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

尝试鉴定博德特氏菌皮肤坏死毒素的细胞受体及其功能域的定位

• 批准号: 13470059
• 负责人: HORIGUCHI Yasuhiro
• 金额: $ 7.55万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470059/
• 关键词: Bordetella pertussis; dermonecrotic toxin; translocation; Rho GTPase; furin; receptor; 細胞内移行

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.

Bordetella dermonecrotic toxin（DNT）是一种由1,464个氨基酸组成的单链多肽，可酶促激活小的GTdR Rho。在毒素分子中，受体结合结构域和催化活性结构域分别位于N-末端和C-末端区域。我们试图剖析DNT作用的早期步骤，如与膜受体结合和转运途径进入细胞质环境。我们发现在DNT序列的Arg 41和Arg 44之间存在一个由哺乳动物内切蛋白酶furin识别的基序。在体外，弗林蛋白酶实际上在该基序处切割DNT。发现弗林蛋白酶处理后所得的两个DNT片段保持彼此缔合。弗林蛋白酶处理的毒素对靶细胞的活性比完整的毒素高出约30倍。另一方面，其中弗林蛋白酶基序被破坏的DNT突变体对细胞没有毒性。弗林蛋白酶处理后产生的DNT的C-末端片段（δ B）具有与人工脂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异性受体结合，然后释放δ B并与细胞膜相互作用，将C端活性结构域转移到细胞质中。为了表达克隆DNT受体编码基因，我们构建了包含绿色荧光蛋白基因和血清反应元件的报告基因，其被作用于Rho激活下游的转录因子识别。该报告基因测定成功地响应于DNT对DNT敏感细胞的作用。我们将利用该系统，用DNT敏感细胞的cDNA文库转染DNT抗性细胞，克隆DNT受体基因。

项目成果

Hong, Y.: "Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum α-toxin"EMBO J.. 21・(19). 5047-5056 (2002)

Hong，Y.：“GPI锚定蛋白上的N-聚糖对气溶素的有效结合的要求，但不与败血梭菌α-毒素结合”EMBO J.. 21・(19) (2002)。

Matsuzawa, T., T. Kashimoto, J. Katahira and Y. Horiguchi: "Identification of a receptor-binding domain of Bordetella dermonecrotic toxin"Infect. Immun.. 70(7). 3427-3432 (2002)

Matsuzawa, T.、T. Kashimoto、J. Katahira 和 Y. Horiguchi：“博德特氏菌皮肤坏死毒素受体结合域的鉴定”感染。

Masuda, M.: "In vivo modifications of small GTPase Rac and Cdc42 by Bordetella dermonecrotic toxin"Infect. Immun.. 70・(2). 998-1001 (2002)

Masuda, M.：“博德特氏菌皮肤坏死毒素对小 GTP 酶 Rac 和 Cdc42 的体内修饰”Infect.. 998-1001 (2002)。

Shime, H., T. Ohnishi, K. Nagao, K. Oka, T. Takao and Y. Horiguchi: "Association of Pasteurella multocida toxin with vimentin"Infect. Immun.. 70(11). 6460-6463 (2002)

Shime，H.，T. Ohnishi，K. Nagao，K. Oka，T. Takao 和 Y. Horiguchi：“多杀性巴斯德氏菌毒素与波形蛋白的关联”感染。

Hong, Y., K. Ohnishi, N. Inoue, J. Y. Kang, H. Shime, Y. Horigucihi, F. G. van der Goot, N. Sugimoto and T. Kinoshita: "Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum α-toxin"EMBO J.. 21(19

Hong, Y., K. Ohnishi, N. Inoue, J. Y. Kang, H. Shime, Y. Horigucihi, F. G. van der Goot, N. Sugimoto 和 T. Kinoshita：“GPI 锚定蛋白上 N-聚糖的需求，以实现高效结合气溶素，但不结合败血梭菌 α-毒素”EMBO J.. 21(19