Crystal structure of the parasporin-2 Bacillus thuringiensis toxin that recognizes cancer cells.

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.

Parasporin-1, a novel cytotoxic protein from Bacillus thuringiensis, induces Ca2+ influx and a sustained elevation of the cytoplasmic Ca2+ concentration in toxin-sensitive cells.

Parasporin-1 is a novel non-insecticidal inclusion protein from Bacillus thuringiensis that is cytotoxic to specific mammalian cells. In this study, we investigated the effects of parasporin-1 on toxin-sensitive cell lines to elucidate the cytotoxic mechanism of parasporin-1. Parasporin-1 is not a membrane pore-forming toxin as evidenced by measurements of lactate dehydrogenase release, propidium iodide penetration, and membrane potential in parasporin-1-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-1-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca(2+) concentration; increases in the intracellular Ca(2+) levels were observed 1-3 min following parasporin-1 treatment. Using four different cell lines, we found that the degree of cellular sensitivity to parasporin-1 was positively correlated with the size of the increase in the intracellular Ca(2+) concentration. The toxin-induced elevation of the intracellular Ca(2+) concentration was markedly decreased in low-Ca(2+) buffer and was not observed in Ca(2+)-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca(2+) buffer and was restored by the addition of Ca(2+) to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca(2+) influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADP-ribose) polymerase was observed. Furthermore, synthetic caspase inhibitors blocked the cytotoxic activity of parasporin-1. These results indicate that parasporin-1 activates apoptotic signaling in these cells as a result of the increased Ca(2+) level and that the Ca(2+) influx is the first step in the pathway that underlies parasporin-1 toxicity.

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis.

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Crystallization of parasporin-2, a Bacillus thuringiensis crystal protein with selective cytocidal activity against human cells.

Bacillus thuringiensis is a valuable source of protein toxins that are specifically effective against certain insects and worms but harmless to mammals. In contrast, a protein toxin obtained from B. thuringiensis strain A1547, designated parasporin-2, is not insecticidal but has a strong cytocidal activity against human cells with markedly divergent target specificity. The 37 kDa inactive protein is proteolytically activated to a 30 kDa active form. The active form of the recombinant protein toxin was crystallized in the presence of ethylene glycol and polyethylene glycol 8000 at neutral pH. The crystals belong to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 134.37, c = 121.24 A. Diffraction data from a native crystal were collected to 2.75 A resolution using a synchrotron-radiation source.

A Bacillus thuringiensis crystal protein with selective cytocidal action to human cells.

Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field.