Translocation Diphtheria toxin

Protein toxins acting intracellularly are often composed of two subunits (A/B model). One subunit is catalytic (A-subunit) and the other is responsible for binding and cell entry (B-subunit). Following binding to an extracellular membrane receptor, the toxins are endocytosed. From the endosomes, the A-subunit is directly (pH dqDendent) transferred into the cytosol (e.g., diphtheria toxin and anthrax toxin) or the toxin is transported in a retrograde manner via the golgi to the ER (e.g., cholera toxin), where translocation into the cytosol occurs [1]. 

In eukaryotic cells, elongation factor-2 (eEF-2) used in translocation is inactivated through ADP-ribosylation by Pseudomonas and Diphtheria toxins. 

C. botulinum toxins belong to the AB group of toxins, which also includes diphtheria toxin, pseudomonas exotoxin A, anthrax toxin, Shiga(like) toxin, cholera toxin, pertussis toxin, and plant toxins, e.g., ricin. Moiety A has an enzymatic activity and usually modified cellular-target entering cytosol. Moiety B consists of one or more components and binds the toxin to surface receptors, and is responsible for translocation of the A component into cells. AB toxins are produced in a non-active form and are activated by a split between two cysteine residues within a region (Falnes and Sandvig, 2000). 

A. General description Denileukin dif-titox is a recombinant, DNA-derived, interleukin-2 receptor specific ligand, cytotoxic fusion protein consisting of diphtheria toxin fragments A and B fused to interleukin-2. It is produced by expression of a recombinant fusion protein in Escherichia coli that contains nucleotide sequences for human interleukin-2, and sequences for the enzymatically active fragment A of diphtheria toxin and the membrane-translocating portion of diph-... 

This reaction is reversible when conducted in vitro, but under the conditions of pH and nicotinamide concentration that exist in the cell, it is irreversible. Thus, diphtheria toxin kills cells by irreversibly destroying the ability of EF-2 to participate in the translocation step of protein synthesis elongation. A number of other protein toxins have subsequently been found to ADP-ribosylate and inactivate cellular proteins involved in other essential cellular pathways. For example, cholera and pertussis toxins ADP-ribosylate and inactivate proteins important to cAMP metabolism. 

The enzymatic specificity of diphtheria toxin deserves special comment. The toxin ADP-ribosylates EF-2 in all eukaryotic cells in vitro whether or not they are sensitive to the toxin in vivo, but it does not modify any other protein, including the bacterial counterpart of EF-2. This narrow enzymatic specificity has called attention to an unusual posttranslational derivative of histidine, diphthamide, that occurs in EF-2 at the site of ADP-ribosylation (see fig. 1). Although the unique occurrence of diphthamide in EF-2 explains the specificity of the toxin, it raises questions about the functional significance of this modification in translocation. Interestingly, some mutants of eukaryotic cells selected for toxin resistance lack one of several enzymes necessary for the posttranslational synthesis of diphthamide in EF-2 that is necessary for toxin recognition, but these cells seem perfectly competent in protein synthesis. Thus, the raison d etre of diphthamide, as well as the biological origin of the toxin that modifies it, remains a mystery. 

Ramon G, Descombey P (1925) Sur 1 immunization antitetanique et sur la production de l antitoxine tetanique Compt Rend Soc Biol 93 508-98 Ratts R, Zeng H, Berg EA, Blue C, McComb ME et al. (2003) The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex. J Cell Biol 160 1139-50... 

Diphtheria Toxin Blocks Protein Synthesis in Eukaryotes by Inhibiting Translocation... 

Figure 29.35. Blocking of Translocation by Diphtheria Toxin. Diphtheria toxin blocks protein synthesis in eukaryotes by catalyzing the transfer of an ADP-ribose unit from NAD+ to diphthamide, a modified amino acid residue in elongation factor 2 (translocase). Diphthamide is formed by a posttranslational modification (blue) of a histidine residue.

The structurally related antibiotics kirromycin and pulvomycin both act upon EF-Tu of most bacteria (and chloroplasts) although not upon its eucaryal (EF-la) counterpart [158,159]. The steroid antibiotic fusidic acid interacts systematically with both the eucaryal (EF-2) and the bacterial (EF-G) translocating factors, including chloroplasts of higher plants. Diphtheria toxin (fragment A) discriminates between bacterial-mitochondrial and eucaryal translocating factors by selectively and irreversibly impairing the eucaryal EF-2 factors [160,161]. 

Unlike diphtheria toxin, little is known about the structures required for the translocation of the enzymatic subunit of PT. In diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the B moiety can be clearly subdivided into two distinct domains, one responsible for receptor binding, composed essentially of (3 sheets, and one responsible for translocation of the A subunits, essentially composed of a helices (Allured etal., 1986 Choe etal., 1992). There is no clear translocation domain in PT, and much less is known about the internalization step of PT, compared to diphtheria toxin and exotoxin A. 

Structural homologies between PFTs and other toxins have not been identified. However, the process of membrane permeabilization may be operative in many cases where proteins have to escape from an intracellular compartment. Well known examples are diphtheria toxin, the neurotoxins and anthrax toxin. Specific domains in many intracel-lularly active toxins have in fact been shown to produce pores in artificial lipid bilayers, and membrane permeabilization is thought to form the basis for translocation of the active moieties from the late endo-some to the cytoplasm (reviewed in Montecucco et ai, 1994). The molecular mechanism of this translocation remains obscure. In the... 

Like diphtheria toxin. Pseudomonas aeruginosa exotoxin A requires low pH to act (FitzGerald ef al., 1980). In spite of this, it has not been possible to induce translocation of Pseudomonas toxin across the surface membrane by exposure to low pH. It appears that the toxin must be transported beyond the endosomes, possibly to the trans-Golgi network or even to the endoplasmic reticulum to find conditions required for translocation (Chaudhary et al., 1990). In fact domain III ends with an amino acid sequence that (after removal of a terminal... 

It has been shown that a number of peptides linked to the N-terminal end of the diphtheria toxin A-fragment are translocated to the cytosol (Stenmark etai, 1991, Stenmark etal., 1992 Ariansen efal., 1993). It is an interesting question to what extent such peptides can subsequently be presented by class I major histocompatibility antigens. If this is the case, fusion proteins of viral or cancer-related peptides and enzymatically inactive mutants of diphtheria toxin could be used for vaccination purposes to expand desired populations of cytotoxic CD T-lymphocytes. 

The Clostridium bofulinum exoenzyme C3 is able to ADP-ribosylote the small G-protein, rho, in a cell free system, but is unable to enter cells (except at very high concentrations) because it lacks a B-moiety. Constructs where exoenzyme C3 was linked to the N-terminal end of diphtheria toxin or to toxin where the N-terminal part of the A-fragment had been deleted, were able to translocate the exoenzyme-containing fusion protein into the cytosol and interfere with the organization of actin filaments (Aullo et a/., 1993). 

Dihydrofolate reductase has been used extensively in translocation experiments. A fusion protein with diphtheria toxin A-fragment was shown to be translocated to the cytosol (Klingenberg and Ols-nes, 1996). The translocation was inhibited by methotrexate, which induces a tight folding of the protein. A fusion with a mutated dihydrofolate reductase that does not bind methotrexate tightly was also translocated, and in this case methotrexate was not able to prevent the translocation. This indicates that not only must the toxin A-fragment be unfolded, but the passenger protein must also be able to unfold for translocation to occur. 

The question whether toxins can be used to translocate nucleic acids into cells is very interesting, but this has not been tested seriously up to now. Toxins are interesting candidates for delivery of anti-sense RNA, ribozymes and genes for transformation and gene therapy. So far there is not convincing evidence that toxins have been effective for such purposes. From what is now known about the translocation of diphtheria toxin, it is likely that the nucleic acid would have to be linked end-to-end to the N-terminus of the A-fragment of this... 

Ariansen SA, Afanasiev BN, Moskaug J0, Stenmark H, Madshus IH, Olsnes S (1993) Membrane translocation of diphtheria toxin A-fragment Role of carbox-yterminal region. Biochemistry 32 83-90. 

Falnes P0, Choe S, Madshus I H, Wilson B, Olsnes S (1994) Inhibition of membrane translocation of diphtheria toxin A-fragment by internal disulfide bridges. J Biol Chem 269 8402-8407. 

Falnes P0, Madshus IH, Sandvig K, Olsnes S (1992) Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B-fragment. Effect on membrane translocation and formation of cation channels. J Biol Chem 267 12284-12290. 

Falnes P0, Olsnes S (1995) Cell-mediated reduction and incomplete membrane translocation of diphtheria toxin mutants with internal disulfides in the A-fragment. J Biol Chem 270 20787-20793. 

Klingenberg O, Olsnes S (1996) Ability of methotrexate to inhibit translocation to the cytosol of dihydrofolate reductase linked to diphtheria toxin. Biochem J 313 647-653. 

Lanzrein M, Garred 0, Olsnes S, Sandvig K (1995) Diphtheria toxin endocytosis and membrane translocation is dependent on the intact membrane anchored receptor (HB-EGF precursor) Studies on the cell-associated receptor cleaved by a metalloprotease in TPA-treated cells. Biochem J 310 285-289. 

Lanzrein M, Sand O, Olsnes S (1996) GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without ion channel activity. EMBOJ... 

Moskaug J0, Sandvig K, Olsnes S (1989) Role of anions in low pH-induced translocation of diphtheria toxin. J Biol Chem 264 11367-11372. 

Papini E, Sandona D, Rappuoli R, Montecucco C (1988) On the membrane translocation of diphtheria toxin at low pH the toxin induces ion channels on cells. EMBO J 7 3353-3359. 

Stenmark H, Moskaug J0, Madshus IH, Sandvig K, Olsnes S (1991) Peptides fused to the amino-terminal end of diphtheria toxin are translocated to the cytosol. J Cell Biol 113 1025-1032. 

Wiedlocha A, Madshus IH, Mach H, Middaugh CR, Olsnes S (1992) Tight folding of acidic fibroblast growth factor prevents its translocation to the cytosol with diphtheria toxin as vector. EMBOJ. 11 4835-4842. 

Diphtheria toxin Catalyzes a reaction between NAD and EF2 to yield an inactive factor. Inhibits translocation. 

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