A novel glycosulfopeptide binds to P-selectin and inhibits leukocyte adhesion to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on le ukocytes that binds selectins, The molecular features of PSGL-1 that determ ine this high affinity binding are unclear. Here we demonstrate the in vitr o synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-select in binding, GSP-6 contains three tyrosine sulfate (TyrSO(3)) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe(x) m otif at a specific Thr residue. GSP-6 binds tightly to immobilized P-select in, whereas glycopeptides lacking either TyrSO(3) or C2-O-sLe(x) do not det ectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GS P-6', which contains sLe(x) on an extended core 1-based O-glycan, does not bind immobilized P-selectin, Equilibrium gel filtration analysis revealed t hat GSP-6 binds to soluble P-selectin with a K-d of similar to 350 nM. GSP- 6 (<5 mu M) substantially inhibits neutrophil adhesion to P-selectin in vit ro, whereas free sLe(x) (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recom binant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide, Thi s approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of no vel drugs to treat inflammatory diseases involving P-selectin-mediated leuk ocyte adhesion.