ADP‐ribosylation of platelet actin by botulinum C2 toxin

Abstract

Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity. In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin. Labelling of the 43-kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half-maximal and maximal ADP-ribosylation occurred at 0.1 pg/ml and 3 pg/ml botulinum C2 toxin, respectively. The K , value of the ADP-ribosylation reaction for NAD was about 1 pM. The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin. The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin. Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin. Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 pM and 10 pM phalloidin, respectively. ADPribosylation of purified actin, induced by botulinum C21 toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.

Clostridium perfringens iota toxin ADP‐ribosylates skeletal muscle actin in Arg‐177

“…ADP-ribosylation of human platelet and rabbit skeletal muscle actin by C. botulinum C2 toxin and C. perfringens iota toxin. Rabbit skeletal muscle G-actin (4pg) (lanes l-3) and human platelet G-actin (2pg) (lanes [4][5][6] were ADP-ribosylated with C2 toxin (1 pg/ml; lanes 1,4), iota toxin (0.4pg/ml; lanes 2,5), and without toxin (lanes 3,6) in the presence of 5 PM [32P]NAD (-0.2 &i) for 30 min as described. Proteins were separated by SDS-polyacrylamide gel electrophoresis and detected by Coomassie staining (A) or autoradiography (B).…”

“…ADP-ribosylation assay ADP-ribosylation was performed as in [5]. Briefly, about 1 mg rabbit skeletal actin (in Gbuffer [14]) was ADP-ribosylated in a medium containing 10 mM thymidine, 10 mM dithiothreitol, 1 mM MgC12, 1 mM EDTA, 1-4~8 iota toxin, 10pM…”

“…Both toxins apparently modify G-but Correspondence address: J. Vandekerckhove, Laboratorium voor Genetica, Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium not F-actin and ADP-ribosylation caused by either toxin severely reduces the ability of actin to polymerize [4,5,7]. However, both toxins are different in their substrate specificity.…”

Stress‐induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to tyrosine‐53 adjacent to the DNase I binding loop

“…Liver and platelet actin is ADP-ribosylated by Clostridium botulinum C2-toxin and this modification impairs polymerization of the actin [1]. Physarum actin is phosphorylated in vitro at threonine residues if it is in a 1 : 1 complex with fragmin [2].…”

Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides

“…These two ADP-ribosylating toxins were found to alter the cytoskeleton network and to cause rounding up of cells (33). C2 toxin ADP-ribosylates non-muscle actin (42 kDa) and blocks actin polymerization (1,2). C3 was found to ADP-ribosylate a 22-kDa protein at the onset of morphological changes, but the mechanism involved in the rounding of the cells is still not clear (33).…”