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J. Biol. Chem., Vol. 282, Issue 10, 7742-7752, March 9, 2007

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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^*

Hideki Katayama¹, Yoshitomo Kusaka, Haruo Yokota, Tetsuyuki Akao, Masayasu Kojima^¶, Osamu Nakamura, Eisuke Mekada, and Eiich Mizuki

From the Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Aikawa, Kurume, Fukuoka 839-0861, Japan, ^¶Institute of Life Science, Kurume University, Hyakunenkouen, Kurume, Fukuoka 839-0864, Japan, and Department of Cell Biology, Research Institute for Microbial Disease, Osaka University, Suita, Osaka 565-0871, Japan

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-Ca²⁺ concentration; increases in the intracellular Ca²⁺ 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 Ca²⁺ concentration. The toxin-induced elevation of the intracellular Ca²⁺ concentration was markedly decreased in low-Ca²⁺ buffer and was not observed in Ca²⁺-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca²⁺ buffer and was restored by the addition of Ca²⁺ to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca²⁺ 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 Ca²⁺ level and that the Ca²⁺ influx is the first step in the pathway that underlies parasporin-1 toxicity.

Received for publication, December 12, 2006

^* This study was supported by special coordination funds for promoting science and technology ("Leading Research Utilizing the Potential of Regional Science and Technology") from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-942-30-6644; Fax: 81-942-30-7244; E-mail: hkatayam{at}fitc.pref.fukuoka.jp.

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