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Journal of Biochemistry 2004 136(5):723-731; doi:10.1093/jb/mvh179
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© 2004 The Japanese Biochemical Society

BIOCHEMISTRY

Development of a Sensitive Separation and Quantification Method for Sialyl Lewis X and Lewis X Involving Anion-Exchange Chromatography: Biochemical Characterization of {alpha}1-3 Fucosyltransferase-VII

Masahiko Miyashiro1, Sachiko Furuya2 and Takahisa Sugita1,*

1 Pharmacology Research Laboratories, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505; and 2 Discovery Research Laboratories, Tanabe Seiyaku Co., Ltd., 2-50 Kawagishi 2-chome, Toda, Saitama, 335-8505

The biosynthesis of the carbohydrate antigen sialyl Lewis X (sLex) in human leukocytes is mediated by {alpha}1-3 fucosyltransferase-VII (FucT-VII), which catalyzes the transfer of fucose from GDP-ß-fucose to the 3-OH of {alpha}2-3 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 NH4OAc (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 (Lex) was eluted in the flow-through fraction and sLex was eluted with 0.1 M 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 {alpha}2-3 SA-LN or N-acetyllactosamine sequences.

* To whom correspondence should be addressed. Tel: +81-6-300-2610, Fax: +81-6-300-2593, E-mail: t-sugita{at}tanabe.co.jp


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