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-sLex) 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-sLex but contains sulfated tyrosine residues (Tyr-SO3)at positions 46, 48, and 51 and sLex in a core 2-based O-glycan (C2-O-sLex) 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-sLex. Leukocytes roll on immobilized GSP-6 in an L-selectin-dependent manner, and rolling is dependent on Tyr-SO3 and C2-O-sLex on GSP-6. The dissociation constant for binding of L-selectin to GSP-6, as measured by equilibrium gel filtration, is approximately 5 microm. Binding is dependent on Tyr-SO3 residues as well as the sialic acid and fucose residues of C2-O-sLex. Binding to an isomeric glycosulfopeptide containing three Tyr-SO3 residues and a core 1-based O-glycan expressing sLex was reduced by approximately 90%. 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-sLex O-glycan.
Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
Anne
A
Leppänen
Tadayuki
T
Yago
Vivianne I
VI
Otto
Rodger P
RP
McEver
Richard D
RD
Cummings
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005643:Fucose / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D019041:L-Selectin / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D019007:P-Selectin / Q000378:metabolism; D010455:Peptides; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D002352:Carrier Proteins; C121374:GSP-6 glycosulfopeptide; D006020:Glycopeptides; D006023:Glycoproteins; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D010455:Peptides; D011994:Recombinant Proteins; D019041:L-Selectin; D005643:Fucose; D014443:Tyrosine; D019158:N-Acetylneuraminic Acid