| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We have recently clarified that Bordetella dermonecrotizing toxin (DNT) is a transglutaminase catalyzing deamidation or polyamination of a specific Gln of Rho GTPases. However, little is known about the early step of DNT action such as binding to a membrane receptor and a translocation pathway to get into cytoplasmic environment. To understand this, we attempted to localize the minimum region of DNT responsible for binding to target cells. The C-terminally truncated mutants of DNT inhibited DNT action on MC3T3-E1 cells. The minimum region with this inhibitory effect was found to consist of the N-terminal 54 amino acids (DNT1-54). ^<125>I-labeled DNT1-54 showed the direct binding to MC3T3-E1 cells. Scatchard analyses revealed that DNT1-54 bound to the cells in a uniform mode with Kd of 2.5 micro molar. Furthermore, ^<125>I-labeled DNT1-54 was found to bind to DNT sensitive C3H10T1/2, Swiss3T3, REF, and NIH3T3 but not to DNT resistant Balb3T3, L929, PAE, COS7, and K562. These results indicate that DNT1-54 contains the binding domain and competes with DNT for a specific receptor which exists on the DNT sensitive cells. DNT1-54 includes the consensus motif for cleavage by a membrane-anchored protease furin between Arg41 and Arg44. Recently we found that DNT was actually cleaved at this motif by a soluble form of furin in vitro. We consider that DNT binds to a specific receptor on the target cells through the N-terminal receptor-binding region, which is then separated from whole DNT molecule after the cleavage by furin or furin-like protease. This process may be essential for the following DNT actions because DNT predigested by furin is about 100 times more efficient than intact DNT in terms of the action on mammalian cells.
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