Model glycosulfopeptides from P-selectin glycoprotein ligand-1 require tyrosine sulfation and a core 2-branched O-glycan to bind to L-selectin

L-selectin expressed on leukocytes is involved in lymphocyte homing to secondary lymphoid organs and leukocyte recruitment into inflamed tissue. L-selectin binds to the sulfated sialyl Lewis x (6-sulfo-sLe(x)) epitope present on O-glycans of various glycoproteins in high endothelial venules. In addition, L-selectin interacts with the dimeric mucin P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes. PSGL-1 lacks 6-sulfo-sLe(x) but contains sulfated tyrosine residues (Tyr-SO3) at positions 46, 48, and 51 and sLe(x) in a core 2-based O-glycan (C2-O-sLe(x)) on Thr at position 57. The role of tyrosine sulfation and core 2 O-glycans in binding of PSGL-1 to L-selectin is not well defined. Here, we show that L-selectin binds to a glycosulfopeptide (GSP-6) modeled after the extreme N terminus of human PSGL-1, containing three Tyr-SO3 and a nearby Thr modified with C2-O-sLe(x). Leukocytes roll on immobilized GSP-6 in an L-selectin-dependent manner, and rolling is dependent on Tyr-SO3 and C2-O-sLe(x) on GSP-6. The dissociation constant for binding of L-selectin to GSP-6, as measured by equilibrium gel filtration, is similar to5 muM. Binding is dependent on Tyr-SO3 residues as well as the sialic acid and fucose residues of C2-O-sLe(x). Binding to an isomeric glycosulfopeptide containing three Tyr-SO3 residues and a core 1-based O-glycan expressing sLe(x) was reduced by similar to90%. All three Tyr-SO3 residues of GSP-6 are required for high affinity binding to L-selectin. Low affinity binding to mono- and disulfated GSPs is largely independent of the position of the Tyr-SO3 residues, except for some binding preference for an isomer sulfated on both Tyr-48 and -51. These results demonstrate that L-selectin binds with high affinity to the N-terminal region of PSGL-1 through cooperative interactions with three sulfated tyrosine residues and an appropriately positioned C2-O-sLe(x) O-glycan.