title: The P-selectin glycoprotein ligand from human neutrophils displays sialylated, fucosylated, O-linked poly-N-acetyllactosamine.
abstract: We previously demonstrated that P-selectin binds with high affinity to a trace, homodimeric glycoprotein ligand on human myeloid cells. The ligand carries the sialyl Lewis x (sLe(x)) epitope, a limited number of N-linked glycans, and clustered, sialylated O-linked glycans. In this study we demonstrate that the polypeptide component of this ligand is identical to that of P-selectin glycoprotein ligand-1 (PSGL-1), a molecule recently identified by expression cloning from a human myeloid cell cDNA library. We have examined the effects of glycosidases on purified, radioiodinated PSGL-1 from human neutrophils to further characterize the structure and function of the attached oligosaccharides. We found that PSGL-1 had poly-N-acetyllactosamine, only some of which could be removed with endo-beta-galactosidase. The majority of the Le(x) and sLe(x) structures were on endo-beta-galactosidase-sensitive chains. Peptide:N-glycosidase F (PNGaseF) treatment removed at least two of the three possible N-linked oligosaccharides from PSGL-1. Expression of Le(x) and sLe(x) was not detectably altered by PNGaseF digestion, indicating that these structures were primarily on O-linked poly-N-acetyllactosamine. Endo-beta-galactosidase-treated PSGL-1 retained the ability to bind to P-selectin, suggesting that some of the oligosaccharides recognized by P-selectin were either on enzyme-resistant poly-N-acetyllactosamine or on chains which lack poly-N-acetyllactosamine. PNGaseF treatment did not affect the ability of PSGL-1 to bind to P-selectin, demonstrating that the oligosaccharides required for P-selectin recognition are O-linked. PSGL-1 also bound to E-selectin, but with at least 50-fold lower affinity than to P-selectin. These data suggest that PSGL-1 from human neutrophils displays complex, sialylated, and fucosylated O-linked poly-N-acetyllactosamine that promote high affinity binding to P-selectin, but not to E-selectin.
authors.affiliation: Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City.
authors.forename: K L
authors.initials: KL
authors.lastname: Moore
authors.forename: S F
authors.initials: SF
authors.lastname: Eaton
authors.forename: D E
authors.initials: DE
authors.lastname: Lyons
authors.forename: H S
authors.initials: HS
authors.lastname: Lichenstein
authors.forename: R D
authors.initials: RD
authors.lastname: Cummings
authors.forename: R P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006224:Cricetinae; D019040:E-Selectin; D005643:Fucose / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009504:Neutrophils / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides / Q000378:metabolism; D019007:P-Selectin; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; D019040:E-Selectin; D006023:Glycoproteins; D007106:Immune Sera; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D019007:P-Selectin; D010455:Peptides; D010980:Platelet Membrane Glycoproteins; D011134:Polysaccharides; D012794:Sialic Acids; D005643:Fucose; C037199:poly-N-acetyllactosamine; D006026:Glycoside Hydrolases; D019158:N-Acetylneuraminic Acid