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Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2001-11-0036.
Submitted November 26, 2001
Accepted August 16, 2002
N-terminal residues in murine P-selectin glycoprotein ligand-1 required for binding to murine P-selectin
Lijun Xia, Vishwanath Ramachandran, J M McDaniel, Kiem N Nguyen, Richard D Cummings, and Rodger P McEver*
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; Department of Biochemistry and Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA
Department of Biochemistry and Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
* Corresponding author; email: rodger-mcever{at}omrf.ouhsc.edu.
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 three 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 Tyr-13 and -15 and for O-glycosylation at Thr-14 and -17. We expressed murine PSGL-1 or constructs with substitutions of these residues in transfected Chinese hamster ovary cells that co-expressed 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 Tyr-13 or Thr-17 markedly diminished, but did not eliminate, binding to P-selectin. In contrast, substitution of Tyr-15 or Thr-14 did not affect binding. Substitution of all four 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 Tyr-13 and O-glycosylation of Thr-17 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.
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