ActinADPribosylating Toxins Cytotoxic Mechanisms of Clostridium botulinum C2 Toxin and Clostridium perfringens lota Toxin

8.1 Introduction

Whereas most of the known bacterial ADP-ribosylating toxins modify GTP-binding proteins (Aktories and Just, 1993) (see chapter 1, 3 and 5), there is a family of clostridial toxins that ADP-ribosylates the ATP-binding protein actin (for review see (Aktories et ai, 1992; Considine and Simpson, 1991; Aktories and Just, 1990; Aktories and Wegner, 1992; Aktories and Wegner, 1989; Ohishi and DasGupta, 1987)). These toxins have proved to be a valuable tool in cell biology because they are most effective agents to induce depoylmerization of actin in intact cells. Therefore, they are used to study the role of the microfilament protein actin in various cell functions. The family of actin ADP-ribosylating toxins comprises Clostridium botulinum C2 toxin, C. perfringens iota toxin, C. spiroforme toxin and a transferase produced by certain strains of C. difficile. Besides their common eukaryotic substrate actin, these toxins are characterized by their binary structure. The toxins are constructed according the A-B model and consist of a binding component and an enzyme component. However, in contrast to other toxins which are A-B toxins, like cholera toxin or pertussis toxin, the components of the actin ADP-ribosylating toxins are separate proteins and are not linked by either covalent or non-covalent bonds.

8.2 Clostridium botulinum C2 Toxin

Bacterial Toxins

Kletus Aktories

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The binding component (C2II) of C. botulinum C2 toxin has a molecu- structure of toxin lar mass of about 100 kDa (Ohishi et al., 1980) and has to be activated by trypsin (Ohishi, 1987). Trypsin treatment cleaves the 100 kDa component and releases a fragment of about 20 kDa. The approx. 80 kDa activated component of C2II binds to the cell surface, thereby inducing a binding site for the enzyme component (C2I). So far, the nature of the eukaryotic cell surface receptor is not known. Most probably the toxin complex (C2I and C2II) is internalized by receptor-mediated endocytosis, followed by translocation of the enzyme component (C2I) into the cytosol (Simpson, 1989; Ohishi and Yanagimoto,

1992). Here, C2I catalyzes the ADP-ribosylation of actin, resulting in dramatic redistribution of F-actin. Recently, the gene of C2I has been reported encoding a protein of 431 amino acids (Mr 49400) with significant similarity to the enzyme component of iota toxin (see below) (Fujii eta/., 1996).

specificity of ADP- ADP-ribosylation of actin by C. botulinum C2 toxin is highly selec-ribosylation five (see below). Neither G proteins, which are substrates for cholera or pertussis toxin nor other cytoskeletal proteins such as tubulin are ADP-ribosylated. As with other bacterial toxins, the enzyme-catalyzed modification is a mono-ADP-ribosylation. Accordingly, the actin-bound ADP-ribose is cleaved by Crotalus durissus phosphodiesterase and releases 5'-AMP. The Km for NAD of the ADP-ribosylation reaction was determined to be about 4 jiM. Using protein chemistry and mutagenesis, arginine-177 has been identified as the acceptor amino acid of ADP-ribose. Typically for an ADP-ribose-arginine bond, ADP-ribose incorporated at arginine-177 by C2 toxin is released by neutral hydroxylamine (0.5M, 2 h) (Aktories et al., 1988). The ADP-ribosylation by C2 toxin is reversible in the presence of a high concentration of nicotinamide (30-50 mM) and in the absence of free NAD (Just eta/., 1990).

8.3 Other actin-ADP-ribosylating Toxins

Clostridium perfringens iota toxin consists of an enzyme component with a molecular mass of about 52317 Da containing a signal sequence of 41 amino acids and a binding component of about 98467 Da with a signal sequence of 39 amino acids (Perelle et ai, 1993; Perelle et al., 1995; Simpson et a/., 1987; Stiles and Wilkens, 1986; Stiles and Wilkins, 1986). The enzyme component shows about 31 % identity and 43 % similarity with the sequence of C2I (Fujii et al., 1996). Interestingly, the binding component has about 55 % similarity to the protective antigen of the anthrax toxin (Perelle et al., 1993). No effect of trypsin on the purified components was observed. However, it has been reported that the toxicity of iota toxin is increased by proteolysis, most probably indicating the action of a bacterial protease (Stiles and Wilkins, 1986). The enzyme component of C. spiroforme toxin appears to be heterogeneous with an Mr ranging from 43 to 47kDa and its binding component is also activated by proteolysis (Simpson et al., 1989). So far no binding component has been reported for the actin-ADP-ribosylating toxin from C. difficile (Popoff et al., 1988) (Note that this transferase is distinct from the C. difficile toxins A and B described in chapter 12). The binding components of iota toxin and C. spiroforme toxin (but not that of C2 toxin) are interchangeable and can even translocate C. difficile ADP-ribosyl-transferase into the cell, suggesting that there is a subgroup of more closely related iota-like actin-ADP-ribosylating toxins (Popoff and Boquet, 1988; Simpson eta/., 1987; Simpson etal., 1989).

8.4 Actin as the Substrate for ADP-ribosylation

Actin is found in all eukaryotic cells, and moreover, it is one of the most abundant proteins in many of them. Besides its role in skeletal muscle characteristics of actin contraction, it is a major component of the microfilament system of the cytoskeleton, and is involved in various cellular motile functions, such as migration, phagocytosis, secretion or intracellular transport. Thus, actin appears to be a crucial target for toxins, because of its essential role for a variety of cellular functions. Actin is a single-chain polypeptide of 375 amino acids that is structurally divided into four domains (I-IV), with a cleft between domain l/ll and lll/IV. Here are localized the high affinity binding sites for ATP/ADP and the divalent cation. In cells, actin has bound Mg2+, whereas actin purified according to the procedure by (Pardee and Spudich, 1982) contains Ca2+. In addition to ATP/ADP-binding, actin possesses ATPase activity. Essential for the physiological role of actin is its ability to polymerize and to form filaments. Actin filaments are polar structures with two nonequivalent ends. Polymerization of actin occurs faster at the plus (barbed) end of filaments than at the minus (pointed) ends. Because barbed ends have a higher apparent affinity for actin monomers than the pointed ends, actin filaments tend to polymerize at the barbed ends and to depolymerize at the pointed ends. In non-muscle cells, about half of the cellular actin is monomeric and half polymeric. A large number of actin-binding proteins are involved in the dynamic regulation of the state of actin.

At least six mammalian actin isoforms have been identified: skeletal muscle a-actin, cardiac muscle a-actin, smooth muscle a- and actin isoforms y-actin and cytoplasmic |3- and y-actin (Vandekerckhove and Weber, 1979; Vandekerckhove and Weber, 1978). All these isoforms are highly homologous, with a maximal difference in the primary structure of about 5% (Vandekerckhove and Weber, 1978). Although arginine at position 177 is conserved in all actin isoforms, C. botulinum C2 toxin specifically ADP-ribosylates cytoplasmic actin and y-smooth muscle actin but not a-actin isoforms (Aktories et a/., 1986b; Mauss et a/., 1990). In contrast to C2 toxin, C. perfringens iota toxin ADP-ribosylates all actin isoforms studied (Mauss et a/., 1990). Additional actin substrates of C2 toxin are Physarum polycephalum actin (unpublished observation), Drosophila indirect flight muscle actin (and also the actin-ubiquitin conjugate) (Just etal., 1993a), and actin from Saccha-romyces cerevisiae, Dictyostelium and the green alga Chara (Grolig et a/., 1996).

ADP-ribosylation of actin depends on the native structure of the protein substrate. In the presence of EDTA, which chelates and removes the actin-bound magnesium ion, resulting in denaturation of actin, ADP-ribosylation is completely blocked (Just et a/., 1990). C. botulinum C2 toxin or C. perfringens iota toxin ADP-ribosylate monomeric G-actin, but not polymerized F-actin (Aktories et a/., 1986b; Schering et a/., 1988). This is due to the fact that the acceptor amino acid arginine-177, which is located in domain III of actin, is at or near

1. Inhibition of aclin polymerization

2. Capping protein function of ADP-ribosylated actin

3. Increase in the critical actin concentration for polymerization

4. Inhibition of actin ATPase activity

5. Increase in ATP exchange rate of actin

6 Inhibition of nucleation activity of the gelsolin-actin complex an actin-actin contact site (Holmes eta/., 1990). Similarly, the position of arginine-177 explains why ADP-ribosylation of monomeric actin inhibits actin polymerization (Aktories et a/., 1986b) most probably by steric hindrance (Table 1). Actin polymerization is inhibited even in the presence of phalloidin, which markedly decreases the critical concentration for polymerization (Aktories et a/., 1986a). However, ADP-ribosylated actin still interacts with unmodified actin and binds like a capping protein to the fast-polymerizing end (barbed or plus end) of F-actin (Wegner and Aktories, 1988; Weigt et a/., 1989). This interaction inhibits further association of monomeric actin at this end. In contrast, ADP-ribosylated actin does not affect polymerization and de-polymerization of unmodified actin at the pointed end of filaments. The equilibrium constant for binding of ADP-ribosylated actin to the barbed end of F-actin was determined to be K<, approx. 108 M~l By capping the barbed end of F-actin, the critical concentration of actin for polymerization increases to values that correspond to the critical actin concentration at the pointed (minus) end of actin filaments.

ADP-ribosylation completely blocks the actin ATPase activity and increases the rate of ATP exchange by about twofold (Geipel et a/., 1990; Geipel et a/., 1989). This effect is not due to inhibition of polymerization, because the basal ATPase activity of G-actin is also inhibited. Moreover, the ATPase activity of actin is blocked even in the quasi-monomeric actin-DNAse I complex after stimulation with the mycotoxin cytochalasin (Geipel et a/., 1990). Thus, by analogy with the ADP-ribosylation of G-proteins by cholera toxin, which inhibits G-protein-associated GTP hydrolysis, the ADP-ribosylation of actin inhibits its intrinsic ATPase activity.

ADP-ribosylation not only blocks actin-actin interaction but also affects the interaction of actin with actin-binding proteins like gelsolin. Gelsolin (Mr approx. 82000) is an actin-binding protein that (i), severs F-actin and increases the number of short filaments, (ii) acts like a barbed-end-capping protein thereby inhibiting fast polymerization of actin, and (iii) binds two actin monomers to form a 1:2 complex with nucleation activity for actin polymerization (Pollard et a/., 1994). As demonstrated with isolated proteins, ADP-ribosylated actin still interacts with gelsolin, however, the nucleation activity of the gelsolin-actin complex was inhibited when ADP-ribosylated actin was bound to the Ca2+-sensitive binding site of gelsolin (Wille etal., 1992) (Fig. 1).

Gelsolin—A

Gelsolin—A

G-Actin r

NAD Toxin

NAD Toxin ir^ Ii

Toxin

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