Abstract:The effect of ă-toxin, a thermolysin-like metalloprotease of Clostridium perfringens, on the inac-

Activation of Clostridium perfringens ⑀-protoxin by tryptic digestion is accompanied by removal of the 13 N-terminal and 22 C-terminal amino acid residues. In this study, we examined the toxicity of four constructs: an ⑀-protoxin derivative (PD), in which a factor Xa cleavage site was generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (⌬N-PD); PD without the 23 C-terminal residues (⌬C-PD); and PD without either the N-or C-terminal residues (⌬NC-PD). A mouse lethality test showed that ⌬N-PD was inactive, as is PD, whereas ⌬C-PD and ⌬NC-PD were equally active. ⌬C-PD and ⌬NC-PD, but not the other constructs formed a large SDS-resistant complex in rat synaptosomal membranes as demonstrated by SDSpolyacrylamide gel electrophoresis. When ⌬NC-PD and ⌬C-PD, both labeled with 32 P and mixed in various ratios, were incubated with membranes, eight distinct high molecular weight bands corresponding to six heteropolymers and two homopolymers were detected on a SDS-polyacrylamide gel, indicating the active toxin forms a heptameric complex. These results indicate that C-terminal processing is responsible for activation of the toxin and that it is essential for its heptamerization within the membrane. ⑀-Toxin produced by Clostridium perfringens types B and D is the third most potent clostridial toxin after botulinum and tetanus toxins, and is responsible for the pathogenesis of fatal enterotoxemia in domestic animals caused by the organisms (1). This toxin exhibits toxicity toward neuronal cells via the glutamatergic system (2, 3) or extravasation in the brain (4) after infection of experimental animals. It has been suggested to be a pore-forming toxin based on the following observations. (i) ⑀-Toxin can form a large complex in the membrane of MDCK 1 cells, and it permeabilizes them (5, 6); (ii) the large complex formed by ⑀-toxin is not dissociated by SDS treatment (6), which is a common feature of pore-forming toxins such as aerolysin (7), Clostridium septicum ␣-toxin (8), and Pseudomonas aeruginosa cytotoxin (9); and (iii) the CD spectrum of ⑀-toxin shows it mainly consists of ␤-sheets (10), as is characteristically observed for pore-forming ␤-barrel toxins.The structures of many bacterial pore-forming toxins or toxin components such as perfringolysin O (11), Bacillus thuringiensis ␦-toxin (12), aerolysin (13), staphylococcal ␣-toxin (14), and protective antigen of anthrax toxin (15) have been determined. These pore-forming toxins are believed to undergo a drastic conformational change upon interaction with a membrane. Since these toxins are inserted into the cytoplasmic membrane without the aid of other proteins such as chaperones or the translocation machinery, characterization of their metamorphosis has been regarded as a novel means for studying membrane-protein interactions (16). A characteristic feature of ⑀-toxin is its potent neurotoxicity, which is not seen for the structurally well defined pore-forming toxins. Thus, it could serve as a useful tool for extending our knowledge o...

Clostridium perfringens ⑀-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled ⑀-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-␤-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When ⑀-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.

The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pzpeptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the fulllength enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the fulllength collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.Collagens are the major protein constituents of the extracellular matrix and the most abundant proteins in all higher organisms (1). The tightly coiled triple helical collagen molecule assembles into water-insoluble fibers or sheets which are cleaved only by collagenases, and are resistant to other proteinases. Various types of collagenases, which differ in substrate specificity and molecular structure, have been identified and characterized. Bacterial collagenases differ from vertebrate collagenases in that they exhibit broader substrate specificity (2, 3). Clostridium histolyticum collagenase is the best studied bacterial collagenase (4) and is widely used as a tissuedispersing enzyme (5, 6). This enzyme is unique in that it can degrade both water-insoluble native collagens and water-soluble denatured ones, can attack almost all collagen types, and can make multiple cleavages within triple helical regions (4). Kinetic studies of collagenases have provided insight into the high-ordered structure of collagens (7,8). However, the structure-function relationship of this unique enzyme is not known.