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.

A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55°C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl 2 indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5 end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.Bacterial spore germination, defined as the irreversible loss of spore characteristics, is triggered by specific germinants and proceeds through a set of sequential steps. Spore germination is essential to allow spore outgrowth and the formation of a new vegetative cell; once triggered, it proceeds in the absence of germinants and germinant-stimulated metabolism. This fact indicates that spore germination is a process controlled by the sequential activation of a set of preexisting germination-related enzymes but not by protein synthesis (10, 26).Among the key enzymes involved in the spore germination of Bacillus subtilis 168, Bacillus cereus IFO 13597, and Clostridium perfringens S40 are a group of cortex-lytic enzymes which degrade spore-specific cortex peptidoglycan. In the spores, at least two cortex hydrolases, spore cortex-lytic enzyme (SCLE) and cortical fragment-lytic enzyme (CFLE), are suggested to cooperatively function for cortex degradation. That is, cortex hydrolysis during germination is initiated by attack of SCLE on intact spore peptidoglycan, which likely leads to un-cross-linking of...

S100A2 and S100A6 interact with several target proteins in a Ca 2؉-regulated manner. However, the exact intracellular roles of the S100 proteins are unclear. In this study we identified Hsp70/ Hsp90-organizing protein (Hop) and kinesin light chain (KLC) as novel targets of S100A2 and S100A6. Hop directly associates with Hsp70 and Hsp90 through the tetratricopeptide (TPR) domains and regulates Hop-Hsp70 and Hop-Hsp90 complex formation. We have found that S100A2 and S100A6 bind to the TPR domain of Hop, resulting in inhibition of the Hop-Hsp70 and Hop-Hsp90 interactions in vitro. Although endogenous Hsp70 and Hsp90 interact with Hop in resting Cos-7 cells, but not with S100A6, stimulation of these cells with ionomycin caused a Hop-S100A6 interaction, resulting in the dissociation of Hsp70 and Hsp90 from Hop. Similarly, glutathione S-transferase pulldown and co-immunoprecipitation experiments revealed that S100A6 binds to the TPR domain of KLC, resulting in inhibition of the KLC-c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP-1) interaction in vitro. The transiently expressed JIP-1 interacts with KLC in resting Cos-7 cells but not with S100A6. Stimulation of these cells with ionomycin also caused a KLC-S100A6 interaction, resulting in dissociation of JIP-1 from KLC. These results strongly suggest that the S100 proteins modulate Hsp70-Hop-Hsp90 multichaperone complex formation and KLC-cargo interaction via Ca 2؉ -dependent S100 protein-TPR protein complex formation in vivo as well as in vitro. Moreover, we have shown that S100A2 and S100A6 interact with another TPR protein Tom70 and regulate the Tom70-ligand interaction in vitro. Thus, our findings suggest a new intracellular Ca 2؉

Edited by Felix WielandKeywords: S100 proteins Tetratricopeptide repeat Cyclophilin 40 FKBP52 Hsp90 a b s t r a c t S100 proteins are a subfamily of the EF-hand type calcium sensing proteins, the exact biological functions of which have not been clarified yet. In this work, we have identified Cyclophilin 40 (CyP40) and FKBP52 (called immunophilins) as novel targets of S100 proteins. These immunophilins contain a tetratricopeptide repeat (TPR) domain for Hsp90 binding. Using glutathione-S transferase pull-down assays and immunoprecipitation, we have demonstrated that S100A1 and S100A2 specifically interact with the TPR domains of FKBP52 and CyP40 in a Ca 2+