Identification of the terminal glycosyltransferases critical in mediating selectin-dependent adhesion in human leukocytes
Buffone, Alexander, Jr.
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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 transgenic mice, accumulating evidence suggests that important differences may exist between humans and mice. To address this, we developed a systematic lentivirus based shRNA delivery system to create human leukocytic cell lines that lack myeloid α1,3 fucosyltransferases. To this end, neutrophils from the FT-IV -/- FT-VII -/- mouse along with human leukocytes deficient in FUT4, FUT7, or both FUT4 and FUT7 were evaluated in their ability to interact with E-, P-, and L-selectin surfaces. Results demonstrate that, in agreement with the mouse FT-IV -/- FT-VII -/- neutrophils, FUT7 and to a lesser extent FUT4 form the selectin-ligand at the N-terminus of human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1). This ligand mediates ~90% of the leukocyte tethering and rolling on P-/L-selectin substrates in both humans and mice. Stark differences were seen in E-selectin mediated adhesion as the FUT4 – 7 – human leukocytes failed to reduce rolling or adherent cells by >20%, whereas a complete loss of E-selectin binding was noted in the FT-IV -/- FT-VII -/- neutrophils on the similar surface. KPL-1 blocking reduced the surface interaction by ∼40% demonstrating that there is at least 1 other PSGL-1 independent E-selectin ligand in human leukocytes. Taken together, the data highlights species-specific differences between human and mice leukocytes. To address this the possibility of another α1,3 FUT being involved in E-selectin ligand synthesis, we utilized our systematic lentivirus based shRNA system to create human leukocytic cell lines that lack up to three myeloid &agr;1,3 fucosyltransferases (FUT4, FUT7 and FUT9). All three FUTs participate in E-selectin ligand biosynthesis, with FUT9 and FUT7 playing major roles. Leukocyte rolling and adhesion was reduced by 45% upon knocking-down FUT9 alone, >70% in dual knock-downs lacking FUT7 and FUT9, and 90% in triple knock-downs. Gene silencing studies were in agreement with gain-of-function experiments where glycoTs were overexpressed in the absence or presence of PSGL-1 in HEK293T cells. Overall, the data from both gain-of-function experiments in HEK293T and loss-of-function in HL-60 reveal a novel role for FUT9 during the biosynthesis of human E-selectin ligands. We then used our lentivirus based shRNA delivery system to create human leukocytic cell lines that lack the another type of terminal glycosyltransferase involved in sLe X formation, Type-II α2,3 sialylsyltransferases. In order to compare differences between the species, neutrophils from the ST3Gal-IV -/- mouse along with human leukocytes deficient in ST3Gal-III, -IV, -VI or both ST3Gal-III and -IV or ST3Gal-IV and -VI were evaluated in their ability to interact with E-, P-, and L-selectin surfaces. Results demonstrate that while ST3Gal-IV completely mediates synthesis of the selectin-ligand for P- and E-selectin in humans, neutrophils from the ST3Gal-IV -/- mouse only disrupt binding 30% and 40% respectively. In L-selectin mediated adhesion, the ST3Gal-IV – human leukocytes reduced adherent cells by >90% and the dual knockdown of ST3Gal-IV – VI – completely removed binding. Stark species -specific differences were noted as there was no loss of L-selectin binding in the ST3Gal-IV -/- mice neutrophils on the similar surface run in parallel. Taken together, the data highlights ST3Gal-IV as the critical mediator of selectin-ligand synthesis in humans, in contrast to the co-dependence of ST3Gal-IV and another as-yet-unidentified ST3Gal in mice leukocytes. Overall, this work identifies a novel method of screening shRNA through use of glycoT-EGFP expressing cells, identifies the roles of all three myeloid FUTs in selectin-ligand synthesis in humans including a previously unreported role for FUT9, demonstrates ST3Gal-IV as the principal sialyltransferases involved in selectin ligand-synthesis, and highlight drastic species-specific differences in glycosyltransferase function, selectin-ligand biosynthesis, and selectin function between human and mice leukocytes.