Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion

Abstract

There is often interest in dissecting the relative contributions of the N-glycans, O-glycans and glycosphingolipids (GSLs) in regulating complex biological traits like cell signaling, adhesion, development and metastasis. To address this, we developed a CRISPR-Cas9 toolkit to selectively truncate each of these commonly expressed glycan-types. Here, O-glycan biosynthesis was truncated by knocking-out Core 1 β3Gal-T Specific Molecular Chaperone (COSMC), N-glycans by targeting the β1,2 GlcNAc-transferase (MGAT1) and GSLs by deleting UDP-glucose ceramide glucosyltransferase (UGCG). These reagents were applied to reveal the glycoconjugates regulating human myeloid cell adhesion to selectins under physiological shear-flow observed during inflammation. These functional studies show that leukocyte rolling on P- and L-selectin is ablated in cells lacking O-glycans, with N-glycan truncation also increasing cell rolling velocity on L-selectin. All three glycan families contributed to E-selectin dependent cell adhesion with N-glycans contributing to all aspects of the leukocyte adhesion cascade, O-glycans only being important during initial recruitment, and GSLs stabilizing slow cell rolling and the transition to firm arrest. Overall, the genome editing tools developed here may be broadly applied in studies of cellular glycosylation.

Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leukocyte Adhesion

“…5). In such studies, PNA, ECL and PHA-L lectin binding to O- and N-glycans is typically augmented upon de-sialylation (Stolfa et al, 2016). However, the MAL-II binding data indicate that the overall expression of α2,3 sialic acid terminated glycans remains unchanged.…”

“…As GlcNAc based S-glycosides truncate the growth of this common motif on different types of glycoconjugates, these small molecules may be broadly useful as inhibitors for a variety of studies, although this may come at the cost of reduced specificity. To demonstrate such biological utility, we tested the ability of SNAP and ONAP to reduce selectin-dependent cell adhesion with focus on E-selectin, an endothelial lectin that binds sialofucosylated glycans like sLe X on all common glycoconjugate-types: O-glycans, N-glycans and GSLs (Stolfa et al, 2016). Here, all S-glycosides dramatically reduced cell surface leukocyte sialyl Lewis-X expression on leukocytes, and E- and L-selectin binding under static and flow conditions.…”

“…HUVECs were cultured in EBM-2 media (Lonza). Isogenic HL-60 clones lacking extended O-glycans ([O]¯ cells), N-glycans ([N]¯ ) and glycolipids ([G]¯ ) were available from a previous study (Stolfa et al, 2016). These cells lack the genes COSMC (core-1 β3 galatoctosyltransferase molecular chaperone), MGAT1 (mannosyl α1,3-glycoprotein β1,2- N -acetylglucosaminyltransferase) and UGCG (UDP-Glucose Ceramide Glucosyltransferase), respectively.…”

“…For example, peracetylated benzyl-α-GalNAc (or ‘GalNAc-OBn’) is added at 2–4 mM into cell culture media to inhibit O-linked glycosylation (Alfalah et al, 1999; Huet et al, 1998; Kuan et al, 1989; Tsuiji et al, 2003), and xylosides are similarly used at 1–2 mM for inhibiting GAG biosynthesis (Fritz et al, 1994; Okayama et al, 1973; Victor et al, 2009). At lower concentrations (~10–100 μM), these decoys have little or no inhibitory activity, and thus are used as molecular probes that report on the cellular O-glycan (Kudelka et al, 2016; Stolfa et al, 2016) or GAG biosynthesis pathways (Victor et al, 2009). …”

“…When added to human leukocytes, SNAP more effectively reduced expression of the sialyl Lewis-X (sLe X , Siaα2–3Galβ14(Fucα1–3)GlcNAc) epitope on cells, compared to ONAP. SNAP also reduced cell adhesion to E-selectin, the major human selectin which binds sialofucosylated epitopes on leukocyte O-glycans, N-glycans and GSLs (Mondal et al, 2016; Stolfa et al, 2016). In mouse, SNAP treated leukocytes exhibited reduced migration to sites of inflammation and bone marrow.…”