Anti-FUT4 monoclonal antibody, clone FQS0632

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 alpha 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 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 alpha 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 FUT7(-)9(-) cells, and similar to 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.

Fucosyltransferases are involved in the last steps of the biosynthesis of ABH and Lewis oligosaccharide antigens. Seven human genes (FUT1 to FUT7) and one pseudogene (Sec 1) have been cloned and localized on different chromosomes (9q34.3; 11q21; 19p13.3 and 19q13.3). Their locations and their high degree of primary sequence identity, suggest that they have appeared by successive duplications followed by translocation and divergent evolution. Their expression is tissue specific and they present a switch during human embryo-foetal development similar to that of hemoglobins. Polymorphic genes FUT1-FUT2 and FUT3-FUT5-FUT6 are organized in two clusters and each gene is partially or totally inactivated by different types of point mutations (nonsense, missense and frame shift), complete gene deletion or a fusion gene. The products of the monomorphic genes FUT4 and FUT7 seem implicated in cell-cell interactions during embryo-foetal development and in the leukocyte adhesion phenomena to endothelial cells in the adult. A phylogenetic tree of the 28 available nucleotide coding sequences of fucosyltransferases has allowed us to situate the duplication events with respect to the separation of species from the main evolutionary path (nematods, birds, mammals, primates and humans). Recently, using a computer approach a general structure of fucosyltransferases has been proposed, inspired from the crystalline structure of the beta-glucosyltransferase of bacteriophage T4. This folding contains two domains with an alternate succession alpha and beta chains. Ln this model the GDP-fucose binding site would be located between the two domains.