Structural and functional characterization of monomeric soluble P-selectin and comparison with membrane P-selectin.
P-selectin is an adhesion receptor for leukocytes on thrombin-activated platelets and endothelial cells. It contains a NH2-terminal carbohydrate-recognition domain, an epidermal growth factor motif, nine consensus repeats, a transmembrane domain, and a cytoplasmic tail. We expressed two soluble forms of P-selectin, one truncated after the ninth repeat (tPS) and the other lacking the transmembrane domain due to alternative RNA splicing (asPS). When visualized by electron microscopy, each was a monomeric rod-like structure with a globular domain at one end, whereas membrane P-selectin (mPS) from platelets formed rosettes with the globular domains facing outward. Sedimentation velocity and equilibrium studies confirmed that tPS and asPS were asymmetric monomers, whereas mPS was oligomeric. HL-60 cells adhered to immobilized tPS and asPS, although less efficiently than to mPS. 125I-Labeled tPS and asPS bound to approximately 25,000 sites/neutrophil and approximately 36,000 sites/HL-60 cell with an apparent Kd of 70 nM. Treatment of HL-60 cells with O-sialoglycoprotease eliminated the binding sites for asPS. We conclude that 1) P-selectin is a rigid, asymmetric protein; 2) monomeric soluble P-selectin binds to high affinity ligands with sialylated O-linked oligosaccharides on leukocytes; and 3) oligomerization of mPS enhances its avidity for leukocytes.
Ushiyama
S
S
W. K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
Laue
T M
TM
Moore
K L
KL
Erickson
H P
HP
McEver
R P
RP
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure; D002478:Cells, Cultured; D004247:DNA; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology* / Q000648:ultrastructure; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D012995:Solubility
D015815:Cell Adhesion Molecules; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D004247:DNA