Binding of glycosulfopeptides to P-selectin requires stereospecific contributions of individual tyrosine sulfate and sugar residues

Abstract

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin on leukocytes that binds to selectins. P-selectin binds to an N-terminal region of PSGL-1 that requires sulfation of at least one of three clustered tyrosines (TyrSO3) and an adjacent core-2-based O-glycan expressing sialyl Lewis x (C2-O-sLex). We synthesized glycosulfopeptides (GSPs) modeled after this region of PSGL-1 to explore the roles of individual TyrSO3 residues, the placement of C2-O-sLex relative to TyrS03, the relative contributions of fucose and sialic acid on C2-O-sLex, and the function of the peptide sequence for binding to P-selectin. Binding of GSPs to P-selectin was measured by affinity chromatography and equilibrium gel filtration. 2-GSP-6, which has C2-O-sLex at Thr-57 and TyrSO3 at residues 46, 48, and 51, bound to P-selectin with high affinity (Kd ∼ 650 nM), whereas an isomeric trisulfated GSP containing C2-O-sLex at Thr-44 bound much less well. Non-sulfated glycopeptide (2-GP-6) containing C2-O-sLex at Thr-57 bound to P-selectin with ∼40-fold lower affinity (Kd∼25 μM). Proteolysis of 2-GP-6 abolished detectable binding of the residual C2-O-sLeX-Thr to P-selectin, demonstrating that the peptide backbone contributes to binding. Monosulfated and disulfated GSPs bound significantly better than non-sulfated 2-GP-6, but sulfation of Tyr-48 enhanced affinity (Kd ∼ 6 μM) more than sulfation of Tyr-46 or Tyr-51. 2-GSP-6 lacking sialic acid bound to P-selectin at ∼10% that of the level of the parent 2-GSP-6, whereas 2-GSP-6 lacking fucose did not detectably bind; thus, fucose contributes more than sialic acid to binding. Reducing NaC1 from 150 to 50 mM markedly enhanced binding of 2-GSP-6 to P-selectin (Kd ∼ 75 nM), demonstrating the charge dependence of the interaction. These results reveal a stereospecific interaction of P-selectin with PSGL-1 that includes distinct contributions of each of the three TyrS03 residues, adjacent peptide determinants, and fucose/sialic acid on an optimally positioned core-2 O-glycan.