Silencing α1,3-Fucosyltransferases in Human Leukocytes Reveals a Role for FUT9 Enzyme during E-selectin-mediated Cell Adhesion

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.

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes

“…During analysis, the interacting cells were defined to travel at velocities less than the theoretical free stream velocity of noninteracting 10-mm diameter spheres at the flow chamber wall. 8 Among these, cells moving ,1 cell diameter in a 10-second period were defined to be "adherent," whereas the remaining were classified to be "rolling." Rolling velocity was calculated by tracking individual cell trajectories.…”

“…HL-60 variants, mouse neutrophils, and transduced hHSCs at 2 3 10 6 cells per mL were perfused at a wall shear stress of 1 to 2 dyne/cm 2 over substrates bearing either physisorbed recombinant L-/P-selectin-immunoglobulin G (IgG), P-selectin-bearing CHO-P cells, E-selectin-bearing L/E cells, or IL-1b-stimulated HUVECs (Buffone et al 8 ; supplemental Methods). Here, the E-selectin density on HUVECs was ;102 sites per mm 2 , the CHO-P cell P-selectin density was ;167 sites per mm 2 , and all recombinant human selectins were adsorbed at ;180 sites per mm 2 .…”

“…4,7 This is particularly relevant for the E-selectin ligands because P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is the major ligand for L-and P-selectin in both humans and mice, and the glycoTs constructing functional selectin ligand(s) on this glycoprotein are similar in both species. 7,8 With regard to E-selectin, however, human but not mouse granulocyte rolling on this selectin is insensitive to pronase treatment. 9,10 Thus, protease-insensitive gangliosides may be physiological E-selectin ligands that are unique to humans.…”

“…11 Among the a1,3fucosyltransferases, the enzyme FUT9 reportedly plays a more significant role during human leukocyte adhesion, compared with FUT7 and FUT4 which are the dominant players in mice. 8 Among additional differences, L-selectin in human but not mouse neutrophils are considered to act as an E-selectin ligand, 12 though its relative roles in direct E-selectin binding vs secondary neutrophil-neutrophil adhesion remains unresolved. 13 ESL-1 is a functional E-selectin ligand on murine but not human myeloid cells.…”

“…25 To quantify the contributions of ST3Gal-3, -4, and -6, these enzymes were specifically disrupted in myeloid cells using either lentivirus-based short hairpin RNA (shRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease-RNA guided genome editing. 8,26 Although a number of studies were performed with HL-60 promyeloid leukemic cells because they resemble primary neutrophils in terms of glycosyltransferase expression and selectin binding phenotype, 27 critical validation was performed using neutrophils derived from CD34 1 human hematopoietic stem cells (hHSCs). The study demonstrates a dominant role for ST3Gal-4 in human leukocyte adhesion to all 3 selectins.…”