Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes

Abstract

The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also.

Keywords: carbohydrate; cell adhesion; endothelial cell; fluid shear; fucosyltransferase; glycosyltransferase; inflammation; leukocyte; selectin; sialyl Lewis-X.

Figure 1. Workflow for the study of fucosyltransferases in human leukocytes. In step 1, CHO cells expressing FUT4-, FUT7-, or FUT9-EGFP fusion proteins were constructed. Various FUT shRNA with puromycin selection cassette were introduced into these cells and their ability to reduce cellular EGFP fluorescence was assessed using flow cytometry. Green fluorescence signal was reduced from the dark shaded histogram without shRNA to light shaded histogram upon addition of efficient shRNA. Empty and dashed histograms represent cells transduced with non-silencing lentivirus and cells lacking EGFP respectively. In step 2, efficient shRNA containing either a puromycin cassette or fluorescence reporter (DsRed or Cerulean) were transduced into HL-60 cells to create stable knockdowns. Cell function was measured under fluid shear using flow chamber substrates bearing either CHO cells expressing P-selectin, recombinant L-selectin, E-selectin expressing L-cells, or IL-1β stimulated HUVECs.

Figure 2. Selectin ligand biosynthetic pathway on PSGL-1: A core-2 O-glycan carrying the sLe^X tetrasaccharide is expressed at the N-terminus of PSGL-1. This is synthesized in the Golgi by the action of various glycosyltransferases (GlycoTs).