Assuntos
Inibidores Enzimáticos/isolamento & purificação , Fucosiltransferases/antagonistas & inibidores , Contagem de Cintilação/métodos , Animais , Cromatografia por Troca Iônica/métodos , Fucosiltransferases/genética , Humanos , Mutação , Proteínas Recombinantes/análise , Spodoptera , Especificidade por SubstratoRESUMO
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.
Assuntos
Epitopos , Fucosiltransferases/metabolismo , Regulação da Expressão Gênica , Oligossacarídeos/biossíntese , Animais , Derivados de Benzeno/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Fucosiltransferases/genética , Humanos , Camundongos , Mifepristona/farmacologia , Oligossacarídeos/genética , Antígeno Sialil Lewis X , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/fisiologia , TransgenesRESUMO
The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe(x)) in human leukocytes is mediated by alpha1-3 fucosyltransferase-VII (FucT-VII), which catalyzes the transfer of fucose from GDP-beta-fucose to the 3-OH of alpha2-3 sialyl N-acetyllactosamine (SA-LN). We developed a simple method for quantitating the reaction product of FucT-VII involving Anion-Exchange Chromatography (AEC). The AEC assay involved the separation of a radio-labeled acceptor from the unreacted nucleotide sugars with 0-0.5 M NH(4)OAc (pH9.0) on QAE-Toyopearl 550C. Furthermore, this assay enabled the separation of the fucosylated products of sialylated and non-sialylated oligosaccharides with this column. Analysis of the FucT-VI reaction mixture showed that Lewis X (Le(x)) was eluted in the flow-through fraction and sLe(x) was eluted with 0.1 M NH(4)OAc, and these products were clearly separated from the fraction of unreacted GDP-[(3)H]fucose. Therefore, this method could be a powerful tool for the characterization of recombinant FucT-VII and for establishing a high-throughput screening system for FucT-VII inhibitors. Beside FucT-VII, this method will be applicable to the assaying of many different glycosyltransferases, including sialyltransferases and glucosaminyltransferases, which are reactive to alpha2-3 SA-LN or N-acetyllactosamine sequences.