Highly sensitive cell-based assay system to monitor the sialyl Lewis X biosynthesis mediated by alpha1-3 fucosyltransferase-VII.

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.