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.     

Secondary structure of diphtheria toxin and its fragments interacting with acidic liposomes studied by polarized infrared spectroscopy

We used infrared attenuated total reflection spectroscopy to study the structure of diphtheria toxin (DT) and its fragments A, B, CB1, and CB4 as a function of the pH in the absence and in the presence of phospholipid vesicles. Binding of DT to asolectin or DL-alpha-dipalmitoylphosphatidylcholine-DL-alpha-dipalmitoylphosphatidic acid liposomes at pH 7.3 results in a 10% increase in its alpha-helix content. At pH 4, in the presence of liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds which did not exist before pH lowering. DT fragment B displays little conformational change upon pH lowering in the presence of liposomes. However, the alpha-helix content of CB1 increases by 10%, and polarization measurements indicate that the alpha-helices of CB1 at pH 4 are oriented parallel to the lipid acyl chains. On the other hand, the alpha-helix content of CB4 decreases dramatically while the low frequency beta-sheet content increases. Dichroism measurements demonstrate that this sheet lies close to a parallel to the bilayer surface. The fragment A of DT experiences a large conformational change upon pH lowering and binds to the liposome membrane even in the absence of DT fragment B. The conformational modification of DT fragment A is fully reversed when pH is brought back to 7.3.     

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)     

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. Copyright © 2009 John Wiley & Sons, Ltd.     

Construction of plasmids coding for diphtheria toxin fragments

Plasmids coding different nontoxic derivatives (toxoids) of the diphtheria toxin were constructed. A secretion of toxoids that carry a signal sequence was found in the periplasmic space of E. coli and Erwinia carotovora. Toxoids without a signal sequence appear in the cytoplasm. We believe that the toxoids secreted in E. coli and E. carotovora cells undergo a limited proteolysis. According to the molecular weights of the fragments there are three targets for proteolysis. One of them being just between A- and B-fragments of the diphtheria toxin. The others are localised in the B-fragment. The role of E. coli signal peptidase in the specific cutting is discussed.     

Systemic toxicity of diphtheria toxin-related fragments (CRM26, CRM45), a hormone-toxin hybrid protein (TRH-CRM45), and ricin A

As a preliminary step in evaluating the use of thyrotropin releasing hormone (TRH) linked to toxin fragments for systemic treatment of pituitary disease, we have examined the metabolic degradation, tissue distribution, renal excretion, and toxicity in rats of TRH-CRM45 which consists of TRH coupled to CRM45, a diphtheria toxin-related polypeptide. For comparison, we have similarly studied CRM45, CRM26 (a smaller diphtheria toxin-related fragment), and ricin A. All four proteins were found to concentrate in the kidney and liver relative to blood (in comparison to bovine serum albumin), tissue:plasma ratios for the kidney being much higher than those observed for the liver. Radioiodinated CRM45 and ricin A were rapidly cleared from the circulation with similar patterns. Systemic toxicity studies showed that at doses greater than 2 micrograms/100 g body wt (bw), CRM45 caused a decrease in growth rate and caused renal damage. CRM45 modified with 2-pyridyldithio(propionate) groups as well as TRH-CRM45 was significantly less toxic than CRM45, as was TRH-CRM45. CRM26 had no discernible effect on the growth rate of the animals. Ricin A, at a dose of 50 micrograms/100 g bw slowed the growth rate of rats, but specific liver or kidney damage could not be detected. These findings define an upper range of doses for possible therapeutic use.     

Interaction of diphtheria toxin fragment A and of elongation factor 2 with Cibacron blue

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10^–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH₂)P are able to displace EF2 from blue Sepharose.     

pH-dependent bilayer destabilization and fusion of phospholipidic large unilamellar vesicles induced by diphtheria toxin and its fragments A and B

The passage by the low endosomal pH is believed to be an essential step of the diphtheria toxin (DT) intoxication process in vivo. Several studies have suggested that this low pH triggers the insertion of DT into the membrane. We demonstrate here that its insertion into large unilamellar vesicles (LUV) is accompanied by a strong destabilization of the vesicles at low pH. The destabilization has been studied by following the release of a fluorescent dye (calcein) encapsulated in the liposomes. The influence of the lipid composition upon this process has been examined. At a given pH, the calcein release is always faster for a negatively charged (asolectin) than for a zwitterionic (egg PC) system. Moreover, the transition pH, which is the pH at which the toxin-induced release becomes significant, is shifted upward for the asolectin LUV as compared to the egg PC LUV. No calcein release is observed for rigid phospholipid vesicles (DPPC and DPPC/DPPA 9/1 mol/mol) below their transition temperature whereas DT induces an important release of the dye in the temperature range corresponding to the phase transition. The transition pH associated to the calcein release from egg PC vesicles is identical with that corresponding to the exposure of the DT hydrophobic domains, as revealed here by the binding of a hydrophobic probe (ANS) to the toxin. This suggests the involvement of these domains in the destabilization process. Both A and B fragments destabilize asolectin and PC vesicles in a pH-dependent manner but to a lesser extent than the entire toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B.

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.     

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.     

Secondary structure changes of diphtheria toxin interacting with asolectin liposomes: an infrared spectroscopy study

Until now, the study of the secondary structure of diphtheria toxin (DT) in the presence of phospholipid vesicles as a function of the pH has been prevented by the optical turbidity of the solution. In the present paper, this problem has been overcome by the use of IR attenuated total reflection (IR-ATR) spectroscopy. Incubation of DT with asolectin liposomes at pH 7.3 results in the binding of DT on to the liposomes and in a 10% increase in its alpha-helix content. At pH 4, in the presence of asolectin liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds, which did not exist before lowering of the pH. This new type of beta-sheet (low frequency beta-sheet) involves 25% of the amino acid residues of the protein.     

Selective killing of subacute sclerosing panencephalitis virus-infected cells by liposomes containing fragment A of diphtheria toxin

A more than 99% reduction in infectivity of cultures infected with subacute sclerosing panencephalitis (SSPE) virus was obtained by treating the cultures with liposomes containing fragment A of diphtheria toxin. In the mixed cultures of human embryonic lung (HEL) cells and SSPE virus-infected HEL cells (SSPE cells), SSPE virus in SSPE cells invaded the surrounding HEL cells successively, forming syncytial giant cells to destroy the whole cultures. By adding liposomes containing fragment A of diphtheria toxin to the mixed cultures, we were successful in killing the SSPE cells selectively and as the result of elimination of SSPE cells from the cultures, the cultures appeared to be cured by uninfected HEL cell growth.     

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.     

Monensin intercalation in liposomes: effect on cytotoxicities of ricin,Pseudomonas exotoxin A and diphtheria toxin in CHO cells

Monensin, a car☐ylic ionophore was intercalated in liposomes (liposomal monensin) and its effect on cytotoxicities of ricin, Pseudomonas exotoxin A and diphtheria toxin in CHO cells was studied. Intercalation of monensin in liposomal bilayer is found to have no effect on its stability and interaction with cells. Liposomal monensin)(1 nM) substantially enhance the cytotoxicities of ricin (62-fold) and Pseudomonas exotoxin A (11.5-fold) while it has no effect on diphtheria toxin. This observed effect is highly dependent on the liposomal lipid composition. The potentiating ability of monensin (1 nM) in neutral vesicles is significantly higher (2.2-fold) as compared to negatively charged vesicles. This ability is drastically reduced by incorporation of stearylamine in liposomes and is found to be dependent on the density of stearylamine as well as on the concentration of serum in the medium. Monensin in liposomes containing 24 mol% stearylamine has a very marginal effect on the cytotoxicity of ricin (7.5-fold) which is further reduced (1.5-fold) in the presence of 20% serum. The uptake of ¹²⁵I-gelonin from neutral vesicles is significantly higher (∼ 2.0-fold) than that from the negative vesicles. The uptake from positive vesicles is highly dependent on the concentration of stearylamine. The reduction in the lag period (30 min) of ricin action by monensin in neutral and negative vesicle is comparable with free monensin. However, monensin in positive vesicle has no effect on it. These studies have suggested that liposomes could be used as a delivery vehicle for monensin for selective elimination of tumor cells in combination with hybrid toxins.     

Interaction of secretory protein precursors with phospholipids in liposomes

The precursors of secretory proteins were synthesized in a reticulocyte lysate system programmed with rat serum albumin or human placental lactogen mRNA and their interaction with phospholipids in liposomes was studied. The precursor proteins could bind to acidic phospholipids that have an exposed phosphate such as dicetyl phosphate and phosphatidic acid or a phosphate that is covered by a small moiety such as phosphatidylglycerol. The binding of precursor proteins was dependent on the mol% of acidic phospholipids in lecithin-liposomes, increased with elevation of temperature in the range of 0 to 45 degrees C, and was not inhibited by the addition of a large excess of mature proteins. Mature proteins or proalbumin showed no significant binding to the liposomes containing acidic phospholipids. About 15% of the acid-precipitable radioactivity bound to the liposomes was resistant to protease digestion. This radioactivity was shown to correspond to methionine-containing peptides with molecular weights of 2,500 to 3,500. These results indicate that the post-translational insertion of a small part of the precursor proteins into the membrane did occur with the present model system, but the post-translational transfer of precursor proteins across the membrane did not.