Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.

Abstract

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p2l1.ADP-ribosylating toxins have proved to be valuable tools for studying their target proteins. The targets generally suffer important changes in function upon modification, and they become labeled when radioactive NAD is used. Most of the known ADP-ribosylating toxins have GTP-binding proteins (G proteins [16]) as substrates and, although the reason is not clear, this association leads to the speculation that undiscovered toxin-G protein pairs may exist. Diphtheria toxin and exotoxin A of Pseudomonas aeruginosa modify elongation factor 2, a 94-kilodalton (kDa) GTP-binding protein, and cholera and pertussis toxins ADP-ribosylate G proteins of about 40 kDa (GS, Gi, G., and transducin). However, no bacterial enzyme has been found that efficiently modifies G proteins of the 21-kDa size class such as the products of genes H-ras, K-ras, and N-ras (13), R-ras (20), rho (21), and ral (6), the proteins YPT1 (29) and Gp (9), or the activator of cholera toxin known as ARF (19) and S (14).In this report, we describe the substrate and some physiological effects of a clostridial enzyme that specifically transfers ADP-ribose to a GTP-binding protein (p21.bot) common to a wide variety of cells and tissues. While the work was in progress the enzyme was described by Aktories et al. (2), who designated it C3. We use the same name. It has several attributes of an enzymatically active portion of a toxin, but since no toxic activity has been demonstrated, we describe it provisionally as an exoenzyme. We show that the reaction catalyzed is indeed an ADP-ribosylation, that the material identified as C3 is in fact responsible, and that ADP-ribosylation of intracellular p21.bot has functional consequences.C3 is secreted only by the C and D types of Clostridium botulinum. These two types also differ from the more commonly studied A, B, and E types in other respects. In particular, the structural information for their Cl and D neurotoxins is carried by lysogenic or pseudolysogenic bacteriophages and nontoxinogenic cured strains are relatively easy to generate (8). Most C and D strains also elaborate C2 toxin that is enterotoxic, cytopathic, and lethal but not neurotoxic. Its enzymatic subunit catalyzes the ADP-ribosy-* Corresponding au...

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…al. 1987Rubin et al 1988). The term C3 indicates the third protein toxin beside the C1 neurotoxin and the binary actin-modifying C2 toxin, which are produced by Clostridium botulinum.…”

A 22 kDa ras‐related G‐protein is the substrate for an ADP‐ribosyltransferase from Clostridium botulinum

“…However, the ADP-ribosylated p22 was not immunoprecipitated by two other ~21" antibodies, Y13-259 and 6B7 (lanes 4,5). Similarly, the BTx substrate has been reported not to be immunoprecipitated by two polyclonal antibodies raised against p21c-'aS [6,17]. Both the 259 and 6B7 antibodies recognized p21c-"s in "S-labeled 1321Nl cells (lanes 7-9 and not shown), and control experiments demonstrated that ADPribosylation by BTx did not interfere with the immunoprecipitation of ~21'~"" by Y13-259 (lanes 7,8).…”

“…The effects of Mg2+ and guanine nucleotides on ADP-ribosylation and the immunological data reported here are consistent with p22 being a Gprotein related to ~21"'. ~21" has been shown to induce differentiation of PC12 cells [24] and Rubin et al [6] recently reported that introduction of purified botulinum exoenzyme C3 into these cells also induced changes in cell morphology.…”

“…Botulinurn toxin types C and D were recently shown to ADP-ribosylate a protein of approx. BTx, botulinum toxin type D preparation; Gprotein, GTP-binding protein; G,, Gi, stimulatory and inhibitory G-proteins of adenylate cyclase; GTPyS, guanosine 5 ' -O-(3-thiotriphosphate); GDP@, guanosine 5 ' -0-(2-thiodiphosphate); ~22, 22 kDa BTx substrate 21 kDa in several tissues [3,4] and this activity has been shown to be due to the presence of a 26 kDa ADP-ribosyltransferase (exoenzyme C3) in the toxin preparations [5,6]. We show here that the 22 kDa protein (~22) ADP-ribosylated by a preparation of botulinurn toxin type D is a Gprotein immunologically related to but distinct from Ha-, Ki-and N-ras.…”

Interaction of recombinant rho A GTP‐binding proteins with photoexcited rhodopsin

“…Three members of the rho protein family, A, B and C, are known to occur in human tissues, with more than 90% sequence homology between each other [1,2]. Although the function of the rho proteins is not known, they have been suggested to be involved in regulation of cell proliferation and differentiation, possibly by affecting the cytoskeletal system [3,4]. By analogy with the related ras proteins [5], it is assumed that rho proteins are inactive in the GDP-bound state and active in the GTP-bound form.…”