globule-like conformation converted the T domain from a shallow membrane-inserted form to its transmembrane form. Fluorescence energy transfer demonstrated that molten globule-like proteins bound to the T domain. Thus, the T domain recognizes proteins that are partly unfolded and may function in translocation of the A chain as a transmembrane chaperone.

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids

The interaction of diphtheria toxin and its enzymatically deficient mutants crm 176 and crm 197 with liposomes has been studied by turbidity measurement and hydrophobic photolabelling with photoactivatable phosphatidylcholines. Diphtheria toxin and crm 176 at neutral pH bind to the surface of lipid bilayers while crm 197 also appears to interact with the fatty acid chains of phospholipids. All proteins undergo a change in conformation over the same range of acidic pH and become able to insert in the lipid bilayer. The tighter lipid interaction of crm 197 may account for its higher cell association constant. The possibility is discussed that the binding of diphtheria toxin to cells is mediated by both a protein receptor and an interaction with the head group of phospholipids.

Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain.

CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.

Interaction of diphtheria toxin with adenylyl-(3',5')-uridine 3'-monophosphate. I. Equilibrium and kinetic measurements.

Purified diphtheria toxin from various sources contains tightly, but noncovalently, bound nucleotides, the major component of which is adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). We used ApUp radiolabeled with 32P to measure equilibrium dissociation constants (KD), and association and dissociation rate constants (k+1 and k-1, respectively) under various conditions. Diphtheria toxin bound 1 molar equivalent of ApUp, regardless of the temperature. Values of KD were 0.2 nM (25 degrees C) and 1.8 nM (37 degrees C) as determined by flow dialysis. No difference in KD was observed between the nicked and intact forms of toxin. The dissociation rate constant showed marked variation with temperature, ranging from 1.8 X 10(-4) s-1 at 5.5 degrees C (t 1/2 of the complex = 64 min) to 2.5 X 10(-2) s-1 at 25 degrees C (t 1/2 of the complex = 28 s). By contrast, k+1 varied only by a factor of 5 over the same temperature range (2.0 X 10(7) M-1 s-1 at 5.5 degrees C; 9.6 X 10(7) M-1 s-1 at 25 degrees C). The KD at 5.5 degrees C, calculated from the ratio of k-1/k+1, is 9 pM, which represents the strongest affinity of a dinucleotide for a protein ever reported. Affinity was maximal in the range of pH 6.5 to 7.1 and was sensitive to ionic strength. Thermodynamic parameters of the system were calculated.

Interaction of diphtheria toxin with adenylyl-(3',5')-uridine 3'-monophosphate. II. The NAD-binding site and determinants of dinucleotide affinity.

Diphtheria toxin (DT) binds NAD with a KD of about 10 microM and adenylyl-(3',5')-uridine 3'-monophosphate (ApUp) with KD values ranging from 9 pM to 1.8 nM, depending on temperature (Collins, C. M., Barbieri, J. T., and Collier, R. J. (1984) J. Biol. Chem. 259, 15154-15158). Here we report experiments to explore relationships between ApUp binding and NAD binding to DT and to identify structural features of ApUp that determine its high affinity for DT. NAD, adenine, and nicotinamide competitively inhibited ApUp binding to DT, and we confirmed that ApUp blocked the binding and hydrolysis of NAD. Binding of P-site ligands to the toxin blocked interactions with ApUp. CRM197, a mutant form of DT defective in NAD binding and hydrolysis, bound ApUp 5,000-fold less tightly than did DT. These results are consistent with models in which the ApUp- and NAD-binding sites on DT overlap or are identical. Various mono-, di-, and oligonucleotides were studied as competitors of ApUp binding or the NAD-glycohydrolase reaction. The results imply that the high affinity of ApUp for DT depends on the presence of the 3'-terminal phosphate and a 3'-5' internucleoside linkage. There was strong specificity for adenine as the 5' base, but only weak specificity for uracil as the 3' base. Oligoribonucleotides containing additional nucleotides at either or both ends of ApUp sequences bound to the toxin 1-3 orders of magnitude less avidly than ApUp. Oligodeoxyribonucleotides containing dApdT sequences bound with still lower affinities. In contrast to the case with whole toxin, ApUp bound to fragment A less avidly than did NAD, and elimination of the 3'-terminal phosphate of ApUp resulted in increased affinity for the protein. These differences may reflect the absence in free fragment A of interactions with the cationic P-site, located on the toxin's B moiety.

Interaction of diphtheria toxin with phosphorylated molecules.

The binding of diphtheria toxin to 125I-labeled cell surface glycoproteins from hamster thymocytes was shown to be inhibited by nucleotides. The relative effectiveness of the nucleotides (at 5 mM) was found to be thymidine triphosphate greater than adenosine triphosphate greater than guanosine triphosphate greater than uridine triphosphate greater than cytidine triphosphate. When adenine-containing compounds were used, the relative effectiveness was determined to be adenosine tetraphosphate greater than adenosine triphosphate greater than adenosine diphosphate greater than adenosine monophosphate. In addition, tetrapolyphosphate, tripolyphosphate, inositol hexaphosphate (phytic acid), and the highly phosphorylated proteins casein and phosvitin were also shown to be potent inhibitors of the binding of diphtheria toxin to 125I-labeled cell surface glycoproteins. Diphtheria toxin was shown to bind directly to 125I-casein; this binding was also inhibited by the highly phosphorylated compounds and was decreased by pretreatment of the 125I-casein with alkaline phosphatase. These results suggest that diphtheria toxin binds to regions of high phosphate density and raise the possibility that the site on the cell surface glycoproteins to which diphtheria toxin binds might be polyanionic in nature.

Effects of lectins on the interaction of diphtheria toxin with mammalian cells

Abstract The effects of concanavalin A (ConA) on diphtheria toxin-cell receptor interactions were studied. ConA had no measurable effect on toxin-receptor binding at 4 °C but increased the total cell-associated toxin 4–5-fold at 37 °C. An assay which distinguishes between cell surface bound and internalized toxin demonstrated that ConA reduces the percentage of internalized toxin while markedly increasing that found on the cell surface. These effects of ConA were blocked by α-methyl mannoside but not by colchicine. Succinyl ConA was as effective as ConA; other lectins, however, were less potent. It is concluded that ConA protects cells from diphtheria toxin by blocking its entry into the cell.

Interaction of diphtheria toxin with mammalian cell membranes.

Uptake of 125I-labeled diphtheria toxin and serologically related proteins by a sensitive human HeLa cell line and by a resistant mouse L929 cell line has been studied. The evidence suggests that there is an initial rapid reaction between a recognition site present on the toxin Fragment B and specific plasma membrane receptors on the sensitive cell (there are approximately 4000/HeLa cell). This initial interaction is followed by a slow irreversible process during which there is a major conformational alteration of the toxin molecule causing the enzymically active 22,000-dalton Fragment A to become exposed to the cytosol. We suggest that it is at this point that cleavage of the NH2-terminal disulfide bond occurs leading to release of Fragment A into the cytoplasm. The toxin Fragment B remains attached to the membrane, probably formed in a complex with receptor, and blocks entry of additional toxin molecules through the same site. Specific membrane receptors are lacking from mouse cells. Both HeLa cells and L929 cells internalize toxin, related nontoxic proteins, and inert molecules such as inulin nonspecifically into endocytotoc vesicles. At 30 degrees the bulk internalization of extracellular fluid is about 1.2% of their cell volume per h for both cell lines. Fragment A does not traverse the plasma membrane by a mechanism that depends on endocytosis. The interaction of diphtheria toxin with the sensitive cell membrane is discussed in relation to other protein toxins and certain glycopeptide tropic hormones in which relatively large, hydrophilic polypeptide fragments or subunits are presumed to traverse the target cell plasma membrane and reach the cytoplasm in biologically active form.

Interaction of diphtheria toxin and its active subunit, fragment A, with toxin-sensitive and toxin-resistant cells.

Diphtheria toxin and purified fragment A, the active subunit of the toxin, were tested on toxin-sensitive and permeability class toxin-resistant cultured mammalian cells. Protein synthesis was inhibited to the same degree in sensitive or resistant cells by active concentrations of purified fragment A. In contrast, resistant cells required a concentration of whole toxin 5 to 6 logs greater than that required by sensitive cells to achieve the same degree of inhibition. On a molar basis, the toxicity of fragment A was equivalent to that of whole toxin on resistant cells. These results are evidence for the existence of two independent mechanisms for the entry of toxin or its active moiety into cells. One mechanism is a highly efficient, toxin-specific entry mechanism, involving surface receptor and fragment B-mediated association, and is active in sensitive cells. The other is a less efficient, nonspecific mechanism, probably related to endocytosis, which is operative in sensitive and resistant cells but is inapparent in sensitive cells when they are exposed to whole toxin because of the action of the specific mechanism.

Activity of diphtheria toxin. II. Early events in the intoxication of HeLa cells.

The initial steps in the interaction of diphtheria toxin with HeLa cells were studied. It was demonstrated that lethal doses of toxin are rapidly adsorbed to the cell. The kinetics of uptake, as measured by lethality, indicated that a single toxin molecule is able to cause cell death. Studies on the effect of pH on intoxication showed that adsorption of toxin occurred over a wide pH range but was partially inhibited at high pH values. Experiments to determine the influence of the ionic environment on intoxication indicated that adsorption of toxin did not take place in the absence of salts and was partially inhibited in the presence of a polyanion. The evidence indicates that the initial binding of toxin to the cell is electrostatic in nature, involving positively charged surface groups. Attempts to demonstrate specific receptors for the attachment of toxin to cells were unsuccessful, suggesting that toxin adsorption may be a nonspecific process. The effect of energy inhibitors on intoxication was examined. Sodium fluoride, an inhibitor of glycolysis, almost completely prevented intoxication in HeLa cells, whereas inhibitors of respiration and oxidative phosphorylation had no effect. Sodium fluoride did not prevent adsorption of toxin but appeared to inhibit a later step in the intoxication process, perhaps the transport of toxin to subsurface or intracellular levels.

INTERACTION OF DIPHTHERIA TOXIN WITH CELL CULTURES FROM SUSCEPTIBLE AND RESISTANT ANIMALS

The cytotoxicity (TD) level of diphtheria toxin for human Chang liver strain was 0.1 guinea pig MLD per ml; for mouse liver NCTC 1469 strain, the TD was 500 MLD per ml. The results of cell culture toxicity correlated well with relative susceptibility of both man and mouse. The initial effects of toxin on the susceptible Chang liver cells were studied at one half the TD level (0.05 MLD per ml). At this low concentration of toxin, the number of cells per culture was reduced slightly below the "0" hr values, whereas the amounts of cell protein, RNA, and DNA were increased. Analysis of the toxin-treated cells indicated an enlargement of the cells. The individual cells contained significantly more protein, RNA, and DNA than the control cells and the cell volume was increased 1.9 times. When purified diphtheria toxin was incubated with the susceptible Chang liver cells and then tested for its biological activity, the results showed an increased diffusion rate in parabiotic culture chambers and definite cytotoxicity to the normally resistant mouse liver cells. The cytotoxicity was neutralizable with antitoxin. The results suggest that the toxin-susceptible cells transform the toxin molecule to a more active derivative which affects the highly resistant mouse liver cell.