Identification of a cell surface receptor for Bordetella dermonecrotic *

我们最近明确了波尔德氏菌皮肤坏死毒素(DNT)是一种转谷氨酰胺酶，催化Rho GTP酶的特定谷氨酰胺脱酰胺或多胺化。然而，关于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位途径，人们知之甚少。为了理解这一点，我们试图定位负责与靶细胞结合的DNT的最小区域。DNT的C端截短突变体可抑制DNT对MC3T3-E1细胞的作用。具有这种抑制作用的最小区域由N端54个氨基酸组成(DNT1-54)。~lt；125&gt；I标记的DNT1-54可与MC3T3-E1细胞直接结合。Scatchard分析表明，DNT1-54以均一的方式与细胞结合，其Kd值为2.5微摩尔。此外，发现^&lt；125&gt；I标记的DNT1-54与DNT敏感的C3H10T1/2、Swiss3T3、REF和NIH3T3结合，但不与DNT抗性的Balb3T3、L929、PAE、COS7和K562结合。这些结果表明，DNT1-54含有结合域，并与DNT竞争存在于DNT敏感细胞上的特定受体。DNT1-54包括在Arg41和Arg44之间被膜锚定的蛋白酶Furin切割的共识基序。最近我们发现，在体外，DNT实际上被一种可溶性的呋喃所切割。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Furin或Furin样酶切割后从整个DNT分子中分离出来。这一过程可能对以下DNT的作用是必不可少的，因为在对哺乳动物细胞的作用方面，经呋喃处理的DNT比完整的DNT的效率高约100倍。

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.