Highly sensitive cell-based assay system to monitor the sialyl Lewis X biosynthesis mediated by α1-3 fucosyltransferase-VII
Highly sensitive cell-based assay system to monitor the sialyl Lewis X biosynthesis mediated by α1-3 fucosyltransferase-VII
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DOI：
10.1016/j.bbrc.2004.09.025
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发表时间：
2004-11-05
影响因子：
3.1
通讯作者：
Sugita, T
中科院分区：
文献类型：
--
作者：
Miyashiro, M;Furuya, S;Sugita, T
白细胞上的sialyl Lewis X （sLe(X)）决定因子作为选择素家族细胞粘附分子的配体，选择素-碳水化合物相互作用被认为在炎症期间白细胞外渗过程中起重要作用。在几种α - 1-3聚焦转移酶（FucTs）中，FucT-VII在sLe(x)-表位的生物合成中起着关键作用。因此，专门设计用于抑制FucT-VII酶的小分子可能具有抗炎剂的潜力。在这里，我们开发了一种多功能的基于细胞的检测系统，使用GeneSwitch系统监测sLe(x)的生物合成。该系统是一个米非司酮（MFP）诱导的哺乳动物表达系统，在MFP存在下，人转染T淋巴细胞在细胞表面以时间依赖的方式表达fft - vii和sLe(x)-表位的mRNA，背景转录率非常低。此外，当转染物用ffuct - vii抑制剂panosialin处理时，诱导细胞上sLe(x)的表达被剂量依赖性地抑制，而ffuct - vii的mRNA水平没有改变。这些结果表明，FucT-VII可能是T淋巴细胞上sLe(x)-表位生物合成的主要调节因子，这种基于细胞的检测可能用于筛选FucT-VII抑制剂系统。(C) 2004爱思唯尔公司版权所有。
The sialyl Lewis X (sLe(x)) determinant on leukocytes serves as a ligand for selectin family cell adhesion molecules, and selectin-carbohydrate interaction is considered to play an important role in the process of leukocyte extravasation during inflammation. Among several alpha1-3 fucosyltransferases (FucTs), FucT-VII plays a critical role in the biosynthesis of sLe(x)-epitopes. Therefore, small molecules specifically designed to inhibit the FucT-VII enzyme may have potential as anti-inflammatory agents. Here, we have developed a versatile cell-based assay system to monitor sLe(x) biosynthesis using the GeneSwitch System. This system is a mifepristone (MFP)-inducible mammalian expression system, and human transfectant T lymphoblasts expressed the mRNA of FucT-VII and the sLe(x)-epitopes on the cell surface in a time-dependent manner in the presence of MFP, with very low background transcription. Furthermore, when the transfectants were treated with the FucT-VII inhibitor panosialin, sLe(x) expression on the induced cells was inhibited dose dependently without alteration at the mRNA level of FucT-VII. These results suggest that the FucT-VII may be a major regulator of the biosynthesis of the sLe(x)-epitopes on T lymphoblasts, and this cell-based assay may be utilized for a screening system of FucT-VII inhibitors. (C) 2004 Elsevier Inc. All rights reserved.