Buffone A Jr, Mondal N, Gupta R, McHugh KP, Lau JT, Neelamegham S. (2013) Silencing α1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 during E-selectin mediated cell adhesion. J Biol Chem. 288(3):1620-1633. PMID: 23192350; PMCID: PMC3548472
Leukocyte adhesion during inflammation is initiated by the binding of sialofucosylated carbohydrates expressed on leukocytes to endothelial E-/P-selectin. While the glycosyltransferases (glycoTs) constructing selectin-ligands have largely been identified using knockout 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 α1,3fucosyltransferases (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 to bone marrow derived neutrophils from Fut4(-/-)Fut7(-/-) dual knockout mice. Results demonstrate that predominantly FUT7, and to a lesser extent FUT4, form 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 α1,3fucosyltransferase FUT9 played an important role since leukocyte adhesion was reduced by 50-60% in FUT9(-)HL-60, 70-80% in dual knock-down FUT7(-)9(-) cells, and ~85% in FUT4(-)7(-)9(-) triple knock-downs. Gene silencing results are in agreement with gain-of-function experiments where all three FUTs conferred E-selectin mediated rolling in HEK293T cells. The manuscript advances new tools to study human glycoT function. It suggests a species-specific role for FUT9 during the biosynthesis of human E-selectin ligands.
Link to journal: http://www.jbc.org/