K. AKTORIES and G. KOCH
ADP-ribosyltransferase C3 is an exoenzyme produced by several strains of Clostridium botulinum. It was serendipitously detected during screening for high producer strains of C. botulinum C2 toxin (Aktories et al., 1987; Aktories et a/., 1988b). The novel ADP-ribosyltransferase was termed C. botulinum ADP-ribosyltransferase C3, because it proved to be distinct from C. botulinum neurotoxin CI and the actin ADP-ribosylating C. botulinum C2 toxin. C3 ADP-ribosylates members of the Rho protein family at asparagine-41 thereby inactivating the GTP-binding protein. Therefore, C3 ADP-ribosyltransferase has become a molecular tool to study the function of Rho proteins.
The C3 ADP-ribosyltransferase gene was shown to be located on a DNA bacteriophage together with neurotoxin CI and D (Popoff eta/., 1991; Hauser et al., 1993). Various isoforms of the C3 transferase have C3 isoforms been described (Popoff et al., 1990; Nemoto et al., 1991; Popoff et al., 1991; Moriishi et al., 1991; Moriishi et al., 1993). Whereas the gene for C3 from C. botulinum strain C468 encodes a protein of 211 amino acids (without signal peptide) with a molecular mass of 23546 Da (Popoff et a I., 1990; Popoff etal., 1991), C3 from C. botulinum strain C 003-9 encodes a protein of 204 amino acids with a molecular mass of 23119 Da (Nemoto etal., 1991) showing about 65% identity with the other C3 isoform. Moreover, C3-related transferases are produced by Clostridium limosum (C. limosum exoenzyme) (Just et al., 1992a), Staphylococcus aureus (EDIN: epidermal differentiation inhibitor) (Inoue etal., 1991), and Bacillus cereus (8. cereus exoenzyme) (Just et al., 1992b; Just etal., 1995). Amino acid sequences of the transferases from C. limosum and S. aureus (EDIN) are about 63 % and 30 % identical with those from C. botulinum strains, respectively. So far, the transferase from B. cereus has not been cloned. Partial amino acid analysis indicates that this transferase is rather distantly related to other C3-like toxins. All C3-like tranferases are 23-28 kDa proteins with a high content of basic amino acids (pi 9-10). C3 transferase is rather heat stable (1 min, 95°C) and is resistant to short-term trypsin treatment (Aktories etal., 1987).
Recently, the active site of C3-like transferases has been identified. It was shown that Glul74 of C. botulium C3 transferase (strain C468; Glu 173 in strain 0003-9) is a catalytic important glutamic acid residue, which is strictly conserved in all bacterial ADP-ribosyltransferases studied so far (Jung et a/., 1993; Aktories et a/., 1995; Böhmer et a/., 1996; Saito et a/., 1995). Interestingly, it was found that C3 like transferases show a significant degree of sequence similarity (especially in the region around the catalytic site) with various eukaryotic ADP-ribosyltransferases like rabbit muscle ADP-ribosyltransferase (Oka-zaki etal., 1994) and the family of RT6-like transferases (Koch-Nolte et al„ 1996).
5.3 Modification of Rho Proteins by C3-like Transferases
Rho proteins belong to a subfamily of low molecular mass GTPases that include RhoA,B,C, Racl,2, Cdc42 (G25K), RhoG and TC10 (for review see references: Nobes and Hall, 1994; Hall, 1994; Takai et al., 1995; Machesky and Hall, 1996). At the amino acid level, Rho subfamily proteins are about 55% identical to each other and about 30% identical to Ras proteins. Like other regulatory GTPases (Bourne etal., 1990; Bourne etal., 1991), Rho subfamily proteins are inactive in the GDP bound form and active with GTP bound (Fig. 1). Activation is induced by nucleotide exchange factors (GEF), also called guanine nucleotide dissociation stimulators (GDS) (Feig, 1994; Quilliam et al., 1995; Overbeck etal., 1995; Quilliam etal., 1995). In the active state, Rho subfamily proteins interact with their specific effectors (more than ten potential effectors have been identified and their number is still growing) which appear to be involved in various regulatory processes (see below). The active state of Rho proteins is terminated by hydrolysis of GTP catalyzed by their endogenous GTPase activity. The basal GTPase activity is very low, but is dramatically increased by GAP proteins (GTPase-stimulating proteins) (Lamarcheand Hall, 1994). Finally, Rho subfamily proteins are regulated by guanine nucleotide dissociation inhibitors (GDI), which inhibit nucleotide exchange. GDI proteins are able to prevent binding of Rho subfamily proteins to membranes where, it is suggested, they act (Isomura et al., 1991). functions of Rho proteins are involved in regulation of the actin cytoskeleton Rho proteins and induce formation of stress fibers and focal adhesions (Paterson et al., 1990; Ridley and Hall, 1992; Tominaga et al., 1993; Laudanna et al., 1996). Most probably independently of their role in actin regulation, Rho proteins have been proposed to participate as molecular switches in the control of phosphoinositide-3-kinase (Zhang et al., 1993), phosphatidylinositol-4-phosphate-5-kinase (Chong et al., signal input
inactive signal input inactive
- stress fiber formation
- ceil adhesion
- cell morphology
- cell motility smooth muscle /if contraction endocytosis cell-cell contact
A PI-3-kinase PIP-5-kinase phospholipase D
transcriptional activation inhibition of Rho dependent processes
Fig. 1. ADP-ribosylation of Rho by C3 transferases. Rho proteins are regulated by a GTPase cycle. The GTP-binding proteins are inactive with GDP bound, and active in the GTP-bound form. GDP/GTP exchange is facilitated by guanine nucleotide dissociation stimulator(s) (GDS) and inhibited by guanine nucleotide dissociation inhibitor(s) (GDI). In the active form, Rho protein interacts with its effector(s) and induces several cellular responses, one of which is polymerization of actin. Rho is ADP-ribosylated by C3 transferases at asparagine-41. Most likely, the modification inhibits the interaction of Rho with its effector(s) which results in inhibition of Rho dependent processes (e.g. F-actin depolymerization)
1994), phospholipase D (Malcolm et al., 1994), myosin phosphatase (Kimura et al., 1996) and smooth muscle contraction (Hirata et al., 1992), cell-cell contact (Tominaga et al., 1993) and endocytosis (Schmalzing eta/., 1995). Furthermore, Rho subtype proteins may play a role in transcriptional activation (Olson eta/., 1995; Hill etai, 1995), and in the transformation of cells induced by the oncogene product Ras (Khosravi-Far eta/., 1995; Qiu etal., 1995).
Rac proteins (Racl,2), which are also members of the Rho sub- functions of Rac and family, are involved in membrane ruffling and lamellipodia formation Cdc42 proteins induced by growth factors (Ridley et al., 1992; Hall, 1994), and it has also been suggested that they are important for Ras-induced transformation (Qiu etal., 1995). Moreover, Rac proteins control NADPH oxidase (Abo et al., 1991; Bokoch, 1994), and may have a role in phospholipase A2 regulation (Peppelenbosch et al., 1995). Cdc42, another member of the Rho family, which occurs in at least two iso-
forms, participates in receptor-induced formation of microspikes and filopodia (Nobes and Hall, 1995; Kozma etal., 1995). substrate specificity of C3 C3 shows a remarkable substrate specificity and modifies RhoA, B,
C (Hall, 1994; Paterson et a/., 1990; Chardin et a/., 1989; Wiegers et a/., 1991; Aktories et a/., 1989; Braun et a/., 1989), while other members of the Rho subfamily are poorly modified (e.g. 5-10% ADP-ribosylation of Rac in the presence of 0.01 % SDS), or not at all (Cdc42) (Just et al., 1992a). Other low molecular mass GTP-binding proteins or heterotrimeric G-proteins are not substrates for C3. The other C3-like transferases appear to share the same substrate specificity. C3-like transferases ADP-ribosylate Rho proteins at asparagine-41 (Sekine etal., 1989). Asparagine-41 is located in the effector region of the small GTP-binding proteins and attachment of the bulky ADP-ribose group by ADP-ribosylation may inhibit the interaction of Rho with its effectors. Alternatively, ADP-ribosylation may affect the activation of the Rho protein by guanine nucleotide exchange factors.
5.4 Characterization of the C. botulinum C3 ADP-Ribosyltransferase Reaction
Like other bacterial ADP-ribosylating toxins (e.g. diphtheria toxin, Pseudomonas aeruginosa exotoxin A, cholera toxin, pertussis toxin, and C botulinum C2 toxin (Aktories and Just, 1993)), C3 is a mono-ADP-ribosyltransferase (Aktories et al., 1988b). Treatment of ADP-ribosylated Rho with phosphodiesterase releases 5'-AMP and not phosphoribosyl-AMP, a cleavage product of poly(ADP-ribose) (Aktories etal., 1988b; Rubin etal., 1988). Accordingly, thymidine, an inhibitor of poly(ADP-ribose)polymerase, does not block C3-like ADP-ribosyltransferases, and can be included in C3 ADP-ribosylation assays to block poly-ADP-ribosylation reactions.
Asparagine is unique as an acceptor amino acid for ADP-ribosylation by C3-like transferases (Sekine et al., 1989). In contrast, pertussis toxin modifies cysteine residues, and cholera toxin and C. botulinum C2 toxin specifically ADP-ribosylate arginine residues (for details see the relevant chapters). The ADP-ribose-asparagine bond formed by C3-like transferases is stable towards neutral hydroxyl-amine (0.5 M, 2h) and mercury ions (2mM, 1 h), whereas cysteine and arginine-specific ADP-ribosylation, respectively, are sensitive towards these agents (Aktories etal., 1988a).
ADP-ribosylation by C3 and its isoenzymes is a reversible reaction, i.e. in the presence of high concentrations of nicotinamide (10mM) and at low pH (pH <7) it can be reversed (Habermann et al., 1991). The de-ADP-ribosylation reaction has been exploited to identify the acceptor amino acid of ADP-ribosylation by C3-like isoforms. Like other ADP-ribosyltransferases, C3-like exoenzymes exhibit NAD gly-cohydrolase activity (Aktories et al., 1988b). However, this enzyme activity is at least 100-fold lower than the transferase activity and most likely has no physiological significance.
In principle, ADP-ribosylation of Rho by C3 needs no other factors in addition to Rho and NAD (for review see also (Aktories and Just, 1995)). Rho is ADP-ribosylated in intact cells, in cell lysate (membranes and cytosol), and as a recombinant protein. Even Rho-gluta-thione S-transferase fusion proteins are substrates for ADP-ribosylation by C3. ADP-ribosylation by C3 is affected by guanine nucleotides, divalent cations, detergents and temperature. Purified endogenous Rho, recombinant Rho proteins, and the membranous Rho protein are better substrates for ADP-ribosylation when bound to GDP rather than GTP (Habermann et a/., 1991). In contrast, ADP-ribosylation of cytosolic Rho is enhanced by addition of GTPyS or GTP (Williamson etal., 1990). It is suggested that the nucleotides facilitate dissociation of RhoGDI, because affinity of GTP-bound RhoA for RhoGDI is lower than of GDP-bound RhoA (Kikuchi et a/., 1992; Regazzi et a/., 1992; Bourmeyster et a/., 1992). Free Rho protein is a better substrate for C3 than Rho complexed with RhoGDI. Similarly, C3 ADP-ribosylation of cytosolic or partially purified Rho protein is increased in the presence of detergents (sodium cholate, deoxycho-late) or phospholipids (phosphatidylinositol bisphosphate). This suggests that detergents and phospholipids dissociate the complex between RhoGDI and Rho protein and provide access for C3-like exoenzymes to ADP-ribosylate Rho (Bourmeyster et a/., 1992; Just et a/., 1993). In the absence of GDI, C3-catalyzed ADP-ribosylation is also affected by various detergents. Sodium cholate (0.2%), deoxy-cholate, dimyristoylphosphatidylcholine (3mM), and SDS (0.01 %) increase C3-catalyzed ADP-ribosylation. In contrast, CHAPS, Lubrol-PX, and SDS (>0.03%) impair ADP-ribosylation (Just et a/., 1992a; Just etal., 1993; Maehama etal., 1990).
5.6 C3-Transferases in Cell Biology and Pharmacology
In intact animals, toxicity of C3 is low compared with other toxins that low toxicity of C3 affect Rho proteins. Intraperitoneal injection of 100 fig of C3 into mice is apparently without obvious consequences. (Note that the minimum lethal dose of C. difficile toxins A and B, which also modify Rho proteins, is about 50 ng per mouse). C3-like transferases appear to lack any specific cell-binding or transport subunits, a fact that calls into question the designation of these exoenzymes as toxins. However, when these bacterial exoenzymes reach the eukaryotic cytosol (e.g. by unspecific uptake or microinjection, see below) they are potent cytotoxins (Wiegers etal., 1991; Didsbury etal., 1989; Chardin et a/., 1989; Paterson et a/., 1990). Thus, the use of C3 toxin as a cell biological tool is hampered by the fact that C3 is not able to enter cells readily. The problem of cell accessibility was first by-passed by using osmotic shock to introduce C3 into cells (Rubin et al., 1988). Many excellent studies from the laboratory of Alan Hall have been performed using the microinjection technique (Paterson et al., 1990; Ridley and Hall, 1992) (see Chapter 6). Other approaches to study the effects of C3 in eukaryotic cells used expression of transfected C3 DNA (Hill et al., 1995), a C3 fusion toxin consisting of C3 and the receptor and translocation domain of diphtheria toxin (Aullo et al.,
1993), and permeabilization of cells before C3 treatment (Koch etal.,
1994). However, most studies exploited the property of C3 of entering most cells when applied at rather high concentrations (30-150 (Ag/ml) (Wiegers etal., 1991 ; Nishiki etal., 1990). Additionally, it appears that some C3 isoforms are somewhat more capable of entering culture cells. In some studies, effects of C3 were even observed at low concentrations (0.5 to 1 ^g/ml) of the transferase (Sugai etal., 1992). So far, it is not clear whether these effects are caused by different C3 isoforms or by specific conditions of cell culture (serum-free treatment).
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