title: Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin.
abstract: P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galbeta1-3GalNAcalpha1-Se r/Thr); they lack core 2 O-linked glycans (Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAcalpha1 -Ser/Thr) because they do not express the core 2 beta1 6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack alpha1 3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha1 3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.
authors.affiliation: W. K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, 73140, USA.
authors.forename: F
authors.initials: F
authors.lastname: Li
authors.forename: P P
authors.initials: PP
authors.lastname: Wilkins
authors.forename: S
authors.initials: S
authors.lastname: Crawley
authors.forename: J
authors.initials: J
authors.lastname: Weinstein
authors.forename: R D
authors.initials: RD
authors.lastname: Cummings
authors.forename: R P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017931:DNA Primers; D019040:E-Selectin / Q000378:metabolism*; D005647:Fucosyltransferases / Q000096:biosynthesis; D006031:Glycosylation; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D019007:P-Selectin / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013737:Testis / Q000378:metabolism; D014162:Transfection
chemical_list: D017931:DNA Primers; D019040:E-Selectin; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D019007:P-Selectin; C084882:P-selectin ligand protein; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D005647:Fucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C031692:galactoside 3-fucosyltransferase