title: Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin.
abstract: P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded, homodimeric mucin ( approximately 250 kDa) on leukocytes that binds to P-selectin on platelets and endothelial cells during the initial steps in inflammation. Because it has been proposed that only covalently dimerized PSGL-1 can bind P-selectin, we investigated the factors controlling dimerization of PSGL-1 and re-examined whether covalent dimers are required for binding its P-selectin. Recombinant forms of PSGL-1 were created in which the single extracellular Cys (Cys(320)) was replaced with either Ser (C320S-PSGL-1) or Ala (C320A-PSGL-1). Both recombinants migrated as monomeric species of approximately 120 kDa under both nonreducing and reducing conditions on SDS-polyacrylamide gel electrophoresis. P-selectin bound similarly to cells expressing either wild type or mutated forms of PSGL-1 in both flow cytometric and rolling adhesion assays. Unexpectedly, chemical cross-linking studies revealed that both C320S- and C320A-PSGL-1 noncovalently associate in the plasma membrane and cross-linking generates dimeric species. Chimeric recombinants of PSGL-1 in which the transmembrane domain in PSGL-1 was replaced with the transmembrane domain of CD43 (CD43TMD-PSGL-1) could not be chemically cross-linked, suggesting that residues within the transmembrane domain of PSGL-1 are required for noncovalent association. Cells expressing CD43TMD-PSGL-1 bound P-selectin. To further address the ability of P-selectin to bind monomeric derivatives of PSGL-1, intact HL-60 cells were trypsin-treated, which generated a soluble approximately 25-kDa NH(2)-terminal fragment of PSGL-1 that bound to immobilized P-selectin. Because N-glycosylation of PSGL-1 hinders trypsin cleavage, a recombinant form of PSGL-1 was generated in which all three potential N-glycosylation sites were mutated (DeltaN-PSGL-1). Cells expressing DeltaN-PSGL-1 bound P-selectin, and trypsin treatment of the cells generated NH(2)-terminal monomeric fragments (<10 kDa) of PSGL-1 that bound to P-selectin. These results demonstrate that Cys(320)-dependent dimerization of PSGL-1 is not required for binding to P-selectin and that a small monomeric fragment of PSGL-1 is sufficient for P-selectin recognition.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Epperson
authors.forename: K D
authors.initials: KD
authors.lastname: Patel
authors.forename: R P
authors.initials: RP
authors.lastname: McEver
authors.forename: R D
authors.initials: RD
authors.lastname: Cummings
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001696:Biomechanical Phenomena; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002448:Cell Adhesion / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D019281:Dimerization; D006031:Glycosylation; D018922:HL-60 Cells; D006801:Humans; D051917:Leukosialin; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism*; D010812:Physical Stimulation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012795:Sialoglycoproteins / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D051917:Leukosialin; D008024:Ligands; D008562:Membrane Glycoproteins; D009077:Mucins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011994:Recombinant Proteins; C465014:SPN protein, human; D012795:Sialoglycoproteins