Anthrax Toxin Protective Antigen Research Articles

Potent antitumor activity of a urokinase-activated engineered anthrax toxin.

The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase in vivo and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor, Pseudomonas exotoxin fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cell-surface urokinase-dependent toxin activation in vivo, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen in vivo, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.

Targeting of Tumor Cells by Cell Surface Urokinase Plasminogen Activator-dependent Anthrax Toxin

Urokinase plasminogen activator receptor (uPAR) binds pro-urokinase plasminogen activator (pro-uPA) and thereby localizes it near plasminogen, causing the generation of active uPA and plasmin on the cell surface. uPAR and uPA are overexpressed in a variety of human tumors and tumor cell lines, and expression of uPAR and uPA is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed mutated anthrax toxin-protective antigen (PrAg) proteins in which the furin cleavage site is replaced by sequences cleaved specifically by uPA. These uPA-targeted PrAg proteins were activated selectively on the surface of uPAR-expressing tumor cells in the presence of pro-uPA and plasminogen. The activated PrAg proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A, thereby killing the uPAR-expressing tumor cells. The activation and cytotoxicity of these uPA-targeted PrAg proteins were strictly dependent on the integrity of the tumor cell surface-associated plasminogen activation system. We also constructed a mutated PrAg protein that selectively killed tissue plasminogen activator-expressing cells. These mutated PrAg proteins may be useful as new therapeutic agents for cancer treatment.

Role of Furin in Delivery of a CTL Epitope of an Anthrax Toxin‐Fusion Protein

Anthrax toxin lethal factor (LF) in combination with anthrax toxin protective antigen (PA) was endocytosed and translocated to the cytosol of mammalian cells. Residues 1-255 of anthrax toxin lethal factor (LFn) was fused to a cytotoxic T lymphocyte (CTL) epitope of an influenza virus. For processing the toxins, PA must be cleaved into a 63-kDa fragment (PA63) by furin, which is a subtilisin-like processing endo-protease expressed by many eukaryotic cells. To test the ability of cells treated with the LFn fusion protein plus PA to deliver the epitope, CTL assay was performed. Two types of cell lines were identified, one was able to deliver CTL epitope while the other failed to efficiently deliver the epitope. To further elucidate the differences between these cells, the role of furin in these cells was examined. Disruption of the furin gene reduced its ability to deliver the CTL epitope. Furin expression in cells capable of efficiently delivering CTL epitope was quantitatively higher than in cells unable to deliver the epitope. The results suggest that furin plays a critical role in delivery of the CTL epitope of LFn fusion protein.

Trp 346 and Leu 352 Residues in Protective Antigen Are Required for the Expression of Anthrax Lethal Toxin Activity

The three separate proteins that make up anthrax toxin-protective antigen (PA), edema factor (EF) and lethal factor (LF) act in binary combinations to produce two distinct reactions in experimental animals: edema (PA+EF) and death (PA+LF). PA is believed to interact with a membrane receptor and, after proteolytic processing, to mediate endocytosis and subsequent translocation of EF or LF into the cytosol. Residues W346, M350, and L352 in loop 3 of domain 2 have been implicated to induce a conformational change when the pH is lowered from 7.4 to 6.5. Modification of the residues Trp (346), Met (350), and Leu (352) to alanine individually and all the three residues together to alanine residues resulted in the loss of cytotoxic activity in combination with LF. The mutant proteins were able to bind to the cell surface receptor, become cleaved by trypsin, bind LF, and oligomerize. These residues might play an important role in the membrane insertion of PA and/or translocation of LF/EF into the cytosol.

Cytosolic delivery and characterization of the TcdB glucosylating domain by using a heterologous protein fusion.

TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB(1-556) coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB(1-556) was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB(1-556) plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 degrees C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.

Oligomerization of anthrax toxin protective antigen and binding of lethal factor during endocytic uptake into mammalian cells.

The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF.

Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

A role for PACE4 in the proteolytic activation of anthrax toxin protective antigen.

Several bacterial protein toxins require activation by eukaryotic proteases. Previous studies have shown that anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are cleaved by furin C-terminal to the sequences RKKR, RQPR, and RVRR, respectively. Because furin-deficient cells retain some sensitivity to PA and DT, it is evident that other cellular proteases can activate these toxins. Whereas furin has been shown to require arginine residues at positions -1 and -4 for substrate recognition, another protease with an activity which could substitute for furin in toxin activation, the furin-related protease PACE4, requires basic residues in the -1, -2, and -4 positions of the substrate sequence. To examine the relative roles of furin and PACE4 in toxin activation, we used furin-deficient CHO cells (FD11 cells) transfected with either the furin (FD11/furin cells) or PACE4 (FD11/PACE4 cells) gene. Mutant PA proteins containing the cleavage sequence RAAR or KR were cytotoxic toward cells expressing only PACE4. In vitro cleavage data demonstrated that PACE4 can recognize RAAR and, to a much lesser extent, KR and RR. When extracts from PACE4-transfected cells were used as a source of proteases, PACE4 had minimal activity, indicating that it had been partially inactivated or did not remain associated with the cell membranes. Cleavage of iodinated PA containing the sequence RKKR or RAAR was detected on the surface of all cell types tested, but cleavage of a dibasic sequence was detected only intracellularly and only in cells that expressed furin or PACE4. The data provide evidence that PACE4 is present at the exterior of cells, that it plays a role in the proteolytic activation of anthrax toxin PA, and that PACE4 can activate substrates at the sequence RAAR or KR.

Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.

The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor.

The protective antigen (PA) component of anthrax toxin contains two sites that are uniquely sensitive to proteolytic cleavage. Cleavage at the sequence RKKR167 by the cellular protease furin is absolutely required for toxicity, whereas cleavage by chymotrypsin or thermolysin at the sequence FFD315 inactivates the protein, apparently by blocking the ability of PA to translocate the catalytic moieties of the toxins, lethal factor (LF) and edema factor (EF), to the cytosol of eukaryotic cells. To specify the role of the chymotrypsin-sensitive site of PA in the translocation of LF, we altered residues 313-315. None of the mutations in this region interfered with the ability of PA to bind to its cellular receptor, be cleaved by cell surface furin, and bind LF. Substitution of Ala for Asp315 or for both Phe313 and Phe314 reduced the ability of PA to intoxicate cells in the presence of LF by 3- and 7-fold, respectively. Substitution of Phe313 by Cys greatly reduced the rate of LF translocation and delayed toxicity. The rate at which the Cys-substituted PA killed cells was increased significantly by blocking the sulfhydryl group with iodoacetamide, suggesting that this added Cys interacts with cellular proteins and slows translocation of LF. Deletion of the 2 Phe rendered PA completely non-toxic. This deleted PA protein lacked the ability shown by native PA to form oligomers on cells and in solution and to induce release of 86Rb from Chinese hamster ovary cells. These results suggest that the chymotrypsin-sensitive site in PA is required for membrane channel formation and translocation of LF into the cytosol. PA double mutants were constructed that cannot be cleaved at either the furin or chymotrypsin sites. These PA proteins were more stable in Bacillus anthracis culture supernatants and may therefore be useful as a replacement for PA in anthrax vaccines.

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).

Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen.

Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA.

Anthrax toxin protective antigen: low‐pH‐induced hydrophobicity and channel formation in liposomes

To probe the role of the protective antigen (PA) component of anthrax toxin in toxin entry into animals cells, we examined the membrane channel-forming properties and hydrophobicity of intact and trypsin-cleaved forms of the protein at various pH values. At neutral pH neither form caused release of entrapped K+ from unilamellar lipid vesicles. At pH values below 6.0, however, K+ was rapidly released upon addition of either the nicked PA (PAN) or the 63 kDa tryptic fragment of PA (PA63), which has been implicated in the toxin entry process. Under the same conditions intact PA exhibited only weak channel-forming activity, and PA20, the complementary tryptic fragment, showed no such activity. Both PA and PA63 exhibited enhanced hydrophobicity at acidic pH values, but the enhancement was greater and the pH threshold higher with PA63. Our findings indicate that proteolytic removal of PA20 from intact PA enables the residual protein, PA63, to adopt a conformation at mildly acidic pH values that permits it to insert readily and form channels in membranes. Thus acidic conditions within endocytic vesicles may trigger membrane insertion of PA63, which in turn promotes translocation of ligated effector moieties, edema factor or lethal factor, across the vesicle membrane into the cytosol.

Immunologic studies of anthrax. IV. Evaluation of the immunogenicity of three components of anthrax toxin.

Summary Three components of anthrax toxin (edema factor, protective antigen and lethal factor) were separated and tested singly at three dose levels and in factorial combination (27 treatments) to determine their efficacy as immunogens in a resistant host (rat) and in a susceptible host (guinea pig). Each treatment was evaluated as an immunogen by five criteria: ( 1 ) protection of the host against challenge, ( 2 ) influence on the number of bacilli/milliliter of blood at death, ( 3 ) change in the units of toxin/milliliter of blood at death, ( 4 ) a development of antibody titer (Ouchterlony) and ( 5 ) units of toxin neutralized/milliliter of blood. These evaluations showed that ( 1 ) the LF component was highly immunogenic in rats against either toxin or spore challenge and in guinea pigs against spore challenge; ( 2 ) the PA component was immunogenic against spore challenge in rats and guinea pigs, but completely ineffective against toxin challenge in rats; ( 3 ) the EF component alone was nonimmunogenic; ( 4 ) the effects of LF and PA combinations interacted significantly with LF to increase resistance in the rat, but was not additive in resistance in the guinea pig. The number of organisms/milliliter of terminal blood decreased as resistance to establishment of disease increased. The units of toxin/milliliter of terminal blood were closely related to the number of bacilli/milliliter of blood at death. Only 17% of the prechallenge serum of guinea pigs, principally among the LF treatments, produced antigen antibody precipitin lines on Ouchterlony plates. The rat sera were all negative in this test. The antigen used to immunize man and animals should contain all the toxin components for maximum efficiency.