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Journal of Biochemistry Advance Access originally published online on November 15, 2007
Journal of Biochemistry 2008 143(2):269-275; doi:10.1093/jb/mvm220
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© 2007 The Japanese Biochemical Society.

Raft-targeting and Oligomerization of Parasporin-2, a Bacillus thuringiensis Crystal Protein with Anti-Tumour Activity

Yuichi Abe, Hiroyasu Shimada and Sakae Kitada*

Department of Chemistry, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan

*To whom correspondence should be addressed: Tel: +81 92 642 4182, Fax: +81 92 642 2607, E-mail: s.kitscc{at}mbox.nc.kyushu-u.ac.jp

Received October 12, 2007; Accepted November 6, 2007


  Abstract

Parasporin-2 is a newly classified Bacillus thuringiensis crystal toxin with strong cytocidal activities toward human liver and colon cancer cells. Similar to other insecticidal B. thuringiensis crystal toxins, parasporin-2 shows target specificity and damages the cellular membrane. However, the mode of parasporin-2 actions toward the cell membrane remains unknown. Here, we show that this anti-tumour crystal toxin targets lipid rafts and assembles into oligomeric complexes in the membrane of human hepatocyte cancer (HepG2) cells. Upon incubation with HepG2 cells, peripheral membrane-bound toxins, which were recovered in a low-density detergent-resistant membrane fraction, i.e. with lipid rafts, were transformed into heat-stable SDS-resistant membrane-embedded oligomers (~200 kDa). The toxin oligomerization was dependent on temperature and coupled with cell lysis. The toxin oligomerization also occurred in a cell-free membrane system and was required for binding to membrane proteins, the lipid bilayer and cholesterols. These results indicate that parasporin-2 is an oligomerizing and pore-forming toxin that accumulates in lipid rafts.

Key Words: Bacillus thuringiensis, Cry protein, lipid raft, parasporin, pore-forming toxin

Abbreviations: Cry, crystal; DRM, detergent-resistant membrane; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MβCD, methyl-β-cyclodextrin; PNS, post-nuclear supernatant; Trf-R, transferrin receptor; DRM, detergent-resistant membrane; LDH, lactate dehydrogenase; BiP, immunoglobulin binding protein; DMEM, Dulbecco's modified Eagle medium; FCS, fetal calf serum; Tom40, translocase of outer membrane of mitochondria


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