Identifying Potential Uses

There are two aspects to the actual and potential uses of the binary toxin: (a) the mechanism of toxin action, and (b) the cells that are vulnerable to this action. The mechanism of C2 action was described in detail in Chapter 18, and will be discussed only briefly in this chapter. This mechanism can be envisioned as a sequence of three events, as follows: binding, productive internalization, and intracellular poisoning. To some extent, the structure-function relationships of the binary toxin have been determined. Thus, the heavy chain {M, approx. 100 000) plays an essential role in binding, and the light chain (A/Ir approx. 50 000) is an enzyme that possesses mono(ADP-ribosyl) transferase activity. These general concepts are easy to grasp when viewed in the context of a simple experiment. Addition of the light chain (viz., the enzymatic component) to intact cells does not cause poisoning, because this chain by itself does not associate with cells. Addition of the heavy chain (viz., the binding component) does not cause poisoning either, because this chain does not express toxicity. However, addition of both chains to intact cells leads to binding and eventual expression of toxicity (Ohishi, 1983a,b; Ohishi and Miyake, 1985). This outcome is intriguing, especially when one takes into account the fact that heavy chain and the light chain do not associate with one another in solution. This appears to indicate that: (a) the heavy chain changes conformation when bound to cell receptors, and the altered conformation leads to exposure of an occult binding site for the light chain, (b) the heavy chain modifies the membrane in such a way as to create a binding site for the light chain, or (c) the heavy chain and the membrane act cooperatively to form a binding site for the light chain. Because this point has not yet been resolved, the binding properties of C2 toxin have not been exploited as research tools. However, this is likely to change. There are experimental and possibly even clinical settings in which the unique properties of a binary toxin could be exploited. One such possibility is explored below.

internalization of toxin The second step in the sequence is productive internalization (Ohishi and Yanagimoto, 1992). Toxin that is associated with cell surface receptors is translocated into the cytosol to reach its substrate. The fact that the substrate is in the cell interior means that the toxin, or at least the enzymatic component of the toxin, must be internalized. There is relatively little information on the underlying mechanism, although at least one study indicates that the toxin reaches the cell interior by receptor-mediated endocytosis (Simpson, 1989b). If this is correct, it would imply that the toxin must penetrate both the cell membrane and subsequently the endosome membrane. A sequence such as this is reminiscent of that utilized by other clostridial toxins, such as botulinum neurotoxin and tetanus toxin, as well as other microbial toxins (i.e., diphtheria toxin). As with the binding step, there is much that remains to be learned about the internalization step. Nevertheless, the fact that toxins possess the ability to penetrate biological membranes suggests that the mechanisms they use could have wide utility. To state the obvious, the ability to achieve efficient penetration of selected cell membranes would be highly advantageous in many areas of drug therapy.

The final step in toxin action is enzymatic modification of certain modification of actin forms of actin (Aktories et a/., 1986; Ohishi and Tsuyama, 1986). The light chain component ADP-ribosylates non-polymerized actin at arginine residue 177 (Vandekerckhove etal., 1988). This has the combined effect of blocking further polymerization and promoting depoly-merization (see Chapter 13), and as a result the cytoskeleton of cells is disrupted and may collapse. This action on the part of the toxin lends itself to the solution of many kinds of problems, but two have emerged as most prominent. First, the toxin has been widely used as a tool to determine the direct or indirect role of the actin-based cytoskeleton in various cell functions. Second, the toxin has been used as one in a battery of techniques to determine how the cell regulates synthesis and utilization of actin.

The potential value of C2 toxin as a research tool depends on the vulnerability of cells. The experimental approach is straightforward when cells are susceptible to natural poisoning, because the two components of C2 toxin can be added to the exterior of cells and the enzymatic component will find its way to the cell interior (see for example Miyake and Ohishi, 1987; Reuner et a/., 1987; Zepeda et a/., 1988). The approach is more problematic when cells are resistant to natural poisoning. In theory, resistance could be due to absence of cell surface receptors, absence of a mechanism for productive internalization, or absence of an intracellular substrate, but thus far only an absence of receptors (Fritz et a/., 1995) and an absence of substrate (Aktories et a/., 1986) have been described. Cells without receptors can be rendered susceptible by using techniques that produce artificial internalization (e.g., permeabilizing the cell membrane or microinjection; see Muller eta/., 1992). Cells that do not have substrate are permanently resistant to poisoning.

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