Attempts for identification of a cell receptor for Bordetella dermonecrotic toxin and localization of its functional domains

博德氏菌皮肤坏死毒素（DNT）是一种由1464个氨基酸组成的单链多肽，它能激活小GTPase Rho。在毒素分子中，受体结合域和催化活性域分别位于n端和c端区域。我们试图剖析DNT作用的早期步骤，如结合膜受体和进入细胞质环境的易位途径。我们发现，在DNT序列的Arg41和Arg44之间存在一个被哺乳动物内源性蛋白酶furin识别的基序。实际上，DNT在体外被furin在这个基序上切割。结果发现，经过呋喃处理后，得到的两个DNT片段仍然相互关联。furin处理过的毒素对靶细胞的活性是完整毒素的30倍。另一方面，破坏furin基序的DNT突变体对细胞没有毒性。经呋喃处理后产生的DNT c端片段（δ B）具有与人工脂质双层膜相互作用的能力，并影响最初对全长毒素具有抗性的细胞。根据这些结果，我们认为DNT通过n端受体结合区与靶细胞上的特定受体结合，然后δ B被释放并与细胞膜相互作用，将c端活性结构域转运到细胞质中。为了克隆编码DNT受体的基因，我们构建了包含绿色荧光蛋白基因和血清应答元件的报告基因，该报告基因由Rho激活下游的转录因子识别。该报告基因试验成功地响应了DNT对DNT敏感细胞的作用。我们将利用该系统克隆DNT受体基因，用DNT抗性细胞转染来自DNT敏感细胞的cDNA文库。

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.