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...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 2004-11, Vol.136 (5), p.723-731
Main Authors: Miyashiro, Masahiko, Furuya, Sachiko, Sugita, Takahisa
Format: Article
Language:English
Subjects:
Catalysis
Chromatography, Ion Exchange - methods
Enzyme Inhibitors - pharmacology
Ethylmaleimide - pharmacology
Fucosyltransferases - antagonists & inhibitors
Fucosyltransferases - chemistry
Guanosine Diphosphate - pharmacology
Humans
Lewis X Antigen - analysis
Lewis X Antigen - chemistry
Oligosaccharides - analysis
Oligosaccharides - chemistry
Recombinant Proteins - antagonists & inhibitors
Recombinant Proteins - chemistry
Sensitivity and Specificity
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Summary: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.
ISSN:0021-924X