Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins

Abstract

Novel strategies to control the binding of adhesion molecules belonging to the selectin family are required for the treatment of inflammatory diseases. We tested the possibility that synthetic monosaccharide analogs can compete with naturally occurring sugars to alter the O-glycan content on human leukocyte cell surface selectin-ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Resulting reduction in the sialyl Lewis-X-bearing epitopes on this ligand may reduce cell adhesion. Consistent with this hypothesis, 50Mper-acetylated 4F-GalNAc added to the growth media of promyelocytic HL-60 cells reduced the expression of the cutaneous lymphocyte associated-antigen (HECA-452 epitope) by 82% within 2 cell doubling cycles. Cell binding to all 3 selectins (L-, E-, and P-selectin) was reduced in vitro. 4F-GalNAc was metabolically incorporated into PSGL-1, and this was accompanied by an approximately 20% reduction in PSGL-1 glycan content. A 70% to 85% reduction in HECA-452 binding epitope and N-acetyl lactosamine content in PSGL-1 was also noted on 4F-GalNAc addition. Intravenous 4F-GalNAc infusion reduced leukocyte migration to the peritoneum in a murine model of thioglycolate-induced peritonitis. Thus, the compound has pharmacologic activity. Overall, the data suggest that 4F-GalNAc may be applied as a metabolic inhibitor to reduce O-linked glycosylation, sialyl Lewis-X formation, and leukocyte adhesion via the selectins. (Blood. 2010;115:1303-1312) IntroductionThe binding of adhesion molecules belonging to the selectin family to carbohydrate ligands facilitates the adhesion of blood leukocytes to activated endothelial cells, platelets, and other leukocytes in the human vasculature. 1,2 Such molecular interactions play an important role in regulating leukocyte recruitment at sites of inflammation, cancer metastasis, and various cardiovascular disorders. 3 Whereas numerous glycoproteins and glycolipids participate in selectin-mediated cell adhesion, interactions with carbohydrate epitopes expressed on the leukocyte glycoprotein P-selectin glycoprotein ligand-1 (PSGL-1, CD162) are particularly important because this ligand binds all 3 members of the selectin family (E-, P-, and L-selectin) with high affinity and under fluid flow conditions. Structural analysis of the glycans of PSGL-1 expressed on human promyelocytic leukemia HL-60 cells reveals that PSGL-1 is predominantly composed of core-2 based O-linked glycans. 4,5 The prototypic selectin-binding carbohydrate structure sialyl Lewis-X (NeuAc␣2,3Gal␤1,4(Fuc␣1,3)GlcNAc-, sLe X ; Figure 1A) is expressed on 2% to 14% of these O-glycans.There is active interest in developing antagonists that control/ block selectin-mediated cell adhesion using either competitive inhibitors or metabolic inhibitors. Competitive inhibitors attempt to block cell adhesion by regulating the ligand-binding epitope of either the selectin or its primary counter-receptor PSGL-1. Antagonists used for such inhibition include the tetrasaccharide sLe X and its glycomimetics, 6 humanized antibodies direc...

Metabolic Inhibition of Sialyl-Lewis X Biosynthesis by 5-Thiofucose Remodels the Cell Surface and Impairs Selectin-Mediated Cell Adhesion

“…Indeed, it is possible that both factors contribute to the observed decreases in sLe X levels. A second mechanism by which sLe X levels on proteins could be indirectly decreased is by 5T-Fuc causing large changes in the N-glycan structures (16,17). To address this question, we subjected both the intracellular and secreted (42) glycoproteins, which we also find show decreased sLe X presentation upon treatment with 5T-Fuc (Fig.…”

“…is a human leukocyte cell line expressing both Le X (48) and sLe X (15,16). 5T-Fuc treatment of these cells also reduced their reactivity toward Fuc (AAL)-, sLe X (CSLEX-1)-, and Le X -specific (FH-2) lectins and mAbs (Fig.…”

“…A promising alternative toward disrupting sLe X biosynthesis involves diverting the efforts of relevant glycosyltransferases by using acceptor decoys (14,15). Finally, metabolic engineering of cells using modified sugar analogues that get incorporated into the growing structure and block completion of the biosynthesis of sLe X have also shown promise (16,17). Such approaches to perturbing glycan struc-ture at early stages or diverting glycosyltransferase activity, however, can significantly alter the structures of glycans produced by cells (16) and, in some cases, induce other metabolic side effects (17).…”

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

“…[46][47][48] In this regard, the current study identifies ST3Gal-4 as a key regulator of human myeloid cell adhesion. Thus, small molecules that can perturb this enzyme activity may be effective anti-inflammatory or antiadhesive drugs.…”

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

“…Programs utilizing ImageJ (National Institutes of Health) and the MATLAB image processing toolbox (Natick, MA) were written for data analysis. Analysis methods were similar to prior work (23,24). Because the number of rolling cells reached steady state within 1 min after starting injection of cells, data analysis was performed in the window between 1 and 3 min.…”