Human L-selectin preferentially binds synthetic glycosulfopeptides modeled after endoglycan and containing tyrosine sulfate residues and sialyl Lewis x in core 2 O-glycans

Abstract

Endoglycan is a mucin-like glycoprotein expressed by endothelial cells and some leukocytes and is recognized by L-selectin, a C-type lectin important in leukocyte trafficking and extravasation during inflammation. Here, we show that recombinant L-selectin and human T lymphocytes expressing L-selectin bind to synthetic glycosulfopeptides (GSPs). These synthetic glycosulfopeptides contain 37 amino acid residues modeled after the N-terminus of human endoglycan and contain one or two tyrosine sulfates (TyrSO(3)) along with a nearby core-2-based Thr-linked O-glycan with sialyl Lewis x (C2-SLe(x)). TyrSO(3) at position Y118 was more critical for binding than at Y97. C2-SLe(x) at T124 was required for L-selectin recognition. Interestingly, under similar conditions, neither L-selectin nor T lymphocytes showed appreciable binding to the sulfated carbohydrate epitope 6-sulfo-SLe(x). P-selectin also bound to endoglycan-based GSPs but with lower affinity than toward GSPs modeled after PSGL-1, the physiological ligand for P- and L-selectin that is expressed on leukocytes. These results demonstrate that TyrSO(3) residues in association with a C2-SLe(x) moiety within endoglycan and PSGL-1 are preferentially recognized by L-selectin.

Fig. 1

The structures of glyco(sulfo)peptides and sugars used in the present study. Tyrosine sulfate and O-glycan attachment sites are numbered according to the sequence of human endoglycan (EG) or PSGL-1 (P). The number in the parenthesis indicates the position of tyrosine sulfate residues, and the last number indicates the number of sugar residues. All peptides contain an extra C-terminal cysteine residue

Fig. 2

Binding of L-sel-Ig to immobilized glyco(sulfo)peptides in a fluorescence-based solid-phase assay at physiologic and low salt buffer. Biotinylated glyco(sulfo)peptides were immobilized on streptavidin-coated microtiter wells (A) 20 pmol/well; (B) 10 pmol/well). (A) L-sel-Ig (40 μg/ml) was incubated with the immobilized GSPs in physiologic salt buffer (20 mM MOPS, pH 7.5, 150 mM NaCl, 1% BSA, 0.05% Tween 20, 0.02% NaN₃) containing 2 mM CaCl₂ and 2 mM MgCl₂ (light gray bars) or 5 mM EDTA (dark gray bars). (B) L-sel-Ig (20 μg/ml) was incubated with the immobilized GSPs in low salt buffer (20 mM MOPS, pH 7.5, 50 mM NaCl, 1% BSA, 0.05% Tween 20, 0.02% NaN₃) containing 2 mM CaCl₂ and 2 mM MgCl₂ (light gray bars) or 5 mM EDTA (dark gray bars). Fluorescently labeled anti-human IgG was used to detect the bound L-sel-Ig. The inset shows binding of L-sel-Ig to immobilized P-GSP-6. All assays were performed in triplicate, and the results represent the mean ± SD of three determinations. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Binding of L-sel-Ig to immobilized glyco(sulfo)peptides and sugars in a fluorescence-based solid-phase assay at low salt buffer. Biotinylated glyco(sulfo)peptides and sugars were immobilized on streptavidin-coated microtiter wells (10 pmol/well). L-sel-Ig (A) 20 μg/ml or (B) 40 μg/ml was incubated with the immobilized ligands in low salt buffer (20 mM MOPS, pH 7.5, 50 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 1% BSA, 0.05% Tween 20, 0.02% NaN₃). Fluorescently labeled anti-human IgG was used to detect the bound L-sel-Ig. All assays were performed in triplicate, and the results represent the mean ± SD of three determinations

Fig. 4

Inhibition of L-sel-Ig binding to immobilized glyco(sulfo)peptides by anti-EG peptide antibodies. Biotinylated glyco(sulfo)peptides were immobilized on streptavidin-coated microtiter wells (5 pmol/well). Anti-QPP and anti-SGF antibodies (100 μg/ml) were first incubated with the immobilized GSPs in PBS containing 1% BSA and 0.05% Tween 20, and unbound antibodies were removed by washing. L-sel-Ig (10 μg/ml) was preincubated with fluorescently labeled anti-human IgG in low salt buffer (20 mM MOPS, pH 7.5, 50 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 1% BSA, 0.05% Tween 20, 0.02% NaN₃) before adding to the wells. Assays with anti-QPP and without an antibody were performed in triplicate, assays with anti-SGF were performed in duplicate, and the results represent the mean ± SD of three or two determinations, respectively

Fig. 5

Affinity chromatography of glyco(sulfo)peptides on immobilized L-sel-Ig at low salt buffer. The indicated radiolabeled glyco(sulfo)peptides were loaded into the L-sel-Ig column in low salt buffer (20 mM MOPS, 50 mM NaCl, 0.02% NaN₃, pH 7.5) containing 2 mM CaCl₂ and 2 mM MgCl₂ (A), (B) and (D) or 1 mM EDTA (C). In (A), dashed lines represent control experiments with the indicated glycosulfopeptides where chromatography was performed in buffer containing 1 mM EDTA. The arrow indicates the fraction number (21) where 10 mM EDTA was used to replace divalent cations in buffer. Experiments shown are representative of three independent experiments

Fig. 6

Equilibrium-binding affinity of L-sel-Ig for P-GSP-6, P-GP-6 and EG-GSP-6 in a fluorescence-based solid-phase assay at low salt buffer. Biotinylated glyco(sulfo)peptides were immobilized on streptavidin-coated microtiter wells (10 pmol/well). Various concentrations of L-sel-Ig were incubated with the immobilized (A) P-GSP-6, (B) P-GP-6 and (C) EG-GSP-6 in low salt buffer (20 mM MOPS, pH 7.5, 50 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 1% BSA, 0.05% Tween 20, 0.02% NaN₃). Fluorescently labeled anti-human IgG (40 μg/ml) was used to detect the bound L-sel-Ig. Assays were performed in triplicate (A) and (B) or in duplicate (C), and the results represent the mean ± standard error of the mean. Experiments shown are representative of three independent experiments

Fig. 7

Binding of human T lymphocytes to immobilized glyco(sulfo)peptides and sugars in a fluorescence-based solid-phase assay at physiologic buffer. Biotinylated glyco(sulfo)peptides and sugars were immobilized on streptavidin-coated microtiter wells (10 pmol/well). Purified, fluorescently labeled human T lymphocytes (A) 190,000 cells/well or (B) 65,000 cells/well were incubated with the immobilized ligands in Hank's balanced salt solution (with Ca² and Mg²) containing 1% BSA (light gray bars). (B) In control experiments, a function-blocking mAb to L-selectin, DREG-56 (20 μg/ml) (medium gray bars), or 5 mM EDTA (dark gray bars) were preincubated with the cells in HBSS before adding to the wells. The inset in (A) shows binding of T lymphocytes to immobilized P-GSP-6. All assays were performed in triplicate, and the results represent the mean ± SD of three determinations. Experiment shown in (A) is a representative of eight independent experiments using T cells isolated from five different donors. *P < 0.05; **P < 0.01

Fig. 8

Binding of P-sel-Ig to immobilized sugars and glyco(sulfo)peptides in a fluorescence-based solid-phase assay at physiologic buffer. Biotinylated sugars and glyco(sulfo)peptides were immobilized on streptavidin-coated microtiter wells (10 pmol/well). P-sel-Ig (20 μg/ml) was incubated with the immobilized GSPs in physiologic salt buffer (20 mM MOPS, pH 7.5, 150 mM NaCl, 1% BSA, 0.05% Tween 20, 0.02% NaN₃) containing 2 mM CaCl₂ and 2 mM MgCl₂ (light gray bars) or 5 mM EDTA (dark gray bars). Fluorescently labeled anti-human IgG was used to detect the bound P-sel-Ig. All assays were performed in triplicate, and the results represent the mean ± SD of three determinations. Experiment shown is a representative of three independent experiments. *P < 0.05; **P < 0.01