Nh

Figure 13.5. Diphthamide residue in eEF2 (in position equivalent to His573 in domain IV of bacterial EF-G). The arrow indicates the site of ADP-ribosylation.

to yield fragments A and B, with molecular masses of 27,000 daltons and 45,000 daltons, respectively, and then fragment A passes into the cytoplasm. Fragment A is an enzyme transferring the ADP-ribose residue from NAD to the same diphthamide residue of eEF2 that was discussed above. This results in inhibition of protein synthesis. There is, however, no immunological cross-reactivity between Pseudomonas aeruginosa toxin and diphtheria toxin; in addition, receptors for these two toxins on the cell membrane are different.

13.5.2.Shiga and Shiga-like Toxins

The toxin produced by Shigella dysenteriae is also a powerful inhibitor of protein synthesis in the cells of a number of vertebrate animals. This protein toxin consists of one polypeptide A chain, with a molecular mass of 30,500 daltons, and six (or seven) relatively short B chains, with a molecular mass of about 5,000 daltons each (A1B6). The B-moiety of the protein appears to be responsible for the interaction of the toxin with the cytoplasmic membrane receptor of the animal cell and for the subsequent entry of the toxin into the membrane. Proteolytic cleavage of the toxin A chain in the membrane yields two fragments, A1 (molecular mass 27,500 daltons) and A2 (molecular mass 3,000 daltons); the A1-fragment passes into the cytoplasm and inhibits the protein synthesis. The inhibition results from the enzymatic activity of fragment A1. (It should be pointed out that enzymatic activity is not displayed prior to cleavage.) The enzymatic action in this case is targeted to the 60S subunit of the eukaryotic 80S ribosome. The enzymatic fragment of the toxin proved to be a glycosidase specifically hydrolyzing N-glycosidic bond of the adenosine residue at position 4324 in mammalian 28S ribosomal RNA (equivalent to A2660 of the E. coli 23S RNA) (Endo et al, 1988). Ribosomes with modified 60S subunits are capable of performing transpeptidation; in other words, the peptidyl transferase center is not impaired. The inhibition of protein synthesis by the A1-fragment of the toxin is due to the damage of the factor-binding site on the 60S ribosomal subunit. The function of eEF1 in aminoacyl-tRNA binding seems to be impaired in a more extent than the function of eEF2 in translocation (Obrig et al., 1987; Furutani et al., 1992).

Shiga-like toxins, called Vero toxins (VT1 and VT2), have been shown to be produced by some pathogenic strains of Escherichia coli. The structure of the toxins is similar to that of the Shiga toxin, and the mechanism of their action is the same.

13.5.3. a-Sarcin a-Sarcin is a toxin produced by the mold Aspergillus giganteus (for review, see Kao & Davies, 1995). It is a small basic protein with a molecular mass of about 16,000 daltons. The protein is an exceedingly potent inhibitor of protein synthesis in eukaryotic cells and cell extracts. It is a highly specific ribonuclease: the inhibition is the result of the hydrolysis of a single phosphodiester bond on the 3'-side of G4325 of mammalian 28S ribosomal RNA (equivalent to G2661 in the E. coli 23S rRNA, see Fig. 9.10 A) (Endo & Wool, 1982). Since the target, a conservative helical structure in domain VI of 28S ribosomal RNA, is involved in the formation of the factor-binding site on the 60S ribosomal subunit (see Section 9.5), the interactions of both elongation factors, eEF1 and eEF2, with the ribosome may be impaired. The predominant effect observed in experiments, however, is the inhibition of the binding of aminoacyl-tRNA:eEF1:GTP complex to the ribosome.

13.5.4.Plant Toxins

A number of powerful toxins that inhibit protein synthesis in target cells are found among plant lectins specifically interacting with the D-galactose residues of the glycoproteins present in the cell membrane of animal cells. These toxins include ricin from castor beans, Ricinus communis; abrin from Abrus precatorius; modeccin from Modecca digitata; and viscumin from the mistletoe, Viscum album (for review, see Olsnes & Pihl, 1982a). These proteins show a striking similarity to the bacterial toxins, as regards their molecular-functional organization.

Ricin is a two-subunit protein (glycoprotein) with a molecular mass of 62,000 daltons. The B-subunit (molecular mass 31,400 daltons) is a lectin in the strict sense of the word and is capable of binding with galactose residues on the external surface of the animal cell membrane. The A-subunit (molecular mass 30,000 daltons) is responsible for the inhibition of protein synthesis in the cytoplasm. The two subunits are linked by a disulfide bridge. The attachment of the toxin molecule to the membrane is followed by entry of the molecule into the membrane, disulfide bridge reduction, and release of the liberated A-subunit into the cytoplasm. The A-subunit possesses a specific N-glycosidase activity: it depurinates the adenosine residue at position 4324 in domain VI of 28S ribosomal RNA (equivalent to A2660 in the E. coli 23S rRNA, see Fig. 9.10 A) (Endo et al., 1987). As a result, the function of the factor-binding site on the 60S ribosomal subunit becomes damaged. Correspondingly, the binding of both aminoacyl-tRNA:eEF1:GTP complex and eEF2:GTP has been reported to be affected by ricin. At the same time, the impairment of the eEF1 interactions with the ribosome is more apparent under in vitro experimental conditions.

Other plant toxins are organized in a similar way and have similar action, although chemically they are different proteins. The so-called pokeweed anti-viral protein (PAP) from Phytalacca americana is particularly interesting. Its molecular mass is 27,000 daltons and it is an analog of the A-subunit of ricin. Correspondingly, it does not possess lectin activity and cannot interact with the cell membrane. Therefore it does not affect intact cells but strongly inhibits protein synthesis in vitro in eukaryotic cell-free systems due to its specific N-glycosidase activity.

13.5.5.Artificial Chimeric Toxins

Thus, many protein toxins of bacterial and plant origin make use of the same principle of cytotoxic action based on a two-subunit or two-domain structure: one subunit (or fragment) interacts with the membrane and is responsible for the transmembrane transport, while the other is released into the cell and exhibits an enzymatic activity there, resulting in the inhibitory modification of some components of the protein-synthesizing system. This principle observed in living nature may be exploited to deliver any enzyme protein inside the cell, if such a protein is artificially conjugated or cross-linked with a suitable membrane-interacting protein (for review, see Olsnes & Pihl, 19826). For example, using a simple procedure of disulfide exchange, it was possible to conjugate the enzymatic A-fragment of diphtheria toxin or the A-subunit of ricin with a nontoxic plant lectin (e.g. with concanavalin A or lectin of Wistaria floribunda) and to obtain a cytotoxic effect; it is clear that the lectin moiety of the chimeric protein was responsible for the delivery of the inhibitory component into the cell. However, just as in the case of the original toxins, the effect was not tissue specific.

High tissue specificity of such a chimeric toxin can be obtained if the A-fragment of diphtheria toxin or the A-subunit of ricin is conjugated with a peptide hormone interacting with the specific receptor of the cell membrane of a given type (e.g. with the chorionic ganadotropin, or epidermal growth factor, or insulin). In such cases, the enzymatic component is delivered into the cell, inhibits protein synthesis, and kills the cell. Furthermore, the membrane-interacting component may be an antibody (or its Fab-fragment) against some surface antigen, which is specific for a membrane of only one cell type. Then, by conjugating the diphtheria toxin A-fragment or ricin A-subunit with such an antibody, an extremely tissue-specific chimeric toxin can be obtained which will selectively kill only specific target cells. When an antibody against the surface antigen of a tumor cell serves as the membrane-binding moiety of the chimeric toxin, such a toxin should selectively kill tumor cells without affecting other cell types.

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