Development of a Sensitive Separation and Quantification Method for Sialyl Lewis X and Lewis X Involving Anion-Exchange Chromatography: Biochemical Characterization of α1-3 Fucosyltransferase-VII

Abstract

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLe^x) in human leukocytes is mediated by α1-3 fucosyltransferase-VII (FucT-VII), which catalyzes the transfer of fucose from GDP-β-fucose to the 3-OH of α2-3 sialyl N-acetyllactosamine (SALN). 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 0-0.5M NH₄OAc (pH 9.0) on QAE-Toyopearl 550 C. 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 0.1M NH₄OAc, and these products were clearly separated from the fraction of unreacted GDP-[³H] 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 α2-3 SA-LN or N-acetyllactosamine sequences.