Parasporin-1, a Novel Cytotoxic Protein to Human Cells from Non-Insecticidal Parasporal Inclusions of Bacillus thuringiensis

• Published in: Journal of biochemistry (Tokyo) 2005, Vol.137 (1), p.17-25
• Main Authors: Katayama, Hideki, Yokota, Haruo, Akao, Tetsuyuki, Nakamura, Osamu, Ohba, Michio, Mekada, Eisuke, Mizuki, Eiichi
• Format: Article
• Language: English
• Subjects: 5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide; Animals; Bacillus thuringiensis; Bacillus thuringiensis - chemistry; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - toxicity; Bacterial Toxins - chemistry; Bacterial Toxins - isolation & purification; Bacterial Toxins - toxicity; Cell Line; Cercopithecus aethiops; cytotoxin; dithiothreitol; DMEM; DTT; Dulbecco’s modified Eagle’s medium; Endotoxins - chemistry; Endotoxins - isolation & purification; Endotoxins - toxicity; FBS; fetal bovine serum; Humans; MALDI; mass spectrometer; matrix-assisted laser desorption/ionization; MEM; membrane permeability; Mice; minimum essential medium; molecular structure; MTT; PAGE; polyacrylamide gel electrophoresis; polyvinylidene difluoride; purification and characterization; PVDF; SDS; sodium dodecyl sulfate; time of flight; TOF
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvi003

Pro-parasporin-1 is a parasporal inclusion protein of the non-insecticidal Bacillus thuringiensis strain A1190. Cytotoxic fragments, named parasporin-1, were generated from pro-parasporin-1 by trypsin digestion. Parasporin-1 was purified by a combination of chromatography procedures based on the cytotoxic activity to HeLa cells. Two different fragments of 15-kDa and 56-kDa were detected in the purified parasporin-1 fraction. These fragments were tightly associated with each other and could not be separated by chromatography under conditions that preserve cytotoxic activity, indicating that the active form of parasporin-1 is a heterodimer of the 15- and 56-kDa fragments. Amino acid sequencing and MALDI-TOF mass spectrometric analysis revealed that parasporin-1 is generated from pro-parasporin-1 by trypsin digestion at Arg 93 and Arg 231. Of 12 human cell lines tested, parasporin-1 showed strong cytotoxicity to four cell lines derived from cancer tissues, but low to no cytotoxicity to the other cell lines. The time-courses of cytotoxicity indicated that the mode of action of parasporin-1 to sensitive cells differs from that shown for previously isolated cytotoxic proteins from Bacillus thuringiensis, Cyt proteins, and other bacterial pore-forming toxins. Thus, parasporin-1 is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis, and may be useful as a tool to recognize and destroy specific cancer cells.