A novel, promoter-based, target-specific assay identifies 2-deoxy-D-glucose as an inhibitor of globotriaosylceramide biosynthesis.
Abnormal biosynthesis of globotriaosylceramide (Gb3) is known to be associated with Gb3-related diseases, such as Fabry disease. The Gb3 synthase gene (Gb3S) codes for alpha1,4-galactosyltransferase, which is a key enzyme involved in Gb3 biosynthesis in vivo. Transcriptional repression of Gb3S is a way to control Gb3 biosynthesis and may be a suitable target for the treatment of Gb3-related diseases. To find a transcriptional inhibitor for Gb3S, we developed a convenient cell-based chemical screening assay system by constructing a fusion gene construct of the human Gb3S promoter and a secreted luciferase as reporter. Using this assay, we identified 2-deoxy-D-glucose as a potent inhibitor for the Gb3S promoter. In cultured cells, 2-deoxy-D-glucose markedly reduced endogenous Gb3S mRNA levels, resulting in a reduction in cellular Gb3 content and a corresponding accumulation of the precursor lactosylceramide. Moreover, cytokine-induced expression of Gb3 on the cell surface of endothelial cells, which is closely related to the onset of hemolytic uremic syndrome in O157-infected patients, was also suppressed by 2-deoxy-D-glucose treatment. These results indicate that 2-deoxy-D-glucose can control Gb3 biosynthesis through the inhibition of Gb3S transcription. Furthermore, we demonstrated the general utility of our novel screening assay for the identification of new inhibitors of glycosphingolipid biosynthesis.
Okuda
Tetsuya
T
Glycolipids Function Analysis Team, Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, Kagawa, Japan. t-okuda@aist.go.jp
Furukawa
Koichi
K
Nakayama
Ken-ichi
K
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000502:physiology; D014281:Trihexosylceramides / Q000096:biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004791:Enzyme Inhibitors; D012097:Repressor Proteins; D014281:Trihexosylceramides; D014409:Tumor Necrosis Factor-alpha; C018549:globotriaosylceramide; D003847:Deoxyglucose; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase