Diphtheria, toxin

Diphtheria toxin is the main pathogenicity factor in diphtheria (Pappen-heimer, 1977). The toxin gene is carried by a bacteriophage, p, which is lysogenic in Corynebacterium diphtheriae. Toxigenic strains of the bacteria cause local infection of the throat. After recovery from the acute phase of the disease, life-threatening organ complications often occur, mainly in the heart, which are due to toxin produced by the bacteria in the throat and released into the circulation. Due to mass vaccination, the disease is now almost extinct in developed countries. 

Aktories (Ed.), Bacterial Toxins. Chapman Hall, Weinheim, 1997. ISBN 3-8261 -0080-8 273 

Diphtheria toxin is produced as a single polypeptide chain (Fig. la) yyhich is easily cleaved ( nicked ) by trypsin and trypsin-like proteases into two disulfide-linked fragments, A and B (Pappenheimer, 1977). The structure of the nicked toxin resembles that of the plant toxins ricin, abrin, modeccin, viscumin and others (Olsnes and Sandvig, 1985). 

Diphtheria toxin inactivates elongation factor 2, an enzyme required for protein synthesis (Pappenheimer, 1977) through catalyzing its ADP-ribosylation, thereby inhibiting protein synthesis and inducing cell death. Elongation factor 2 contains a unique amino acid, diph-thamide, which is formed by posttranslational modification of a histidine residue (Van Ness et ai, 1980). The ADP-ribose binds covalently to this unusual amino acid. 

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Page 1170 (Figure 28 5) is adapted from crystallographic coordinates deposited with the Protein Data Bank PDB ID IDDN White A Ding X Vanderspek J C Murphy J R Ringe D Structure of the Metal Ion Activated Diphtheria Toxin Re pressor/Tox Operator Complex Nature 394 p 502 (1998)... 

A different kind of enzyme, translocase [80700-39-6], which transfers a fragment of NAD to the protein—synthesis factor (elongation factor 2), is catalyzed by diphtheria toxin, thereby inhibiting protein synthesis (43). In tumor cells, the rate of protein synthesis is 100 to 1000 times more sensitive to diphtheria toxin than the analogous process in normal cells (41) therefore, diphtheria toxin is selectively toxic to tumor cells. 

Protective and exploitive proteins Immunoglobulins Thrombin Eibrinogen Antifreeze proteins Snake and bee venom proteins Diphtheria toxin Rtcin... 

FIGURE 10.32 The structures of (a) S-eudotoxiu (two views) from Bacillus thuringiensis and (b) diphtheria toxin from Cmynehacterium diphtheriae. Each of these toxins possesses a bundle of a-hehces which is presumed to form the trausmembraue channel when the toxin Is Inserted across the host membrane. In S-endotoxln, helix 5 (white) Is surrounded by 6 helices (red) In a 7-hellx bundle. In diphtheria toxin, three hydrophobic helices (white) lie at the center of the transmembrane domain (red). 

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]. 

Diphtheria toxin, Pseudomonas exotoxin A Elongation factor 2 ADP-ribosylation Inhibition of protein synthesis (diphtheria, Pseudomonas infection)... 

Diphtheria toxin, an exotoxin of Corynebacterium diphtheriae infected with a specific lysogenic phage, catalyzes the ADP-ribosylation of EF-2 on the unique amino acid diphthamide in mammalian cells. This modification inactivates EF-2 and thereby specifically inhibits mammalian protein synthesis. Many animals (eg, mice) are resistant to diphtheria toxin. This resistance is due to inability of diphtheria toxin to cross the cell membrane rather than to insensitivity of mouse EF-2 to diphtheria toxin-catalyzed ADP-ribosylation by NAD. 

Microbial toxins such as diphtheria toxin and activated serum complement components can produce large pores in cellular membranes and thereby provide macromolecules with direct access to the internal miheu. 

Diphtheria antitoxin Neutralization of the erythrogenic effect of diphtheria toxin in the skin of guinea-pigs lOOOlUmI" if prepared in horses 5001U mM If prepared in other species... 

In the early 1900s, a balanced mixture of diphtheria toxin and antitoxin was found to produce active immunity in both animals and humans. This preparation gained widespread acceptance and protected approximately 85% of recipients. Several years later, diphtheria toxoid was developed by treating the toxin with small amounts of formalin. This process caused the toxin to lose its toxic properties while maintaining its immunogenic properties. In the mid-1920s, the addition of an alum precipitate enhanced the immunogenic properties of the toxoid. 

Denileukin diftitox is a combination of the active sections of interleukin 2 and diphtheria toxin. It binds to high-affinity interleukin 2 receptors on the cancer cell (and other cells), and the toxin portion of the molecule inhibits protein synthesis to result in cell death. The pharmacokinetics of denileukin diftitox are best described by a two-compartment model, with an a half-life of 2 to 5 minutes and a terminal half-life of 70 to 80 minutes. Denileukin diftitox is used for the treatment of persistent or recurrent cutaneous T-cell lymphoma whose cells express the CD25 receptor. Side effects include vascular leak syndrome, fevers/chills, hypersensitivity reactions, hypotension, anorexia, diarrhea, and nausea and vomiting. 

The entry of CL into cells may be essential for the cellular entry [232] or secretion [233] of some macromolecules such as diphtheria toxin and modeccin. Sandvig and Olsnes [232] studied the entry of diphtheria toxin and modeccin into Vero cells in pH 7.2 media containing 20 mM Hepes, 1 mM Ca(OH)2, 5 mM glucose,a sufficient amount of mannitol to ensure isotonicity, and varying concentrations of NaCl. The cellular uptake of 0.1 nM diphtheria toxin at the end of 50 min was strongly dependent on CL concentration. It was 0% at 0 mM NaCl, 25% of the 140 mM NaCl control at 2 mM NaCl, and 60% of the control at 70 mM NaCl. A similar trend was observed for modeccin, i.e., no transport at 0 mM NaCl, 20% of control at 0.05 mM NaCl, 60% of control at 0.1 mM NaCl, 80% of control at 0.5 mM NaCl, and 100% of control at 2 mM NaCl. 

K Sandvig, S Olsnes. (1982). Entry of toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. I. Requirement for Ca2+. J Biol Chem 257 7495-7503. 

Using this approach, EGF has been successfully conjugated by disulfide exchange to the A chain of diphtheria toxin (Shimisu et al., 1980). A cystaminyl derivative of insulin also could be conjugated to the A chain of diphtheria toxin by this method (Miskimins and Shimizu, 1979). Other references to disulfide exchange using cystamine include Oeltmann and Forbes (1981) and Bacha et al. (1983) who prepared antibody-toxin and peptide-toxin conjugates, respectively. 

Bacha, P., Murphy, J.R., and Reichlin, S. (1983) Thyrotropin-releasing hormone-diphtheria toxin-related polypeptide conjugates./. Biol. Chem. 258, 1565. 

Collier, R.J., and Cole, H.A. (1969) Diphtheria toxin subunit active in vitro. Science 164, 1179. 

Colliei R.J., and Kandel, J. (1971) Structure and activity of diphtheria toxin./. Biol. Chem. 246, 1496-1503. 

Colombatti, M., Greenfield, L., and Youle, R.J. (1986) Cloned fragment of diphtheria toxin linked to T cell-specific antibody identifies regions of B chain active in cell entry./. Biol. Ghem. 261, 3030. 

Dell Arciprete, L., Colombatti, M., Rappuoli, R., and Tridente, G. (1988) A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity./. Immunol. 140, 2466-2471. 

Gill, D.M., Pappenheimer Jr, A.M, Brown, R., and Kurnick, J.T. (1969) Studies on the mode of action of diphtheria toxin VII. Toxin-stimulated hydrolysis of nicotinimide adenine dinucleotide in mammalian cell extracts./. Exp. Med. 129, 1-21. 

Honjo, J., Nishizuka, Y., Hayaishi, O., and Kato, I. (1968) Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J. Biol. Cbem. 243, 3553-3555. 

Keen, J.H., Maxfield, F.R., Hardegree, M.C., and Habig, W.H. (1982) Receptor-mediated endocytosis of diphtheria toxin by cell in culture. Proc. Natl Acad. Sci. USA 79, 2912. 

Masuho, Y., Hara, T., and Noguchi, T. (1979) Preparation of hybrid of fragment Fab of antibody and fragment A of diphtheria toxin and its cytotoxicity. Biochem. Biophys. Res. Comm. 90, 320. 

Miskimins, W.K., and Shimizu, N. (1979) Synthesis of cytotoxic insulin cross-linked to diphtheria toxin fragment a capable of recognizing insulin receptors. Biochem. Biophys. Res. Comm. 91, 143. 

Oeltmann, T.N. (1985) Synthesis and in vitro activity of a hormone-diphtheria toxin fragment A hybrid. Biochem. Biophys. Res. Comm. 133, 430. 

Oeltmann, T.N., and Forbes, J.T. (1981) Inhibition of mouse spleen cell function by diphtheria toxin fragment A coupled to anti-mouse Thy-1.2 and by ricin A chain coupled to anti-mouse IgM. Arch. Biochem. Biophys. 209, 362. 

Sandvig, K., and Olsnes, S. (1981) Rapid entry of nicked diphtheria toxin into cells at low pH. Characterization of the entry process and effects of low pH on the toxin molecule. /. Biol. Cbem. 256, 9068. 

Replacement of most Virtually all of the murine amino acid sequences with sequences found in human antibodies Fusion protein consisting of the diphtheria toxin linked to interleukin-2 (IL-2)... 

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