ADP-ribosylation of Actin

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 (M, 49400) with significant similarity to the enzyme component of iota toxin (see below) (Fuji 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 ef al., 1990). C. botulinum C2 toxin or C. perfringens iota toxin ADP-ribosylate monomeric G-actin, but not polymerized F-actin (Aktories et al., 1986b Schering et al., 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... 

ADP-ribosylation completely blocks the actin ATPase activity and increases the rate of ATP exchange by about twofold (Geipel ef al., 1990 Geipel ef al., 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 ef al., 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. 

Fig. 1. Toxin-catalyzed ADP-ribosylation inhibits nucleation activity of the gelsolin-octin complex. In the presence of Ca, gelsotin forms a 1 1 and a 1 2 complex with octin monomers at the so-called EGTA-resistant (a) and Ca -sensitive (b) binding site, respectively. Gelsolin-actin complexes act as nuclei for actin polymerization. Actin bound to both sites (a, b) can be ADP-ribosylated. Whereas ADP-ribosylation of actin bound to the EGTA-resistant site has no effect on nucleation, ADP-ribosylation of actin bound to the Ca " -sensitive site inhibits nucleation activity of the gelsolin-actin complex...

Aktories K, Wegner A (1989) ADP-ribosylation of actin by clostridial toxins. InJ. Cell Biol. 109 1385-7... 

Geipel U, Just I, Sobering B etal. (1989) ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis. In Eur. J. Biochem. 179 229-32... 

Grolig F, Just I, Aktories K (1996) ADP-ribosylation of actin from the green alga Chara corollino by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. In Protoplasma in press... 

Just I, Geipel U, Wegner A et al. (1990) De-ADP-ribosylation of actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin. In Eur. J. Biochem. 192 ... 

Just I, Wille M, Chaponnier C et al. (1993b) Gelsolin-actin complex is target for ADP-ribosylation by Clostridium botulinum C2 toxin in intact human neutrophils. In Eur. J. Pharmacol. Mol. Pharmacol. 246 293-7 Kiefer A, Lerner M, Sehr P et al. (1996) Depolymerization of F-actin by microinjection of ADP-ribosylated skeletal muscle G-actin in PtK2 cells in the absence of the ADP-ribosylating toxin. In A/ted. Microbiol. Immunol. 184 175-80 Mauss S, Chaponnier C, Just I et al. (1990) ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin. In Eur. J. Biochem. 194 237-41... 

If an action of C2 toxin can be documented (i.e., ADP-ribosylation of actin disaggregation of the cytoskeleton), but there is no change in the cellular response under study, then this particular response is not immediately dependent on integrity of the cytoskeleton. 

To quantify the amount of toxin-induced ADP-ribosylation of actin in intact cells, the indirect method of differential ADP-ribosylation is appropriate. Assuming that in intact cells C2 modifies cellular actin, subsequent pP]ADP-ribosylation of the lysates should result in a decreased incorporation of pP]ADP-ribose. Because C2I shows transferase activity on ice, although less than at 37°C, the cells should be lysed in a buffer containing [ P]NAD and C2I. 

ADP-ribosylation of actin in cell lysates is performed in the following way ... 

The extent of ADP-ribosylation is checked by separating modified and unmodified actin using non-denaturing gel electrophoresis, whereas SDS gel electrophoresis is not efficient. ADP-ribosylation changes the isoelectric point of actin to more acid values, resulting in increased migration. This method is also appropriate to study double or triple ADP-ribosylation of actin, e.g., in the presence of iota toxin and/or turkey erythrocyte transferase (Just ef al., 1995). The latter transferase modifies actin even at two arginine residues. 

Fig. 1. Time dependency of ADP-ribosylation of actin by C2 toxin in rot basophilic leukemia (RBL) cells. Rbl cells were incubated with C2 holotoxin (lOOng/ml C2I + 200ng/ml C2II) for the times indicated. Controls were incubation with the enzyme component C2I (lOOng/ml) alone, or with the binding component C2II (200ng/ml) alone, for 120 min. Another control (-) was not treated with toxin. After incubation, the medium was removed and cells were scraped off in the presence of lysis buffer (2 mM MgCb, 0.1 mM phenylmethylsulfonyl fluoride, 10 ig/ml leupeptin, 25 mM triethanolamine, pH 7.5), sonicated five times for 5sec on ice and centrifuged for 10 min at 1000 g. The supernatants were incubated with 1 [ig/ml C2I and 5nM [ P]NAD for 15 min at 30°C. The proteins were separated by 12.5% SDS-polyacrylamide gel electrophoresis and analyzed by phosphorimaging...

Terashima, M., Mishima, K., Yamada, K., Tsuchiya, M., Wakutani, T., Shimoyama, M. (1992). ADP-ribosylation of actins by arginine-specific ADP-ribosyltransferase purified from chicken heterophils. Eur. J. Biochem. 204, 306-311. 

Clancy, R. M., Leszczynska-Piziak, J, and Abramson, S. B. (1993). Nitric oxide stimulates the ADP-ribosylation of actin in human neutrophils. Biochem. Biophys. Res. Commun. 191, 847-852. 

Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J (1988) Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol 107 307-319. Ronnov-Jessen L, Petersen OW (1996) ADP-ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin influence on microfilament morphology and migratory behavior. Electrophoresis 17 1776-1780. 

Clancy, R., Leszczynska, J., Amin, A., Levartovsky, D., and Abramson, S.B. (1995). Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymerization in human neutrophils. J. Leukoc Biol. 58(2), 196-202. 

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