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

Alexander Buffone, Nandini Mondal, Rohitesh Gupta, Kyle P. McHugh, Joseph T.Y. Lau, Sriram Neelamegham

Research output: Contribution to journalArticlepeer-review

69 Scopus citations

Abstract

Background: During inflammation, the selectins engage glycosylated macromolecules expressed on blood leukocytes under fluid shear conditions. Results: Although all three myeloid 1,3-fucosyltransferases FUT9, FUT7, and FUT4 regulate human E-selectin ligand biosynthesis, FUT7 and FUT4 are sufficient to form L/P-selectin ligands. Conclusion: FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Significance: This study identifies potential(1,3)FUTs regulating inflammation in humans. 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 1,3-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 FUT47 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 1,3-fucosyltransferase FUT9 played an important role because leukocyte adhesion was reduced by 50-60% in FUT9-HL-60, 70-80% in dual knockdown FUT79 cells, and 85% in FUT479 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 forFUT9during the biosynthesis of human E-selectin ligands.

Original languageEnglish (US)
Pages (from-to)1620-1633
Number of pages14
JournalJournal of Biological Chemistry
Volume288
Issue number3
DOIs
StatePublished - Jan 18 2013
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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