Characterization of the Catalytic Site of the ADP-Ribosyltransferase Clostridium botulinum C2 Toxin by Site-directed Mutagenesis

Barth, Holger; Preiß, J; Hofmann, Fred; Aktories, Klaus

doi:10.1074/jbc.273.45.29506

Cited by 98 publications

(113 citation statements)

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“…Rhodamine-or Oregon green 488-linked phalloidin (Molecular Probes, R-415; O-7466) was mixed with injection buffer (250 mM K acetate͞10 mM Hepes, pH 7.4) at 300 units͞250 l. Synapsin antibodies [G-304 (23)] were labeled with Alexa Fluor 594 (Molecular Probes, A-10239). The catalytic C2I component of Clostridium botulinum C2 toxin was obtained from H. Barth, University of Freiburg, Freiburg, Germany, and tested in an ADP-ribosylation assay (24). In both lamprey spinal cord and rat brain extracts, a single band with a molecular weight corresponding to actin was ADP-ribosylated (see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The effects of phalloidin indicated that the actin-containing cytomatrix participates in synaptic vesicle recycling. To examine this possibility further, we tested the effect of disrupting actin polymerization with the enzymatic C2I subunit of C. botulinum C2 toxin (24). The effect of the toxin was studied in synapses either maintained at rest or after 30 min stimulation at 5 Hz.…”

Section: Perturbation Of Actin Filaments With C2 Toxin and Myosin Framentioning

confidence: 99%

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Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Shupliakov

Bloom

Gustafsson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

208

229

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Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster. S ynaptic communication depends on local recycling of synaptic vesicles in nerve terminals. A major recycling pathway involves retrieval by means of clathrin-coated pits (1-3). The basic features of this process are shared with that of clathrin-dependent endocytosis in nonneuronal cells (4). It involves recruitment of clathrin by adaptors to the membrane followed by polymerization of the clathrin coat. A clathrin-coated pit grows progressively along with an invagination of the coated membrane. After a deeply invaginated coated pit has formed, the base of the pit is cut off resulting in formation of a free coated vesicle. A range of accessory proteins have been identified, which are thought to regulate different aspects of the endocytic process (2). Many of these proteins interact with components of the actin-regulating machinery, which has pointed to a coupling between endocytosis and the actin cytoskeleton (2, 5, 6). Such a coupling is also supported by the observation of actin tails at moving endocytic vesicles (7-10). Disruption of actin function has been shown to inhibit endocytosis in some but not all cell types (11)(12)(13)(14).In nerve terminals clathrin-mediated budding occurs in an endocytic zone of the plasma membrane, which surrounds the release site (1, 3). Several observations indicate that actin filaments are present in this synaptic compartment. Studies of the denervated frog neuromuscular junction have shown that phalloidin labeling accumulates around rather than within release sites (15). In cell-free synaptosome preparations actin was identified by immunogold labeling in regions containing endocytic intermediates a...

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Section: Methodsmentioning

confidence: 99%

Section: Perturbation Of Actin Filaments With C2 Toxin and Myosin Framentioning

confidence: 99%

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Shupliakov

Bloom

Gustafsson

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

208

229

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“…Taq polymerase was from Roche Molecular Biochemicals. Recombinant C. botulinum toxin C2I and C2II were expressed in Escherichia coli as glutathione S-transferase fusion proteins, and the toxin components were liberated using thrombin as described recently (20). C2II was activated by incubation with 0.2 g of trypsin/g of C2II for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Lysates were diluted 10-fold with 50 mM Hepes (pH 7.4) and subjected to an ADP-ribosylation assay as described (20). Briefly, samples (50 g of protein) were incubated with 300 ng of C2I in 35 mM Hepes (pH 7.4), 0.1 mM dithiothreitol, and 0.5 M [␣-32 P]NAD for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Binding of Clostridium botulinum C2 Toxin to Asparagine-linked Complex and Hybrid Carbohydrates

Eckhardt

Barth

Blöcker

et al. 2000

Journal of Biological Chemistry

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114

104

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Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic subunit (C2I) capable of ADPribosylating actin and a binding subunit (C2II) that is responsible for interaction with receptors on eukaryotic cells. Here we show that binding of C2 toxin depends on the presence of asparagine-linked carbohydrates. A recently identified Chinese hamster ovary cell mutant (Fritz, G., Schroeder, P., and Aktories, K. (1995) Infect. Immun. 63, 2334 -2340) was found to be deficient in Nacetylglucosaminyltransferase I. C2 sensitivity of this mutant was restored by transfection of an N-acetylglucosaminyltransferase I cDNA. C2 toxin sensitivity was reduced after inhibition of ␣-mannosidase II. In contrast, Chinese hamster ovary cell mutants deficient in sialylated (Lec2) or galactosylated (Lec8) glycoconjugates showed an increase in toxin sensitivity compared with wild-type cells. Our results show that the GlcNAc residue linked ␤-1,2 to the ␣-1,3-mannose of the asparagine-linked core structure is essential for C2II binding to Chinese hamster ovary cells.Clostridium botulinum C2 toxin is one of several binary toxins that recruit a binding component to deliver the enzymatic active component to the interior of eukaryotic cells (1). The 49-kDa catalytically active component (C2I) exhibits ADP-ribosyltransferase activity toward actin (2) and is translocated into cells through interaction of its N-terminal domain with the binding component C2II (3). Proteolytic cleavage by trypsin removes an N-terminal 20-kDa fragment, thereby activating the C2II binding component (4). The activated C-terminal 60-kDa fragment is then capable of binding both the enzymatic component and its receptor on eukaryotic cell membranes (5). After binding, the C2I-C2II complex is internalized by receptor-mediated endocytosis (6). Inside the cell, probably in an acidic endosomal compartment, C2I is translocated into the cytosol. In the cytosol, C2I ADP-ribosylates monomeric G-actin at arginine 177 (2, 7). ADP-ribosylated G-actin is not able to polymerize but binds to the growing end of actin filaments in a capping protein-like manner, resulting in depolymerization of microfilaments (8). At the morphological level, the breakdown of F-actin polymers is accompanied by the rounding up of toxin-treated cells. Toxins that act in a similar manner have been cloned from Clostridium spiroforme (9), Clostridium perfringens (iota toxin) (10), and Clostridium difficile CD196 (11). All are of binary structure, modifying G-actin, although they show some variations with respect to their substrate specificity. While C2 only ADP-ribosylates ␤-and ␥-actin, iota toxin can modify all actin isoforms (12). The binding components of these toxins exhibit about 40% sequence homology to C2II (13), although the former three are more similar to each other than to C2II. The receptor structure for none of these toxins has been defined yet. Recently, a Chinese hamster ovary (CHO) 1 cell mutant (RK14) resistant to C2 toxin has been isolated (14). The resistance of this mutant could be...

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“…ETA, a mono-ADP-ribosyltransferase (ADPRT) enzyme, is related to several other bacterial toxins composed of four groups classified according to their protein substrates: (i) elongation factor-2 ADP-ribosylating toxins (diphtheria toxin (DT) and ETA), (ii) heterotrimeric G-protein ADP-ribosylating toxins (cholera and pertussis toxins), (iii) toxins that ADP-ribosylate small GTPases (Clostridium botulinum C3 exoenzyme), and (iv) ADP-ribosyltransferases with actin as a substrate (clostridial ADP-ribosyltransferases and Clostridium perfringens toxin) (3). ETA is also catalytically related to the DNA repair enzyme family, poly(ADP-ribose) polymerases, which are involved in several physiological events such as chromatin decondensation, DNA replication and repair, gene expression, malignant transformation, cellular differentiation, and apoptosis (4,5).…”

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confidence: 99%

A Catalytic Loop within Pseudomonas aeruginosa Exotoxin A Modulates Its Transferase Activity

Yates

Merrill

2001

Journal of Biological Chemistry

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Mutagenesis techniques were used to replace two loop regions within the catalytic domain of Pseudomonas aeruginosa exotoxin A (ETA) with functionally silent polyglycine loops. The loop mutant proteins, designated polyglycine Loops N and C, were both less active than the wild-type enzyme. However, the polyglycine Loop C mutant protein, replaced with the Gly 483 -Gly 490 loop, showed a much greater loss of enzymatic activity than the polyglycine Loop N protein. The former mutant enzyme exhibited an 18,000-fold decrease in catalytic turnover number (k cat ), with only a marginal effect on the K m value for NAD ؉ and the eukaryotic elongation factor-2 binding constant. Furthermore, alanine-scanning mutagenesis of this active-site loop region revealed the specific pattern of a critical region for enzymatic activity. Binding and kinetic data suggest that this loop modulates the transferase activity between ETA and eukaryotic elongation factor-2 and may be responsible for stabilization of the transition state for the reaction. Sequence alignment and molecular modeling also identified a similar loop within diphtheria toxin, a functionally and structurally related class A-B toxin. Based on these results and the similarities between ETA and diphtheria toxin, we propose that this catalytic subregion represents the first report of a diphthamide-specific ribosyltransferase structural motif. We expect these findings to further the development of pharmaceuticals designed to prevent ETA toxicity by disrupting the stabilization of the transition state during the ADP-ribose transfer event.

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Characterization of the Catalytic Site of the ADP-Ribosyltransferase Clostridium botulinum C2 Toxin by Site-directed Mutagenesis

Cited by 98 publications

References 40 publications

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Binding of Clostridium botulinum C2 Toxin to Asparagine-linked Complex and Hybrid Carbohydrates

A Catalytic Loop within Pseudomonas aeruginosa Exotoxin A Modulates Its Transferase Activity

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