N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin.
P-selectin binds to the N-terminal region of human P-selectin glycoprotein ligand-1 (PSGL-1). For optimal binding, this region requires sulfation on 3 tyrosines and specific core-2 O-glycosylation on a threonine. P-selectin is also thought to bind to the N terminus of murine PSGL-1, although it has a very different amino acid sequence than human PSGL-1. Murine PSGL-1 has potential sites for sulfation at Tyr13 and Tyr15 and for O-glycosylation at Thr14 and Thr17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that coexpressed the glycosyltransferases required for binding to P-selectin. The cells were assayed for binding to fluid-phase P-selectin and for tethering and rolling on P-selectin under flow. In both assays, substitution of Tyr13 or Thr17 markedly diminished, but did not eliminate, binding to P-selectin. In contrast, substitution of Tyr15 or Thr14 did not affect binding. Substitution of all 4 residues eliminated binding. Treatment of cells with chlorate, an inhibitor of sulfation, markedly reduced binding of wild-type PSGL-1 to P-selectin but did not further decrease binding of PSGL-1 with substitutions of both tyrosines. These data suggest that sulfation of Tyr13 and O-glycosylation of Thr17 are necessary for murine PSGL-1 to bind optimally to P-selectin. Because it uses only one tyrosine, murine PSGL-1 may rely more on other peptide components and O-glycosylation to bind to P-selectin than does human PSGL-1.
Xia
Lijun
L
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Ramachandran
Vishwanath
V
McDaniel
J Michael
JM
Nguyen
Kiem N
KN
Cummings
Richard D
RD
McEver
Rodger P
RP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D005647:Fucosyltransferases / Q000378:metabolism; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D013431:Sulfates; D013912:Threonine; D014162:Transfection; D014443:Tyrosine
D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D013431:Sulfates; D013912:Threonine; D014443:Tyrosine; D005647:Fucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase; C031692:galactoside 3-fucosyltransferase