title: Silencing α1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 enzyme during E-selectin-mediated cell adhesion.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, State University of New York, Buffalo, New York 14260, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Buffone
authors.forename: Nandini
authors.initials: N
authors.lastname: Mondal
authors.forename: Rohitesh
authors.initials: R
authors.lastname: Gupta
authors.forename: Kyle P
authors.initials: KP
authors.lastname: McHugh
authors.forename: Joseph T Y
authors.initials: JT
authors.lastname: Lau
authors.forename: Sriram
authors.initials: S
authors.lastname: Neelamegham
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002450:Cell Communication; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020868:Gene Silencing; D018922:HL-60 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019041:L-Selectin / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism*; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D013045:Species Specificity; D014162:Transfection
chemical_list: D019040:E-Selectin; D016256:Lewis X Antigen; D019007:P-Selectin; D034741:RNA, Small Interfering; D019041:L-Selectin; C066693:FUT4 protein, human; C502597:FUT7 protein, human; C519500:FUT9 protein, human; D005647:Fucosyltransferases; C518194:fucosyltransferase 9, mouse