Silencing α1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 enzyme during E-selectin-mediated cell adhesion.
Leukocyte adhesion during inflammation is initiated by the binding of sialofucosylated carbohydrates expressed on leukocytes to endothelial E/P-selectin. Although the glycosyltransferases (glycoTs) constructing selectin-ligands have largely been identified using knock-out mice, important differences may exist between humans and mice. To address this, we developed a systematic lentivirus-based shRNA delivery workflow to create human leukocytic HL-60 cell lines that lack up to three glycoTs. Using this, the contributions of all three myeloid α1,3-fucosyltransferases (FUT4, FUT7, and FUT9) to selectin-ligand biosynthesis were evaluated. The cell adhesion properties of these modified cells to L-, E-, and P-selectin under hydrodynamic shear were compared with bone marrow-derived neutrophils from Fut4(-/-)Fut7(-/-) dual knock-out mice. Results demonstrate that predominantly FUT7, and to a lesser extent FUT4, forms the selectin-ligand at the N terminus of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) in humans and mice. Here, 85% reduction in leukocyte interaction was observed in human FUT4(-)7(-) dual knockdowns on P/L-selectin substrates. Unlike Fut4(-/-)Fut7(-/-) mouse neutrophils, however, human knockdowns lacking FUT4 and FUT7 only exhibited partial reduction in rolling interaction on E-selectin. In this case, the third α1,3-fucosyltransferase FUT9 played an important role because leukocyte adhesion was reduced by 50-60% in FUT9-HL-60, 70-80% in dual knockdown FUT7(-)9(-) cells, and ∼85% in FUT4(-)7(-)9(-) triple knockdowns. Gene silencing results are in agreement with gain-of-function experiments where all three fucosyltransferases conferred E-selectin-mediated rolling in HEK293T cells. This study advances new tools to study human glycoT function. It suggests a species-specific role for FUT9 during the biosynthesis of human E-selectin ligands.
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CHEMICAL CHEMICAL GENE-N GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-N GENE-Y GENE-N GENE-N GENE-N GENE-N GENE-Y GENE-N GENE-Y GENE-Y GENE-N GENE-N GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-Y GENE-N GENE-N GENE-N GENE-Y GENE-Y
carbohydrates N PSGL-1 human FUT4(-)7 P/L-selectin Fut4 Fut7 FUT4 FUT7 E-selectin α1,3-fucosyltransferase FUT9 E/P-selectin FUT9 FUT7(-)9 FUT4(-)7(-)9 fucosyltransferases glycosyltransferases E-selectin human glycoT FUT9 human E-selectin glycoTs selectin glycoTs α1,3-fucosyltransferases FUT4 FUT7 FUT9 selectin L-, E-, and P-selectin Fut4 Fut7 FUT7 FUT4 selectin P-selectin glycoprotein ligand-1 α1,3-fucosyltransferases FUT9 E-selectin