SUMMARY Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell-surface N- and O-linked glycan biosynthesis at 10–100μM concentrations. The >10 fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell-surface sialyl Lewis-X expression, and leukocyte rolling on selectin-substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to murine infusion inhibited neutrophil homing to sites of acute inflammation and bone marrow by ~80–90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors/decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation.
Objective Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids (GSLs) expressed on human myeloid cells in regulating E-selectin mediated cell adhesion. Approach and Results A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase (UGCG) using either lentivirus delivered shRNA or CRISPR-Cas9 based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UGCG disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics based flow chamber studies demonstrated that both the UGCG knockouts and knockdowns display ~60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells (ECs), without altering cell adhesion to P-selectin. Consistent with the concept that the GSLs support slow rolling and the transition to firm arrest, inhibiting UGCG activity resulted in frequent leukocyte detachment events, skipping motion and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. Conclusions GSLs likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.
There is a critical need to develop small molecule inhibitors of mucin-type O-linked glycosylation. The best known reagent currently is peracetylated benzyl-GalNAc, but it is only effective at millimolar concentrations. This manuscript demonstrates that Ac5GalNTGc, a peracetylated C-2 sulfhydryl substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast and prostate cells, Ac5GalNTGc increased cell surface VVA-binding by ~10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry and Western blot analysis of HL-60 promyelocytes demonstrate that 50-80µM Ac5GalNTGc prevented elaboration of 30-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac5GalNTGc resulted in 50-80% reduction in leukocyte sialyl-Lewis-X expression, and L-/P-selectin mediated rolling under flow. Ac5GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ~60%. Overall, Ac5GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.
SUMMARYThere is a critical need to develop small molecule inhibitors of mucin-type O-linked glycosylation. The best known reagent currently is peracetylated benzyl-GalNAc, but it is only effective at millimolar concentrations. This manuscript demonstrates that Ac5GalNTGc, a peracetylated C-2 sulfhydryl substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast and prostate cells, Ac5GalNTGc increased cell surface VVA-binding by ~10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry and Western blot analysis of HL-60 promyelocytes demonstrate that 50-80μM Ac5GalNTGc prevented elaboration of 30-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac5GalNTGc resulted in 50-80% reduction in leukocyte sialyl-Lewis-X expression, and L-/P-selectin mediated rolling under flow. Ac5GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ~60%. Overall, Ac5GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.