Development of a sensitive separation and quantification method for sialyl Lewis X and Lewis X involving anion-exchange chromatography: biochemical characterization of alpha1-3 fucosyltransferase-VII.
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.
Miyashiro
Masahiko
M
Pharmacology Research Laboratories, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan.
Furuya
Sachiko
S
Sugita
Takahisa
T
D002384:Catalysis; D002852:Chromatography, Ion Exchange / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006801:Humans; D016256:Lewis X Antigen / Q000032:analysis / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012680:Sensitivity and Specificity; D000080506:Sialyl Lewis X Antigen
D004791:Enzyme Inhibitors; D016256:Lewis X Antigen; D009844:Oligosaccharides; D011994:Recombinant Proteins; D000080506:Sialyl Lewis X Antigen; D006153:Guanosine Diphosphate; C502597:FUT7 protein, human; D005647:Fucosyltransferases; D005033:Ethylmaleimide