Silencing α1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 enzyme during E-selectin-mediated cell adhesion

J Biol Chem. 2013 Jan 18;288(3):1620-33. doi: 10.1074/jbc.M112.400929. Epub 2012 Nov 28.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Adhesion
  • Cell Communication
  • E-Selectin / genetics
  • E-Selectin / metabolism*
  • Fucosyltransferases / antagonists & inhibitors
  • Fucosyltransferases / genetics
  • Fucosyltransferases / metabolism*
  • Gene Expression
  • Gene Silencing
  • HL-60 Cells
  • Human Umbilical Vein Endothelial Cells / cytology
  • Human Umbilical Vein Endothelial Cells / metabolism*
  • Humans
  • Inflammation / genetics
  • Inflammation / metabolism
  • Inflammation / pathology
  • L-Selectin / genetics
  • L-Selectin / metabolism
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / metabolism*
  • Lewis X Antigen / genetics
  • Lewis X Antigen / metabolism
  • Mice
  • Mice, Knockout
  • P-Selectin / genetics
  • P-Selectin / metabolism
  • RNA, Small Interfering
  • Species Specificity
  • Transfection

Substances

  • E-Selectin
  • Lewis X Antigen
  • P-Selectin
  • RNA, Small Interfering
  • L-Selectin
  • FUT4 protein, human
  • FUT7 protein, human
  • Fucosyltransferases
  • fucosyltransferase 9, mouse
  • fucosyltransferase IX, human