Diphtheria Toxin Fragment Research Articles

Immunoliposomes with different acid sensitivities as probes for the cellular endocytic pathway

By combining dioleoylphosphatidylethanolamine (DOPE) with oleic acid (OA), palmitoylhomocysteine (PHC) or dipalmitoylsuccinylglycerol (DPSG) we have prepared pH-sensitive liposomes with different acid sensitivities. DOPE/OA liposomes are the most acid sensitive, while DOPE/DPSG liposomes are the least acid sensitive. Incubation of DOPE/OA liposomes with mouse L929 cells reduces the pH-sensitivity of these liposomes by altering the lipid composition. Using diphtheria toxin fragment A as a marker for cytoplasmic delivery, we find that the delivery kinetics of pH-sensitive immunoliposomes closely correlates with the modified acid sensitivities of the liposomes. Immunoliposomes encounter pH 6-6.2 with a t1/2 of 5-15 min after internalization. By contrast, acidification of the endosomes to pH 5.0 takes longer (t1/2 approximately 25 min). We also used a whole cell null point technique (Yamishiro and Maxfield (1987) J. Cell Biol. 105, 2713-2721) to directly determine the average pH encountered by the endocytosed immunoliposomes. We find that acidification determined by the null point method proceeds less rapidly than that estimated from DTA delivery data. This is likely due to the fact that the measured DTA delivery is done by those liposomes which first arrive at the endosomes with sufficient acidity. Our data suggests that DOPE/PHC immunoliposomes deliver at the early endosome while DOPE/DPSG immunoliposomes deliver at the late endosomes. The DOPE/OA immunoliposomes, with the altered composition and acid sensitivity, deliver with a kinetics intermediate between the other two immunoliposomes. Thus, pH-sensitive liposomes represent useful probes for studying the kinetics of endosome acidification.

Diacytosis of asialoglycoprotein in isolated hepatocytes is dependent on the structure of ligand and cellular distribution of the receptors

Diacytosis, degradation and retention of 125I-labeled asialoorosomucoid (ASOR), its reduced and car☐ymethylated N-terminal cyanogen bromide-cleaved fragment (RC-ASCNBr-I) and asialofetuin preloaded into isolated rat or rabbit hepatocytes for various periods of time were compared. In rat hepatocytes preloaded with a saturating concentration (3 · 10 −8 M) of the ligands, the proportion of the preloaded ligands distributed to degradation and diacytosis was fairly constant during 4 h of preincubation. In addition, a small portion of the preloaded ligands was neither diacytosed nor degraded, but was retained intracellularly. Diacytosis of 125I-ASOR (29%) was greater than that of either 125I-RC-ASCNBr-I (23%) or 125I-asialofetuin (15%). Diacytosis of 125I-ASOR, when preloaded in the presence of 5 μM colchicine, was significantly enhanced by 79% (increasing from 29% to 52%), whereas those of 125I-RC-ASCNBr-I and 125I-asialofetuin were not significantly altered (with average increases of 14% and 19%, respectively). The fraction of the preloaded 125I-asialofetuin (69%) and 125I-RC-ASCNBr-I (68.6%) that was degraded was slightly higher than that of 125I-ASOR (64%) and all was decreased by colchicine. The fraction of all three ligands retained by the cells was increased 2- to 4-fold by colchicine. The extents of retention of 125I-asialofetuin and 125I-RC-ASCNBr-I were greater than that of 125I-ASOR, particularly after preloaded for more than 2 h. Preloading of the cells with ligands at a non-saturating concentration (6.5 · 10 −10 M) did not change these patterns of ligand distribution. Conjugation of diphtheria toxin fragment A (DTA) to ASOR or RC-ASCNBr-I also did not significantly alter the pattern of ligand distribution. In rabbit hepatocytes containing more asialoglycoprotein receptors than rat cells, 125I-ASOR was diacytosed to a greater extent (50%, −colchicine; 61%, +cholchicine) but degraded to a lesser extent (33%, −colchicine; 13%, +colchicine) than was observed in rat cells. The extent of retention of 125I-ASOR in rabbit cells was also greater than that in rat cells. A similar pattern of differences between rabbit and rat hepatocytes was observed for 125I-DTA-ASOR. These results indicate that intracellular sorting of internalized asialglycoproteins between diacytosis and degradation is dependent on both the structure of the ligand and the distribution of the cellular receptors.

Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution and toxic binding chain

Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys. Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC 50) of protein synthesis occurring at 4 ± 3·10 −11 M ( n = 7) which was much lower than that of DT (7.8 ± 9.8·10 −9 M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC 50 = 5.4 ± 4.9·10 −10 M, n = 7) was higher than that of DT (IC 50 = 5 ± 4·10 −11 M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10 −8 M, although DT was highly toxic (IC 50 = 1.8 ± 1.4·10 −10, n = 3). In the presence of 5 μM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC 50 of 1.2 ± 0.8·10 −9 M ( n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.

The effect of immunotoxin against human glioma cells.

The authors produced immunotoxin by coupling the diphtheria toxin fragment A to the monoclonal antibody against a human glioma cell line. This toxin selectively inhibited protein synthesis of glioma cells in culture.

X-ray Grade Crystals of the Enzymatic Fragment of Diphtheria Toxin

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.

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.

Elongation factor 2 as the major substrate for ca 2+/calmodulin-dependent protein kinase in rat adrenal glomerulosa cells

We have previously shown the existence of the major substrate protein of Mr 100,000 (substrate 100 K protein) for Ca 2+/calmodulin (CaM)-dependent protein kinase in rat adrenal glomerulosa cells. In the present study, the identity of the substrate 100 K. protein to elongation factor 2 (EF-2) was investigated. In a 105,000 g-supernatant fraction (cytosol), the protein of Mr 100,000 with the pI (isoelectric point) value of 6.7 was phosphorylated in the presence of calcium and CaM. The optical densities of this phosphorylated band were greatly enhanced in the presence of the EF-2 purified from pig liver (1 μg) [20–23-fold, n = 5] when compared with those in the absence of the component. In the presence of the purified EF-2, the phosphorylation of Mr 100,000 was detected only in the presence of calcium alone or calcium plus CaM. This phosphorylation in the presence of calcium alone was completely inhibited in the presence of the CaM antagonist pimozide (500 μM), showing the existence of endogenous CaM in the cytosol. In the same fraction, the ADP-ribosylated protein of Mr 100,000 was detected in the presence of diphtheria toxin (fragment A) and (adenylate- 32P) NAD, indicating the presence of EF-2 in the cytosol from rat adrenal glomerulosa cells. These results suggest that the substrate 100 K protein may be identical to EF-2 in rat adrenal glomerulosa cells.

Prolongation of cardiac allograft survival in murine recipients treated with a diphtheria toxin-related interleukin-2 fusion protein.

A diphtheria toxin-related IL-2 fusion gene has been constructed that encodes a 68KD recombinant toxin in which the diphtheria toxin receptor-binding domain has been replaced with amino acids 2-133 of IL-2. This chimeric IL-2 toxin is cytotoxic for cells expressing the high-affinity IL-2 receptor but not for cells lacking this receptor. The ability of this IL-2 toxin to prolong allograft survival was examined in a murine vascularized, heterotopic heart transplant model in the strain combination B10.BR into C57B1/10. When given at a dose of 1.0 micrograms/day for 10 days, the IL-2 toxin significantly prolonged allograft survival in all recipients. CRM-45, a fragment of diphtheria toxin missing the binding domain, was ineffective, confirming the specificity of the therapy. The results demonstrate that this IL-2 toxin, which targets activated T cells expressing the IL-2 receptor, will prolong allograft survival, offering a new option for immunosuppressive therapy.

Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells.

Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [32P]orthophosphate and 10% (vol/vol) fetal bovine serum. A 32P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [32P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [32P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32P label was lost from the EF-2 within 30 min.

4830962 Recombinant diphtheria toxin fragments: David H Gelfand, Lawrence I Greenfield, Frances C Lawyer assigned to Cetus Corporation

4830962 Recombinant diphtheria toxin fragments: David H Gelfand, Lawrence I Greenfield, Frances C Lawyer assigned to Cetus Corporation

4830962 Recombinant diphtheria toxin fragments: David H Gelfand, Lawrence I Greenfield, Frances C Lawyer assigned to Cetus Corporation

4830962 Recombinant diphtheria toxin fragments: David H Gelfand, Lawrence I Greenfield, Frances C Lawyer assigned to Cetus Corporation

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.

Conformation and model membrane interactions of diphtheria toxin fragment A.

Low pH is believed to play a critical role in the penetration of membranes by diphtheria toxin in vivo. In this report, the pH dependence of the conformation of fragment A of diphtheria toxin has been studied using fluorescence techniques. As pH is decreased, fragment A in solution undergoes a reversible conformational change beginning below pH 5. The conformational change occurs rapidly upon exposure to low pH. It involves both an increase in the exposure of tryptophanyl residues to solution and a switch from a hydrophilic state to a hydrophobic state as judged by fragment A binding to micelles of a mild detergent (Brij 96). At low pH fragment A also rapidly and tightly binds to and penetrates model membranes. Binding is reversed when pH is neutralized. The transition pH, the apparent midpoint of the change between the hydrophilic state and the membrane-penetrating hydrophobic state, occurs at about pH 3.5 in the presence of Brij 96 micelles, pH 4 in the presence of small unilamellar vesicles (SUV) composed of zwitterionic phosphatidylcholine, and pH 5 in the presence of SUV composed of 25 mol % anionic phosphatidylglycerol and 75% phosphatidylcholine. The effects of high temperature provide an important clue as to the nature of the changes at low pH. At neutral pH and high temperature, i.e. in the thermally denatured state, a conformational change similar to that observed at low pH occurs, although fragment A does not become hydrophobic. In addition, the effects of low pH and high temperature on the stability of the native state are cumulative. This indicates that the changes in fragment A both at high temperature and at low pH involve denaturation, although there appears to be only partial unfolding under these conditions. Based on the results of this study, the role of fragment A in diphtheria toxin membrane penetration and translocation is evaluated.

Expression of a biologically active diphtheria toxin fragment B in Escherichia coli.

The toxB gene of Corynebacterium diphtheriae bacteriophage beta encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed in Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphtheriae-derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor-2, thereby indicating that the recombinant fragment B had retained its biological activity.

Interleukin 2 receptor-targeted cytotoxicity. Interleukin 2 receptor-mediated action of a diphtheria toxin-related interleukin 2 fusion protein.

The IL-2 toxin-mediated inhibition of protein synthesis in high affinity IL-2-R-positive murine and human T cell lines has been examined. Both excess free IL-2 and mAb to the Tac epitope of the p55 subunit of IL-2-R are shown to block the action of IL-2 toxin; whereas, agents that interact with other receptors or antigens on the T cell surface have no effect. We show that IL-2 toxin, like diphtheria toxin, must pass through an acidic vesicle in order to intoxicate target T cells. Finally, we demonstrate that the IL-2 toxin-mediated inhibition of protein synthesis in both human and murine T cells that bear the high affinity IL-2-R is due to the classic diphtheria toxin fragment A-catalyzed ADP ribosylation of elongation factor 2.

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 1. Liposome aggregation and fusion.

The interaction of diphtheria toxin and its cross-reacting mutants crm 45,228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing. The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5-6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.

High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli.

ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone. When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded. When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble. We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis. Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases. Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E. coli. We also showed that the solubility of tox gene products expressed in E. coli was directly related to the growth temperature of the culture. Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E. coli. This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level. Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.

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 1:1 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.

Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.

Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell Tf receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors.

Effect of 3-aminobenzamide on the frequency of antigen switching in Trypanosoma brucei in vitro

Previous studies on chemical modifications of diphtheria toxin (DT) fragment A have suggested that the Trp153 amino acid residue is essential for the ADP ribosylation of elongation factor 2. We verified this experimentally after replacing Trp153 by Phe or Ala residues through in vitro mutagenesis of a cloned toxin gene fragment. Each of the mutant fragment A forms were found to reveal a reduced ADP ribosyl transferase (ADPRT) activity as well as lower affinity for NAD. Both ADPRT activity and NAD affinity of DT fragment A were only partially destroyed by nearly synonymous Trp153 => Phe153 substitution, but dramatically destroyed by Ala153 substitution. At the same time, each of the mutant fragment A forms appeared to be thermostable, suggesting that the mutations do not dramatically destroy the structure of the protein. These results clearly demonstrate that Trp153 is not highly specific for DT fragment A structure maintenance, but is highly specific for the key toxin functions such as ADP ribosylation of elongation factor 2 and NAD binding. We suggest that the Trp153 role in DT functioning may be that of binding the ribose moiety of NAD, which is crucial for DT catalytic activity and hence for toxicity.