Sampathkumar, S.-G. | bioRxiv Science

bioRxiv2021

Glycan engineering using synthetic ManNAc analogues enhances sialyl-Lewis-X expression and adhesion of leukocytes

Sialyl-Lewis-X (sLeX/CD15s) epitopes regulate cell adhesion via interactions with selectins. High levels of sLeX are associated with chronic inflammation. Intense efforts have been devoted for the development of sLeX mimetics for the prevention of atherosclerosis. By contrast, low levels of sLeX are associated with leukocyte adhesion deficiency (LAD) disorders. In this context, we employed metabolic glycan engineering to alter the fine structures of sialoglycans. Treatment of HL-60 (human acute myeloid leukemia) cells with peracetyl N-cyclobutanoyl-D-mannosamine (Ac4ManNCb) resulted in a four-fold increase in both sLeX levels and adhesion to E-selectin-Fc chimera. Enhanced sLeX levels on CD162/PSGL-1, CD43, and CD44 were observed through immunoprecipitation. Molecular dynamics (MD) simulations on interactions with E-selectin revealed dramatic differentials in the conformational dynamics of sLeX-Cb compared to sLeX, highlighting the significance of remote N-acyl side chains of sialic acids in facilitating bio-active conformations. These results provide opportunities for pharmacological interventions in both LAD and chronic inflammation. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/473788v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@172ab54org.highwire.dtl.DTLVardef@14e45acorg.highwire.dtl.DTLVardef@1c05c65org.highwire.dtl.DTLVardef@1f2056e_HPS_FORMAT_FIGEXP M_FIG C_FIG Remote substituents on the tetrasaccharide sialyl-Lewis X (sLeX / CD15s) influence ensembles of bio-active conformations and alter biological outcomes. Treatment with peracetyl N-cyclobutanoyl-D-mannosamine (Ac4ManNCb) results in four-fold enhancement in the expression of sialyl-Lewis-X (sLeX / CD15s) and adhesion to E-selectin (CD62E). Molecular dynamics simulations show that the sLeX-Cb prefers a flatter bio-active conformation (gray) compared to wild-type sLeX (cyan). Institute and/or researcher Twitter usernames: @NImmunology @Gops_GlycoIndia

biochemistry↗

bioRxiv2020

Efficient Inhibition of O-glycan biosynthesis using the hexosamine analog Ac5GalNTGc

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 50-80M Ac5GalNTGc prevented elaboration of 30-60% of the O-glycans beyond the Tn-antigen (GalNAc1-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.

biochemistry↗