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1.
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. 相似文献
2.
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. 相似文献
3.
Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol. 总被引:4,自引:0,他引:4
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. 相似文献
4.
Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin 总被引:24,自引:0,他引:24
C B Siegall V K Chaudhary D J FitzGerald I Pastan 《The Journal of biological chemistry》1989,264(24):14256-14261
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. 相似文献
5.
Mutational analysis of domain I of Pseudomonas exotoxin. Mutations in domain I of Pseudomonas exotoxin which reduce cell binding and animal toxicity 总被引:16,自引:0,他引:16
Y Jinno V K Chaudhary T Kondo S Adhya D J FitzGerald I Pastan 《The Journal of biological chemistry》1988,263(26):13203-13207
Pseudomonas exotoxin (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three structural domains. Domain I is responsible for cell recognition, II for translocation of PE across membranes and III for ADP ribosylation of elongation factor 2. Treatment of PE with reagents that react with lysine residues has been shown to lead to a reduction in cytotoxic activity apparently due to a modification of domain I (Pirker, R., FitzGerald, D. J. P., Hamilton, T. C., Ozols, R. F., Willingham, M. C., and Pastan, S. (1985) Cancer Res. 45, 751-757). To determine which lysine residues are important in cell recognition, all 12 lysines in domain I were converted to glutamates by site-directed mutagenesis. Also, two deletion mutants encompassing almost all of domain I (amino acids 4-252) or most of domain I (amino acids 4-224) were studied. The mutant proteins were produced in Escherichia coli, purified, and tested for their cytotoxic activity against Swiss 3T3 cells and in mice. The data indicate that conversion of lysine 57 to glutamate reduces cytotoxic activity towards 3T3 cells 50-100-fold and in mice about 5-fold. Deletion of amino acids 4-224 causes a similar reduction in toxicity towards cells and mice. Deletion of most of the rest of domain I (amino acids 4-252) causes a further reduction in toxicity toward cells and mice indicating this second region between amino acids 225 and 252 of domain I is also important in the toxicity of PE. Competition assays indicated that the ability of PEGlu57 to bind to 3T3 cells was greatly diminished, accounting for its diminished cytotoxic activity. 相似文献
6.
Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A 总被引:2,自引:0,他引:2
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. 相似文献
7.
Domain II mutants of Pseudomonas exotoxin deficient in translocation 总被引:16,自引:0,他引:16
Y Jinno M Ogata V K Chaudhary M C Willingham S Adhya D FitzGerald I Pastan 《The Journal of biological chemistry》1989,264(27):15953-15959
Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2. PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation. To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity. Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant. Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment. 相似文献
8.
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 相似文献
9.
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. 相似文献
10.
R J Kreitman C B Siegall V K Chaudhary D J FitzGerald I Pastan 《Bioconjugate chemistry》1992,3(1):63-68
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) 相似文献
11.
V K Chaudhary D J FitzGerald I Pastan 《Biochemical and biophysical research communications》1991,180(2):545-551
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. 相似文献
12.
Cell-mediated cleavage of Pseudomonas exotoxin between Arg279 and Gly280 generates the enzymatically active fragment which translocates to the cytosol. 总被引:16,自引:0,他引:16
M Ogata C M Fryling I Pastan D J FitzGerald 《The Journal of biological chemistry》1992,267(35):25396-25401
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. 相似文献
13.
Pseudomonas aeruginosa causes life-threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96-219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234-453) encodes a 14-3-3-dependent ADP-ribosyltransferase domain which transfers ADP-ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high-affinity substrates for ADP-ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP-ribosyltransferase activity. MALDI-MS and site-directed mutagenesis studies determined that ExoS ADP-ribosylated moesin at three C-terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP-ribosylated-moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP-ribosylation directly inhibited ERM phosphorylation. Expression of dominant active-moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP-ribosylation. These data explain how the expression of the ADP-ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton. 相似文献
14.
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. 相似文献
15.
Erin L. Boland Crystal M. Van Dyken Rachel M. Duckett Andrew J. McCluskey Gregory M.K. Poon 《Journal of molecular biology》2014
The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions—supplied exogenously as genetic elements or purified protein fragments—had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes. 相似文献
16.
Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides. 总被引:6,自引:1,他引:5
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Clones expressing regions of the 100-kDa Bacillus sphaericus SSII-1 mosquitocidal toxin (Mtx) as fusion proteins with glutathione S-transferase were constructed, and the toxin-derived peptides were purified. The in vitro ADP-ribosylation activities of these peptides and their effects on larvae and cells in culture were studied. Mtx25 (amino acids 30 to 493) was found to ADP-ribosylate two proteins with molecular masses of 38 and 42 kDa, respectively, in Culex quinquefasciatus (G7) cell extracts, in addition to ADP-ribosylating itself. Mtx21 (amino acids 30 to 870; or a combination of Mtx25 and Mtx26 (amino acids 259 to 870) caused mortality in C. quinquefasciatus larvae. Mtx25, Mtx26, or Mtx24 (amino acids 30 to 276) alone and Mtx24 in combination with Mtx26 were not toxic to larvae. Mtx21 and Mtx26 produced marked morphological changes in G7 cells and to a lesser extent in Aedes aegypti cells but had no effect on Anopheles gambiae or HeLa cells. Thus, a domain in the N-terminal region of the Mtx protein is sufficient for ADP-ribosylation of C. quinquefasciatus cell protein, and a domain in the C-terminal region is sufficient for toxicity to cultured C. quinquefasciatus cells; however, both regions are necessary for toxicity to mosquito larvae. 相似文献
17.
Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL 总被引:23,自引:0,他引:23
S Seetharam V K Chaudhary D FitzGerald I Pastan 《The Journal of biological chemistry》1991,266(26):17376-17381
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. 相似文献
18.
G J Chaudry R B Wilson R K Draper R C Clowes 《The Journal of biological chemistry》1989,264(25):15151-15156
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. 相似文献
19.
Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity. 总被引:5,自引:0,他引:5
J E Wedekind C B Trame M Dorywalska P Koehl T M Raschke M McKee D FitzGerald R J Collier D B McKay 《Journal of molecular biology》2001,314(4):823-837
Exotoxin A of Pseudomonas aeruginosa asserts its cellular toxicity through ADP-ribosylation of translation elongation factor 2, predicated on binding to specific cell surface receptors and intracellular trafficking via a complex pathway that ultimately results in translocation of an enzymatic activity into the cytoplasm. In early work, the crystallographic structure of exotoxin A was determined to 3.0 A resolution, revealing a tertiary fold having three distinct structural domains; subsequent work has shown that the domains are individually responsible for the receptor binding (domain I), transmembrane targeting (domain II), and ADP-ribosyl transferase (domain III) activities, respectively. Here, we report the structures of wild-type and W281A mutant toxin proteins at pH 8.0, refined with data to 1.62 A and 1.45 A resolution, respectively. The refined models clarify several ionic interactions within structural domains I and II that may modulate an obligatory conformational change that is induced by low pH. Proteolytic cleavage by furin is also obligatory for toxicity; the W281A mutant protein is substantially more susceptible to cleavage than the wild-type toxin. The tertiary structures of the furin cleavage sites of the wild-type and W281 mutant toxins are similar; however, the mutant toxin has significantly higher B-factors around the cleavage site, suggesting that the greater susceptibility to furin cleavage is due to increased local disorder/flexibility at the site, rather than to differences in static tertiary structure. Comparison of the refined structures of full-length toxin, which lacks ADP-ribosyl transferase activity, to that of the enzymatic domain alone reveals a salt bridge between Arg467 of the catalytic domain and Glu348 of domain II that restrains the substrate binding cleft in a conformation that precludes NAD+ binding. The refined structures of exotoxin A provide precise models for the design and interpretation of further studies of the mechanism of intoxication. 相似文献
20.
Pseudomonas exotoxin: chimeric toxins 总被引:17,自引:0,他引:17
Pseudomonas exotoxin binds to and enters cells by receptor-mediated endocytosis. Within the cell it requires exposure to low pH to enable it to translocate to the cell cytoplasm where it inhibits protein synthesis by ADP-ribosylating elongation factor 2. The toxin has three main structural domains whose functions are: Ia, cell binding; II, translocation; and III, ADP-ribosylation. Key amino acids have been identified within each domain that are required for the function of the toxin. Chimeric toxins were made originally by using chemical cross-linking reagents to couple Pseudomonas exotoxin (or other toxins) to cell-binding proteins. More recently, a variety of Pseudomonas exotoxin-related chimeric toxins have been made by gene fusion technology. These chimeric toxins may be useful clinically for treating various diseases and experimentally for understanding receptor function. 相似文献