Single Molecule Characterization of P-selectin/Ligand Binding

• Published in: The Journal of biological chemistry Vol. 278; no. 12; pp. 10556 - 10561
• Main Authors: Hanley, William, McCarty, Owen, Jadhav, Sameer, Tseng, Yiider, Wirtz, Denis, Konstantopoulos, Konstantinos
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 21-03-2003
• Subjects: Actins - metabolism; Colonic Neoplasms - metabolism; Humans; Ligands; Membrane Glycoproteins - metabolism; Monte Carlo Method; Neutrophils - metabolism; P-Selectin - metabolism; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M213233200

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes
(PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall
adhesiveness ( i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands
is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic
colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells
rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin
ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate
of 0.20 s −1 . The tensile strength of P-selectin binding to a novel O- linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate
was 2.78 s −1 . Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T
ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences
in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas
to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.