Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Binding properties of Clostridium botulinum type C progenitor toxin to mucins

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.     

Structure and action of the binary C2 toxin from Clostridium botulinum

C2 toxin from Clostridium botulinum is composed of the enzyme component C2-I, which ADP-ribosylates actin, and the binding and translocation component C2-II, responsible for the interaction with eukaryotic cell receptors and the following endocytosis. Three C2-I crystal structures at resolutions of up to 1.75 A are presented together with a crystal structure of C2-II at an appreciably lower resolution and a model of the prepore formed by fragment C2-IIa. The C2-I structure was determined at pH 3.0 and at pH 6.1. The structural differences are small, indicating that C2-I does not unfold, even at a pH value as low as 3.0. The ADP-ribosyl transferase activity of C2-I was determined for alpha and beta/gamma-actin and related to that of Iota toxin and of mutant S361R of C2-I that introduced the arginine observed in Iota toxin. The substantial activity differences between alpha and beta/gamma-actin cannot be explained by the protein structures currently available. The structure of the transport component C2-II at pH 4.3 was established by molecular replacement using a model of the protective antigen of anthrax toxin at pH 6.0. The C-terminal receptor-binding domain of C2-II could not be located but was present in the crystals. It may be mobile. The relative orientation and positions of the four other domains of C2-II do not differ much from those of the protective antigen, indicating that no large conformational changes occur between pH 4.3 and pH 6.0. A model of the C2-IIa prepore structure was constructed based on the corresponding assembly of the protective antigen. It revealed a surprisingly large number of asparagine residues lining the pore. The interaction between C2-I and C2-IIa and the translocation of C2-I into the target cell are discussed.     

Observations on toxin and hemagglutinin produced by Clostridium botulinum type C.

In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.     

Observations on toxin and hemagglutinin produced by Clostridium botulinum type C.

In the culture fluid of a hemagglutinin-positive strain of Clostridium botulinum type C, two toxins of different molecular size, hemagglutinin positive and negative, were separated by sucrose density gradient centrifugation.     

Sporulation and C2 toxin production by Clostridium botulinum type C strains producing no C1 toxin.

All of the 8 strains that were previously assumed to be nontoxigenic Clostridium botulinum type C were re-examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions. One per cent glucose added to trypticase peptone medium enhanced C2 toxin production. The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 10(4)/ml, no C2 toxicity was demonstrated. The C2 toxin was produced only during sporulation and not during vegetative growth.     

The actin-ADP-ribosylating Clostridium botulinum C2 toxin

Clostridium botulinum C2 toxin is the prototype of actin-ADP-ribosylating toxins. The toxin consists of the enzyme component C2I and the separated binding/translocation component C2II. C2II is proteolytically activated to form heptamers, which bind the enzyme component. After endocytosis of the receptor-toxin complex, the enzyme component enters the cytosol from an acidic endosomal compartment to modify G-actin at arginine177. Recent data indicate that chaperons are involved in the translocation process of the toxin.     

Immunochemical study of Clostridium botulinum type B toxin

Fractionation of type B. Cl. botulinum toxin, a protein complex, was carried out; as a result, 3 fractions, alpha, beta, and gamma, were isolated in a pure form, alpha-fraction, or neurotoxin, is highly toxic (5-10.10(7) LDm per 1 mg of protein), beta-fraction showed hemagglutinating activity (64-128 HAU per 1 mg of protein), gamma-fraction was not biologically active. The molecular weight of alpha and gamma-fractions was 150,000. All these fractions had antigenic properties. alpha-fraction was serologically specific. beta- and gamma-fractions showed incomplete serologic identity.