Identification of N-terminal Residues on P-selectin Glycoprotein Ligand-1 Required for Binding to P-selectin

Abstract

The major high affinity ligand for P-selectin on human leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). To bind P-selectin, PSGL-1 must be modified with tyrosine sulfate and sialylated, fucosylated, core-2 O-glycan(s). The required sites for these modifications on full-length PSGL-1 have not been defined. The N-terminal region of mature PSGL-1, which begins at residue 42, includes tyrosines at residues 46, 48, and 51, plus potential sites for Thr-linked O-glycans at residues 44 and 57. We expressed full-length PSGL-1 constructs with substitutions of these residues in transfected Chinese hamster ovary cells. The cells were co-transfected with cDNAs for the glycosyltransferases required to construct sialylated and fucosylated, core-2 O-glycans on PSGL-1. The transfected cells were assayed for their abilities to bind fluid-phase P-selectin and to support rolling adhesion of pre-B cells expressing P-selectin under hydrodynamic flow. In both assays, substitution of Thr-57 with alanine eliminated binding of PSGL-1 to P-selectin without affecting sulfation of PSGL-1, whereas substitution with serine, to which an O-glycan might also be attached, did not affect binding. Binding was not altered by substituting alanines for the two amino acids on either side of Thr-57, or by substituting alanine for Thr-44. Substitution of all three tyrosines with phenylalanines markedly reduced sulfation and prevented binding to P-selectin. However, all constructs in which one or two tyrosines were replaced with phenylalanines bound P-selectin. These results suggest that full-length PSGL-1 requires an O-glycan attached to Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.The selectins are Ca 2ϩ

Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region

“…Since substitution of Y26 with F or A virtually abolished sulfation of the receptor under experimental conditions, it is likely that Y28 is not sulfated. Substitution of one of three sulfated tyrosines of P-selectin glycoprotein ligand-1 did not lead to lack of sulfation of two other tyrosines taking part in interaction with Pselectin, and only substitution of all these tyrosines prevented binding of the ligand (60).…”

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

“…Regulation of human myeloid cell binding to P-and L-selectin A core 2-based O-linked glycan bearing the sLe X epitope, located at the N terminus of PSGL-1, at Thr-57 in humans, 35,39 is the primary ligand for L-and P-selectin. Studies with knockout mice show that the biosynthesis of this O-glycan is initiated by the formation of the core 1 structure (Galb1,3GalNAca1-Thr) catalyzed by the core-1 b1,3galactosyltransferase/T-synthase.…”

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

“…Cells-Transfected Chinese hamster ovary (CHO) cells coexpressing human PSGL-1 with FTVII or with FTVII and core-2 ␤1-6-N-acetylglucosaminyltransferase I (core2GlcNAcT-I) were prepared as described (34 -36). Transfected CHO cells expressing human PSGL-1 in which Thr-16 was substituted with alanine (T16A, also known as T57A in an earlier numbering system) were prepared as described (35). HL-60 cells were maintained in RPMI 1640 containing 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin.…”