Analysis of sequences in domain II of Pseudomonas exotoxin A which mediate translocation

Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.     

Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL

Pseudomonas exotoxin (PE) is a 66,000 molecular weight protein secreted by Pseudomonas aeruginosa. PE is made up of three domains, and PE40 is a form of PE which lacks domain Ia (amino acids 1-252) and has very low cytotoxicity because it cannot bind to target cells. The sequence Arg-Glu-Asp-Leu-Lys (REDLK) at the carboxyl terminus of Pseudomonas exotoxin has been shown to be important for its cytotoxic activity (Chaudhary, V. K., Jinno, Y., FitzGerald, D. J., and Pastan, I. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 308-312). In this study, we tested the effect of altering the carboxyl sequence of PE from REDLK to the characteristic endoplasmic reticulum retention sequence, KDEL, or to KDEL repeated three times (KDEL)3. We also made similar changes at the carboxyl terminus of two chimeric toxins in which domain I of PE (amino acids 1-252) was either replaced with transforming growth factor alpha (TGF alpha) to make TGF alpha-PE40 or with a single chain antibody (anti-Tac) reacting with the human interleukin 2 receptor to make anti-Tac(Fv)-PE40. Statistical analyses of our results demonstrate that PE and its derivatives ending in KDEL or (KDEL)3 are significantly more active than PE or derivatives ending in REDLK. We have also found that brefeldin A, which is known to perturb the endoplasmic reticulum, inhibits the cytotoxic action of PE. Our results suggest that the altered carboxyl terminus may enable the toxin to interact more efficiently with a cellular component involved in translocation of the toxin to the cytosol.     

Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A

The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D. J., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE. Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity. These modified PEs were also examined for their ability to block PE cytotoxicity. Our results shown that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity. Furthermore, extracts containing PE fragments without ADP-ribosylation activity were able to block the cytotoxic activity of intact PE. Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE. The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.     

A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing.

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.     

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain

 We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain, of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(Δ34–220), which had most of the Ia domain deleted but retained amino acid residues 1–33 and 221–252 of this domain, was constructed. EGF-PE(Δ34–220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(Δ34–220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(Δ1–252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(Δ34–220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(Δ34–220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(Δ34–220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells. Received : 20 May 1994 / Received last revision : 9 September 1994 / Accepted : 28 September 1994     

Alanine scanning mutagenesis identifies surface amino acids on domain II of Pseudomonas exotoxin required for cytotoxicity, proper folding, and secretion into periplasm.

Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2. Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present. To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II. Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells. Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol. Several of the mutations resulted in altered protease sensitivity of the toxin. Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded. Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency.     

Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.     

Properties of chimeric toxins with two recognition domains: interleukin 6 and transforming growth factor alpha at different locations in Pseudomonas exotoxin.

Pseudomonas exotoxin (PE) is a potent cytotoxic agent that is composed of 613 amino acids arranged into three major domains. We have previously identified two positions where ligands can successfully be placed in PE to direct it to cells with specific surface receptors. One site is at the amino terminus and the other is close to but not at the C-terminus. To examine the possibility of constructing oncotoxins with two different recognition elements that will bind to two different receptors, we have placed cDNAs encoding either transforming growth factor alpha (TGF alpha) or interleukin 6 (IL6) at the 5' end of a PE gene and also inserted a cDNA encoding TGF alpha near the 3' end of the PE gene. The plasmids encoding these chimeric toxins were expressed in Escherichia coli and the chimeric proteins purified to near homogeneity. In all the new toxins, the TGF alpha near the C-terminus was inserted after amino acid 607 of PE and followed by amino acids 604-613 so that the correct PE C-terminus (REDLK) was preserved. For each chimera, the toxin portion was either PE4E, in which the cell binding domain (domain Ia) is mutated, PE40, in which domain Ia is deleted, or PE38, in which domain Ia and part of domain Ib are deleted. These derivatives of PE do not bind to the PE receptor and allow 607, 355, or 339 amino acids, respectively, between the two ligands.(ABSTRACT TRUNCATED AT 250 WORDS)     

A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly

Pseudomonas exotoxin A (PE) is a cytotoxin composed of three structural domains. Domain I is responsible for cell binding, domain II for membrane translocation enabling access to the cytosol, and domain III for the catalytic inactivation of protein synthesis, which results in cell death. To investigate the role of the six alpha-helices (A-F) that form the translocation domain, we deleted them successively one at a time. All mutants showed native cell-binding and catalytic activities, indicating that deletions specifically affected translocation activity. This step of the intoxication procedure was examined directly using a cell-free translocation assay, and indirectly by monitoring cytotoxicity. Translocation activity and log(cytotoxicity) were highly correlated, directly indicating that translocation is rate limiting for PE intoxication. Deletion of B, C and D helices resulted in non-toxic and non-translocating molecules, whereas mutants lacking the A or E helix displayed significant cytotoxicity albeit 500-fold lower than native PE. We concluded that B, C and D helices, which make up the core of domain II, are essential, whereas the more peripheral A and E helices are comparatively dispensable. The last helix (F) is inhibitory for translocation because its deletion produced a mutant displaying a translocation activity 60% higher than PE, along with a three- to sixfold increase in cytotoxicity in all tested cell lines. This toxin is the most in vitro active PE mutant obtained until now. Finally, partial duplication of domain II did not give rise to a more actively translocated PE, but rather to a threefold less active molecule.     

Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol.

Pseudomonas exotoxin (PE) is a three-domain toxin which is cleaved by a cellular protease within cells and then reduced to generate two prominent fragments (Ogata, M., Chaudhary, V. K., Pastan, I., and FitzGerald, D. J. (1990) J. Biol. Chem. 265, 20678-20685). The N-terminal fragment is 28 kDa in size and contains the binding domain. The 37-kDa C-terminal fragment, which translocates to the cytosol, contains the translocation domain and the ADP-ribosylation domain. Cleavage followed by reduction is essential for toxicity since mutant forms of the toxin that cannot be cleaved by cells are nontoxic. Previous results with these mutants suggest that cleavage occurred in an arginine-rich (arginine residues are at positions 274, 276, and 279) disulfide loop near the beginning of the translocation domain, but the exact site of cleavage was not determined. Since very few molecules of the 37-kDa fragment are generated within cells it was not possible to determine the site of cleavage by performing a conventional N-terminal sequence analysis of the 37-kDa fragment. Two experimental approaches were used to overcome this limitation. First, existing amino acids near the cleavage sites were replaced with methionine residues; this was followed by the addition of [35S]methionine-labeled versions of these toxins to cells. The pattern of radioactive toxin fragments recovered from the cells indicated that the toxin was cleaved either just before or just after Arg279. Second, [3H]leucine-labeled toxin was produced and added to the cells. Sequential Edman degradations were performed on the small amount of radioactive 37-kDa fragment that could be recovered from toxin-treated cells. A peak of radioactivity in the fifth fraction indicated that leucine was the 5th amino acid on the C-terminal side of the cleavage site. This result confirmed that cleavage was between Arg279 and Gly280.     

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro , furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5–10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli , purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0–5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.     

A dipeptide insertion in domain I of exotoxin A that impairs receptor binding

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.     

Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin

The Rho-GTPases-activating toxin CNF1 (cytotoxic necrotizing factor 1) delivers its catalytic activity into the cytosol of eukaryotic cells by a low pH membrane translocation mechanism reminiscent of that used by diphtheria toxin (DT). As DT, CNF1 exhibits a translocation domain (T) containing two predicted hydrophobic helices (H1-2) (aa 350-412) separated by a short peptidic loop (CNF1-TL) (aa 373-386) with acidic residues. In the DT loop, the loss of charge of acidic amino acids, as a result of protonation at low pH, is a critical step in the transfer of the DT catalytic activity into the cytosol. To determine whether the CNF1 T domain operates similarly to the DT T domain, we mutated several ionizable amino acids of CNF1-TL to lysine. Single substitutions such as D373K or D379K strongly decreased the cytotoxic effect of CNF1 on HEp-2 cells, whereas the double substitution D373K/D379K induced a nearly complete loss of cytotoxic activity. These single or double substitutions did not modify the cell-binding, enzymatic or endocytic activities of the mutant toxins. Unlike the wild-type toxin, single- or double-substituted CNF1 molecules bound to the HEp-2 plasma membrane could not translocate their enzymatic activity directly into the cytosol following a low pH pulse.     

Influence of deletions within domain II of exotoxin A on its extracellular secretion from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium that secretes many proteins into the extracellular medium via the Xcp machinery. This pathway, conserved in gram-negative bacteria, is called the type II pathway. The exoproteins contain information in their amino acid sequence to allow targeting to their secretion machinery. This information may be present within a conformational motif. The nature of this signal has been examined for P. aeruginosa exotoxin A (PE). Previous studies failed to identify a common minimal motif required for Xcp-dependent recognition and secretion of PE. One study identified a motif at the N terminus of the protein, whereas another one found additional information at the C terminus. In this study, we assess the role of the central PE domain II composed of six alpha-helices (A to F). The secretion behavior of PE derivatives, individually deleted for each helix, was analyzed. Helix E deletion has a drastic effect on secretion of PE, which accumulates within the periplasm. The conformational rearrangement induced in this variant is predicted from the three-dimensional PE structure, and the molecular modification is confirmed by gel filtration experiments. Helix E is in the core of the molecule and creates close contact with other domains (I and III). Deletion of the surface-exposed helix F has no effect on secretion, indicating that no secretion information is contained in this helix. Finally, we concluded that disruption of a structured domain II yields an extended form of the molecule and prevents formation of the conformational secretion motif.     

Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A

The promising arena of DNA-based vaccines has led us to investigate possible candidates for immunization against bacterial pathogens. One such target is the opportunistic pathogen Pseudomonas aeruginosa which produces exotoxin A (PE), a well-characterized virulence factor encoded by the toxA gene. In its native protein form, PE is highly cytotoxic for susceptible eukaryotic cells through ADP-ribosylation of elongation factor-2 following internalization and processing of the toxin. To study the biologic and immunological effects of PE following in situ expression, we have constructed eukaryotic plasmid expression vectors containing either the wild-type or a mutated, non-cytotoxic toxA gene. In vitro analysis by transfection of UM449 cells suggests that expression of the wild-type toxA gene is lethal for transfected cells whereas transfection with a mutated toxA gene results in the production of inactive PE which can be readily detected by immunoblot analysis of cell lysates. To investigate the effects resulting from the intracellular expression of potentially cytotoxic gene products in DNA vaccine constructs, we immunized mice with both the wild-type and mutant toxA plasmid constructs and analyzed the resulting humoral and cellular immune responses. Immunization with the mutated toxA gene results in production of neutralizing antibodies against native PE and potentiates a T(H)1-type response, whereas only a minimal humoral response can be detected in mice immunized with wild-type toxA. DNA-based vaccination with the non-cytotoxic toxA(mut) gene confers complete protection against challenge with the wild-type PE. Therefore, genetic immunization with genes encoding potentially cytotoxic gene products raises concern with regard to the selection of feasible gene targets for DNA vaccine development.     

A proper amino terminus of diphtheria toxin is important for cytotoxicity

A series of deletions and substitutions were made at the 5' end of the gene fusion between the first 388 codons of diphtheria toxin (DT) and a cDNA encoding human IL2. The chimeric protein (DT388-IL2) was expressed and purified from E. coli and found to be very cytotoxic to a human T cell line, HUT 102, that expresses a large number of IL2 receptors. Deletion of the first five amino acids of DT resulted in a non-cytotoxic chimeric protein that had both ADP-ribosylation activity and IL2 receptor binding activity. Deletion of the first two amino acids of DT had little effect on cytotoxicity, while deletion of the first four amino acids or of two acidic residues at positions 3 and 4 greatly reduced cytotoxicity. Unexpectedly, a mutant containing a single leucine in place of the first two amino acids (gly, ala) was 2-3 fold more active. The amino terminus of DT may participate in the translocation of the A chain to the cytosol in a manner similar to Pseudomonas exotoxin (PE) in which a specific C-terminal sequence has been proposed to be involved in its cytotoxicity.     

Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin

Pseudomonas exotoxin is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The substitution of the cell recognition domain (domain Ia) with a growth factor such as transforming growth factor alpha (TGF alpha), creates a cell-specific cytotoxic agent, TGF alpha-PE40, which kills cells bearing epidermal growth factor (EGF) receptors. We have used TGF alpha-PE40 to define the role of sequences in domains II, Ib, and III. Various mutations were made in these domains and mutant forms of TGF alpha-PE40 expressed in Escherichia coli. Mutant proteins were then tested for their ADP-ribosylation, EGF receptor-binding, and cell-killing activities. Additionally, the amino boundary of domain III, which contains the ADP-ribosylation activity, was determined by deletion analysis. Data indicate that (i) the functional amino terminus of domain III is near amino acid 400; (ii) deletion of various regions in domain II or conversion of cysteines 265 and 268 to serines results in a loss of cytotoxicity which ranged from 10-fold to more than 150-fold, indicating that domain II is essential for full expression of cytotoxicity; (iii) deletion of the amino terminus of domain Ib results in a molecule with somewhat increased cytotoxic activity, indicating that domain Ib is not essential for the cytotoxic effect of TGF alpha-PE40; and (iv) TGF alpha-PE40, produced by denaturing and refolding of insoluble material from inclusion bodies, binds better to EGF receptors and is about 10-fold more cytotoxic to cells bearing EGF receptors than is the secreted form of soluble TGF alpha-PE40.     

Verapamil enhances the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin and antitransferrin receptor with Pseudomonas exotoxin

Verapamil, a clinically important calcium channel blocker, has been found to cause a 40-fold enhancement of killing of the human KB cell line by a cytotoxic conjugate of epidermal growth factor with Pseudomonas exotoxin (EGF-PE). Synergistic effects of verapamil and EGF-PE are also seen on HeLa D98 cells and a human epidermal carcinoma cell line, A431. Verapamil also potentiates the effect of a toxic conjugate formed between Pseudomonas exotoxin and a monoclonal antibody to the human transferrin receptor (anti-TFR-PE) and enhances the effect of Pseudomonas exotoxin (PE) alone. Two other calcium antagonists were tested. Diltiazem enhances the cytotoxic effect of EGF-PE, but nifedipine does not. Verapamil does not affect the binding and uptake of 125I-EGF by KB cells, but it significantly delays the disappearance of internalized 125I-EGF from the cells. Density gradient fractionation studies using cell homogenates suggest that 125I-EGF accumulates in an undegraded form in lysosomes when cells are treated with verapamil. By immunofluorescence microscopy using an antibody to PE, EGF-PE was found to accumulate in lysosomes; by electron microscopy the lysosomes had an abnormal appearance. The effects of verapamil on toxicity of EGF-PE and lysosomal function appear to be related. However, it is not known whether the enhanced toxicity of EGF-PE in the presence of verapamil is due to its delayed degradation in lysosomes or some more general effect of verapamil on membrane permeability.     

IL2-PE664Glu, a new chimeric protein cytotoxic to human-activated T lymphocytes

To produce a molecule that will kill activated T cells as well as lymphomas and leukemias expressing interleukin 2 (IL2) receptors, we have created a recombinant chimeric protein in which IL2 is attached in peptide linkage to a truncated mutant form of Pseudomonas exotoxin (PE) (Lorberboum-Galski, H., FitzGerald, D.J.P., Chandhary, V.K., Adhya, S., and Pastan, I. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1922-1926). Although this molecule was very active on rodent cells, it had lower activity on some human cell types. A new chimeric protein termed IL2-PE664Glu has been constructed that is extremely toxic to both phytohemagglutinin blasts and mixed leukocyte reaction blasts prepared from monkey and human lymphocytes. The chimeric gene encoding this protein was constructed by fusing a cDNA clone for human interleukin 2 to the 5' end of a mutated cDNA encoding a full-length PE molecule. Four amino acids in domain I of PE were changed thus decreasing its nonspecific toxicity. IL2-PE664Glu is a much more active cytotoxic molecule for primate and human-activated T cells than IL2-PE40 which is a chimeric protein that was found to be an effective immunosuppressive agent in rodent models. Our results indicate that IL2-PE664Glu should be evaluated as an immunosuppressive agent for the treatment of human immune disorders in which activated T cells expressing the IL2 receptor are prominent.