title: Metabolic inhibition of sialyl-Lewis X biosynthesis by 5-thiofucose remodels the cell surface and impairs selectin-mediated cell adhesion.
abstract: Sialyl-Lewis X (sLe(X)) is a tetrasaccharide that serves as a ligand for the set of cell adhesion proteins known as selectins. This interaction enables adhesion of leukocytes and cancer cells to endothelial cells within capillaries, resulting in their extravasation into tissues. The last step in sLe(X) biosynthesis is the α1,3-fucosyltrasferase (FUT)-catalyzed transfer of an L-fucose residue to carbohydrate acceptors. Impairing FUT activity compromises leukocyte homing to sites of inflammation and renders cancer cells less malignant. Inhibition of FUTs is, consequently, of great interest, but efforts to generate glycosyltransferase inhibitors, including FUT inhibitors, has proven challenging. Here we describe a metabolic engineering strategy to inhibit the biosynthesis of sLe(X) in cancer cells using peracetylated 5-thio-L-fucose (5T-Fuc). We show that 5T-Fuc is taken up by cancer cells and then converted into a sugar nucleotide analog, GDP-5T-Fuc, that blocks FUT activity and limits sLe(X) presentation on HepG2 cells with an EC(50) in the low micromolar range. GDP-5T-Fuc itself does not get transferred by either FUT3 or FUT7 at a measurable rate. We further demonstrate that treatment of cells with 5T-Fuc impaired their adhesive properties to immobilized adhesion molecules and human endothelial cells. 5T-Fuc, therefore, is a useful probe that can be used to modulate sLe(X) levels in cells to evaluate the consequences of inhibiting FUT-mediated sLe(X) formation. These data also reveal the utility of using sugar analogues that lead to formation of donor substrate analogues within cells as a general approach to blocking glycosyltransferases in cells.
authors.affiliation: Department of Chemistry, Simon Fraser University, 8888 University Dr., Burnaby V5A 1S6, Canada.
authors.forename: Wesley F
authors.initials: WF
authors.lastname: Zandberg
authors.forename: Jayakanthan
authors.initials: J
authors.lastname: Kumarasamy
authors.forename: B Mario
authors.initials: BM
authors.lastname: Pinto
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
mesh_terms: D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005643:Fucose / Q000031:analogs & derivatives* / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D060847:Metabolic Engineering / Q000379:methods; D009844:Oligosaccharides / Q000096:biosynthesis*; D019087:Selectins / Q000378:metabolism*; D000080506:Sialyl Lewis X Antigen
chemical_list: C119490:5-thiofucose; D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D019087:Selectins; D000080506:Sialyl Lewis X Antigen; D005643:Fucose; C502597:FUT7 protein, human; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase