| Budget Amount *help |
¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 2002: ¥5,600,000 (Direct Cost: ¥5,600,000)
Fiscal Year 2001: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Research Abstract |
Bordetella dermonecrotic toxin (DNT) which enzymatically activates the small GTPase Rho is a single chain polypeptide consisting of 1,464 amino acids. In the toxin molecule, the receptor-binding domain and the catalytically active domain are localized to the N-terminal and the C-terminal regions, respectively. We attempted to dissect the early step of DNT action such as binding to a membrane receptor and a translocation pathway to enter cytoplasmic environment. We found that a motif recognized by furin, a mammalian endoproteinase, exists between Arg41 and Arg44 in the DNT sequence. DNT was actually cleaved at this motif by furin in vitro. The resultant two fragments of DNT after furin treatment were found to remain associated with each other. The furin-treated toxin was about 30 times more active on target cells than intact one. On the other hand, mutants of DNT in which the furin motif was destroyed showed no toxicity on the cells. The C-terminal fragment of DNT yielded after furin treatment (delta B), had the ability to interact with artificial lipid bilayer membrane and to affect cells which are originally resistant to the full-length toxin. From these results, we consider that DNT binds to a specific receptor on target cells through the N-terminal receptor-binding region, and then delta B is liberated and interacts with cellular membrane to translocate the C-terminal active domain in to the cytoplasm.
For expression cloning of a gene encoding the DNT receptor, we constructed a reporter gene including green fluorescent protein gene and serum responsive elements, which is recognized by transcription factors acting downstream of Rho activation. This reporter gene assay successfully worked in response to DNT action on DNT sensitive cells. We are going to clone the DNT receptor gene by using this system with DNT resistant cells which are transfected with cDNA library from the DNT sensitive cells.
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