Microbiol. Immunol., 39(3), 161-168, 1995

Minireview

Structure and Function of Clostridium botulinum Toxins

Keiji Oguma*, Yukako Fujinaga, and Kaoru Inoue

Department of Bacteriology, Okayama University Medical School, Okayama, Okayama 700, Japan

Received December 9, 1994

Key words: Clostridium botulinum, Botulinum toxin, Tetanus toxin, Toxin gene, Progenitor toxin

I. Introduction

Clostridium botulinum strains produce seven immuno-logically distinct neurotoxins, types A to G. The neu-rotoxins inhibit the release of acetylcholine (Ach) at the neuromuscular junctions and synapses, and cause botulism in humans and animals. The molecular mass (Mr) of all types of neurotoxins are approximately 150 kDa. The neurotoxins associate with nontoxic compo-nents in culture, and become large complexes varying from 300 kDa (12S) to 900 kDa (19S), which are des-ignated as progenitor toxins. Recently, the genes cod-ing for type A to G neurotoxins have been cloned, and their whole nucleotide sequences have been deter-mined. Furthermore, it has become clear that the neu-rotoxins are Zn2+ -binding proteins and possess pro-tease activities. Structures of the nontoxic components of the progenitor toxins have also been investigated genetically in types C, E and F.

In this paper, the gene organization of the neurotox-ins and the nontoxic components is summarized, and their structure and function are discussed.

2. Structure and Function of Neurotoxins

Type A to G neurotoxins are synthesized as single-chain polypeptides with approximately 150-kDa Mr. The single-chain toxins are nicked by a protease(s) at about one-third of their length from their N-terminals to yield dichain form, which is joined by a single disul-fide bond. After reduction, these nicked toxins can be separated into light (L) chain (Mr, ca. 50 kDa) and heavy (H) chain (Mr, ca. 100 kDa) components (12, 38, 57, 66, 68). The toxin blocks Ach release from the target cells by proceeding through three reactions,

including an extracellular binding step, an internaliza-tion step and an intracellular lytic step (6, 7, 52). It has been postulated that the 50-kDa C-terminal of the H chain is involved in neurospecific cell binding (1, 2, 28, 50, 67), and that the remaining 50-kDa N-terminal of the H chain forms channels in membranes to induce penetration (translocation) of the L chain into the cytosol (10, 51), which blocks neurotransmitter release (Fig. 1). Since the monoclonal antibodies recognizing an amino acid sequence of less than 50 residues of the C-terminal of the type C neurotoxin (which is unique to type C toxin) neutralized the toxin-lethal activity in mice and inhibited the toxin-binding to the neuronal cells (NG108-C15), it was suggested that this limited C-terminal region is essential for the type C toxin-binding to the receptor (25). As candidate receptors of the toxins, gangliosides (26, 53) and a glycoprotein (34, 67) have been proposed. Recently, data showing that synaptotagmin, one of the membrane-associated proteins of the neurons, is the receptor for type B toxin was reported (32).

The genes of type A to G neurotoxins have been cloned, and their whole nucleotide sequences have been determined (Table 1). Homology of the amino acid sequences among these toxins are approximately

30-60%. Distance matrix trees depicting the relation-ship of L and H chains, reported by Campbell et al (11), are shown in Fig. 2. As is obvious from the fact that several monoclonal antibodies cross-react with dif-ferent types of neurotoxins (25, 37, 61), there exist sev-eral highly conserved regions among the different types of botulinum neurotoxins (Fig. 1). One of the most conserved segments of the neurotoxins is located in the central region of the L chain, which includes the

*Address correspondence to Dr. Keiji Oguma, Department of Bacteriology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, Okayama 700, Japan.

Abbreviations: Ach, acetylcholine; cha, type C hemaggluti-

nin component gene; cnt, type C nontoxic-nonHA component

gene; ctx, type C neurotoxin gene; HA, hemagglutinin; Mr, molecular mass; H chain, heavy chain; L chain, light chain;

ORF, open reading frame.

161

162 K. OGUMA ET AL

HExxH zinc binding motif of metalloendopeptidases.

It has become clear that the botulinum and the tetanus

toxins contain a zinc atom coordinated by two his-

tidines in this motif, and possess a proteolytic activity

specific for the synaptic vesicle membrane proteins,

VAMP (or synaptobrevin-1 and/or -2), and the presy-

naptic plasma membrane proteins, SNAP-25 (synapto-

somal-associated protein of Mr 25K) and syntaxin (8,

30, 45, 47, 64). These proteins are considered to be

crucial in constitutive vesicle-exocytosis in neurons;

they are implicated in a docking/fusion process by

interacting with a NSF (N-ethylmaleimide-sensitive

factor)/ƒ¿- or ƒÀ-SNAP (soluble NSF attachment pro-

tein)/y-SNAP complex (Fig. 3). Now, it is concluded

that botulinum and tetanus toxins inhibit the neuro-

transmitter release by cleaving the membrane-associat-

ed/transmembrane proteins involved in the docking/

fusion processes. The target proteins and the cleavage

sites of each type of toxin are summarized in Table 2.

3. Structure and Function of Progenitor Toxins

Sakaguchi et al found that the toxin molecules exist-ing in type A to G strain cultures with acid conditions are larger than the neurotoxins, and designated them as progenitor toxins (27, 29, 31, 33, 38, 39, 55). Type A

strain produces three different-sized progenitor toxins, 900 kDa (19S), 500 kDa (16S) and 300 kDa (12S). Type B, C, and D strains produce 500- and 300-kDa toxins. Whereas, type E and F, and type G produce only 300 and 500-kDa toxins, respectively. All these

progenitor toxins consist of a neurotoxin with Mr of approximately 150 kDa and the nontoxic components. The progenitor toxins of 900 and 500 kDa show hemag-glutinin (HA) activity, but the 300-kDa toxin does not. It was postulated that the 300-kDa toxin is formed by association of a neurotoxin with a nontoxic component having no HA activity which is designated here as nontoxic-nonHA. Whereas, the 500- and the 900-kDa toxins are formed by conjugation of the 300-kDa toxin with HAs (Fig. 4). Molecular masses (Mr) of the non-toxic-nonHAs of all types of progenitor toxins were determined to be approximately 140 kDa by SDS-PAGE. Since the Mr of the neurotoxins are 150 kDa, Mr of the HAs of 900- and 500-kDa progenitor toxins are speculated to be approximately 600 and 200 kDa, respectively. Mr of HAs, however, are not clear because nobody has yet succeeded in separating and purifying the HAs from the progenitor toxins. On SDS-PAGE, the purified type A, B and C 500-kDa progenitor toxins demonstrated several bands besides the neurotoxin and the nontoxic-nonHA bands, indicating that the HA

Fig. 1. Schematic structure of clostridial neurotoxins. The neurotoxin is formed as a single-chain polypeptide . After cleavage and

reduction, the neurotoxin separates into L and H chains. A highly homologous region existing in the L chain which contains a

HExxH motif, and that existing in the N-terminal side of the H chain which contains many hydrophobic amino acid residues are con-

sidered to be important for protease activity and channel formation in membranes, respectively. The C-terminal of the toxin seems to

be crucial for binding to the receptors. •¡ , highly homologous region; •¬, relatively highly homologous region .

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consists of several subcomponents (54, 59). We clarified the structure of HA of the type C 500-

kDa progenitor toxin. The purified progenitor toxin was analyzed by SDS-PAGE using 4-20% acrylamide gradient gel, and we reached the conclusion that the HA consists of 53-, 33-, 22- to 23-, and 17-kDa subcom-ponents as in the case of type A and B progenitor tox-ins. The 22-23-kDa subcomponent consists of at least four proteins showing slightly different Mr (19).

These findings are confirmed by genetical analyses (19, 20). Since toxin and HA production is governed by specific bacteriophages in type C and D strains (16,

23, 35, 36), genes for the neurotoxin and the nontoxic components were cloned from the phage DNAs. Genes for the neurotoxin (ctx), the nontoxic-nonHA (cnt) and the HA (cha) occur as a cluster, and the following con-clusions have been drawn (Fig. 5): 1) cha lies just upstream of cnt, and ctx lies just downstream of cnt, 2) cha consists of three ORFs designated as cha-33, cha-17 and cha-70, 3) cnt and ctx are in a single transcrip-tion unit, transcribed from a promoter located in the 5'- untranslated region of cnt, 4) cha (cha-33, -17, -70) is in the opposite orientation from cnt and ctx, and cha-33, -17, -70 are also in a single transcription unit, tran-

Table 1. Structural genes of C. botulinum neurotoxin and nontoxic components

Fig. 2. Distance matrix trees. Distance matrix trees reported by Campbell et al (Biochim. Biophys. Acta 1216: 487-491, 1993).

164 K. OGUMA ET AL

Table 2. Target proteins of clostridial neurotoxins

Fig. 3. Target proteins of clostridial neurotoxin. The proteins considered to be crucial in the docking/fusion process of vesicle exocy-tosis are shown. Botulinum and tetanus toxins cleave the SNAP receptors in presynaptic membrane (SNAP-25 and syntaxin) and in vesicle membrane (synaptobrevin).

Fig. 4. Structure of Clostridium botulinum progenitor toxins. Progenitor toxins consist of a neurotoxin and nontoxic components. A 300-kDa progenitor toxin is formed by association of a neurotoxin with a nontoxic component having no HA activity (nontoxic-nonnA). 500-kDa and 900-kDa toxins are formed by conjugation of the 300-kDa toxin with HA. These complexes dissociate into the neurotoxin and the nontoxic components in an alkaline condition.

MINIREVIEW 165

scribed from a promoter located in the 5 '-untranslated region of cha-33. However, the primary extension experiment of the neurotoxin gene, and the existence of possible terminator-like structures around the 3' end of each HA gene did not completely exclude the mono-cistronic transcription of the neurotoxin gene, and monocistronic or bicistronic transcription of HA genes (20), 5) Mr of the products of ctx, cnt, cha-33, cha-17, and cha-70 were calculated to be 149.0, 138.8, 33.8, 16.7 and 70.6 kDa, respectively (Table 1).

Based on the results of N-terminal analyses of each subcomponent of HA, it was also concluded that the gene product of the cha-70 gene, 70.6 kDa, is cleaved at Lys192 and at different sites of its N-terminal to give 48.5-kDa and 20.4- to 22.5-kDa proteins, which corre-spond to HA-53 and HA-22 to -23, respectively. The cleaved sites of the proteins are the carboxyl side of Lys or Tyr. Therefore, it was speculated that these pro-tein processings are mediated by a protease(s) having trypsin- and chymotrypsin-like activities. Furthermore, Hauser et al identified a gene, ORF-22, at the 5' end of this gene cluster (20). Since the product of this gene, 22 kDa, displays significant similarity to a regulatory protein of C. perfringens (uvi A gene product), it was presumed that this protein may be involved in the regu-lation of expression of the neurotoxin and HA genes

(Fig. 5). Recently, we found that gene organization of type D is the same as that of type C (unpublished data).

The genetic analyses are also performed in types E and F (13, 18). Type E and F strains produce only 300- kDa toxin, showing no HA activity. As expected, the genes for neurotoxin and nontoxic-nonHA were identi-fied, but the gene for HA was not (Table 1). The genes

of neurotoxin and nontoxic-nonHA also seem to be in a single transcription unit as in type C. The amino acid sequences of the nontoxic-nonHAs of types C, E, and F are more highly conserved than their neurotoxins; 58- 71% identity, and 75-84% similarity. In addition, the existence of the nontoxic-nonHA genes was proposed in types A, B, G and toxigenic strains of C. baratii and C. butyricum by using PCR with oligonucleotide primers designed to correspond with regions of the nontoxic-nonHA gene of type C (13).

As for the function of nontoxic components, the fol-lowing conclusion was drawn. The nontoxic compo-nents of the progenitor toxins protect the neurotoxin from the gauntlet of acidity and proteases in the stom-ach. Thus, the oral toxicity of the progenitor toxins is much higher than that of the neurotoxin alone (39, 40, 42). After passing through the stomach, the progenitor toxins are absorbed, and then dissociate into the neuro-toxins and the nontoxic components in the small intes-tine because the binding between the neurotoxins and the nontoxic-nonHA is dissociated in an alkaline con-dition, but that between nontoxic-nonHA and HA is not. Thereafter, the neurotoxins react with the target organs through the lymph and the blood vessels.

Despite the above findings, the following questions still remain to be answered. Why do 900-, 500-, 300- kDa progenitor toxins exist in the same culture? Does the HA of the 900-kDa toxin have the same subcompo-nents as those of the 500-kDa toxin? How do those subcomponents form the HA (molecular ratio of each subcomponent)? Is the function of nontoxic-nonHA and HA same? Do the nontoxic components have some functions other than protecting the neurotoxin in the

Fig. 5. Molecular construction of progenitor toxins and their gene organization. The structural genes for type C neurotoxin (ctx), non-toxic-nonHA (cnt), and HA (cha) are indicated by boxes. The predicted ways of transcription, translation, and post-translation-protein

processing are indicated by arrows. In types E and F, structure genes for HA were not identified.

166 K. OGUMA ET AL

stomach? One region each of HA-22 to -23 and HA-53 showed significant amino acid sequence homology to the same region of C. perfringens type A enterotox-in, which is considered to be important for insertion of the toxin into the target cells. Also, HA-22-23 has an Arg-Gly-Asp (RGD) sequence, which is found in fibronectin, and is crucial for its interaction with the cell surface receptor, an integrin (19). These data sug-gest that HA is involved in the adhesion of the progeni-tor toxins to the intestinal tissue before the progenitor toxins dissociate into the neurotoxin and the nontoxic components in the small intestine. These questions need to be clarified in the near future.

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