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Identification of a cell surface receptor for Bordetella dermonecrotic *

皮肤坏死博德特氏菌细胞表面受体的鉴定*

基本信息

批准号：
11670264
负责人：
HORIGUCHI Yasuhiko
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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.

我们最近阐明，博德特氏菌皮肤坏死毒素（DNT）是一种转氨酶催化脱酰胺或多胺化的一个特定的谷氨酰胺的Rho GTP酶。然而，对DNT作用的早期步骤如与膜受体结合和进入细胞质环境的转运途径知之甚少。为了理解这一点，我们试图定位DNT负责结合靶细胞的最小区域。DNT的C端截短突变体抑制DNT对MC 3 T3-E1细胞的作用。发现具有这种抑制作用的最小区域由N-末端54个氨基酸（DNT 1 -54）组成。标记<125>的DNT 1 -54可与MC 3 T3-E1细胞直接结合。Scatchard分析表明，DNT 1 -54以均匀模式与细胞结合，Kd为2.5微摩尔。此外，<125>发现标记的DNT 1 -54与DNT敏感的C3 H10 T1/2、Swiss 3 T3、REF和NIH 3 T3结合，但不与DNT抗性的Balb 3 T3、L929、PAE、COS 7和K562结合。这些结果表明，DNT 1 -54含有结合结构域，并与DNT竞争存在于DNT敏感细胞上的特异性受体。DNT 1 -54在Arg 41和Arg 44之间包括用于被膜锚定蛋白酶弗林蛋白酶切割的共有基序。最近，我们发现，DNT实际上是切割在这个基序的可溶性形式的弗林蛋白酶在体外。我们认为DNT通过N末端受体结合区与靶细胞上的特定受体结合，然后被弗林蛋白酶或弗林蛋白酶样蛋白酶切割后与整个DNT分子分离。这一过程可能是必要的，因为DNT预消化弗林蛋白酶是约100倍的效率比完整的DNT在哺乳动物细胞上的行动。