Glycosulfopeptides with O-Glycans Containing Sialylated and Polyfucosylated Polylactosamine Bind with Low Affinity to P-selectin

Abstract

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO 3 ؊ ) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe x ). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO 3 ؊ residues and a short, monofucosylated C2-OsLe x bound to P-selectin with high affinity (K d ϳ650 nM). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6 and GSP-6؆, with three TyrSO 3 ؊ residues and either difucosylated polylactosamine (C2-O-Le x -sLe x ) or trifucosylated polylactosamine (C2-O-Le x -Le x -sLe x ). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6 and GSP-6؆ bound to P-selectin with low affinity (K d ϳ37 M for GSP-6 and K d ϳ50 M for GSP-6؆). Binding of GSP-6 and GSP-6؆ to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6 and GSP-6؆. These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.

Dimeric Galectin-1 Binds with High Affinity to α2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans

“…We developed a fluorescence-based solid-phase assay for P-and L-selectin binding to immobilized synthetic glyco(sulfo)peptides modeled after human PSGL-1 (51,80) and observed that the relative binding affinities between selectins and glyco(sulfo)peptides as determined by the solid-phase assay were in agreement with the binding affinities determined by other methods. Our results predict that these differences in lectin behavior probably reflect the need of dGal-1 to have extended glycans for recognition when the glycans are immobilized, and this need may reflect the biological situation where PL chains are probably extended from cell-surface glycoconjugates.…”

“…The core 2-based O-glycans of GP-4, GP-4Ј, GP-4Љ, and GP-4ٞ were synthesized enzymatically as described (50,51). The purified glycopeptides were biotinylated through their N terminus using EZ-Link TM NHS-LC-LC-Biotin (succinimidyl 6Ј-(biotinamido)-6-hexanamidohexanoate; Pierce).…”

“…In any case, the ability of dGal-1 to bind directly to core 2-based O-glycans containing simple and extended PL units has not been studied. Here, we used a series of glycopeptides modeled after the N terminus of human PSGL-1 containing an O-glycan at a single Thr residue to study the role of core 2 O-glycan and extended core 2 structures in Gal-1 binding (51). Glycopeptides GP-4, GP-4Ј, GP-4Љ, and GP-4ٞ contain one, two, three, and four linear LN repeats on the core 2 branch, respectively.…”

α2,3‐Sialyltransferase‐IV is essential for L‐selectin ligand function in inflammation

“…These findings suggest that lack of a2,3-sialylation of murine PSGL-1 by ST3Gal-IV does not affect P-selectin binding to PSGL-1, but severely curtails the L-selectin ligand function of PSGL-1. In vitro studies investigating P-selectin-and L-selectin-IgG chimera binding to a synthetic glycosulfopeptide modeled after the N terminus of human PSGL-1 containing three clustered tyrosine sulfates and a short core 2 decorated and monofucosylated sLe x demonstrated that desialylation of the glycosulfopeptide was followed by a considerable reduction of glycosulfopeptide binding to P-selectin-and L-selectin-IgG [31,32]. Since the requirements for P-selectin and L-selectin binding to the N-terminal binding site on PSGL-1 are probably similar in the murine system, it is likely that other ST3Gal such as ST3Gal-VI contribute to the synthesis of the crucial sLe x containing core 2-dependent O-glycan at the N terminus of PSGL-1 in the absence of ST3Gal-IV.…”

Structurally Distinct Requirements for Binding of P-selectin Glycoprotein Ligand-1 and Sialyl Lewis x to Anaplasma phagocytophilum and P-selectin

“…Biotinylation of Glycoconjugates and Proteins-2-GP-1, 2-GSP-1, 2-GP-6, 2-GSP-6, and sPSGL-1 (20 g) were biotinylated at their Cterminal cysteine residues by incubation with 4-mM N- [6-(biotinamido)hexyl]-3-(2Ј-pyridyldithio)propionamide (Pierce) as described (22). Biotinylated sPSGL-1 was dialyzed against PBS to remove free biotin (32).…”