Single Molecule Characterization of P-selectin/Ligand Binding

Abstract

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 bloodborne 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 Pselectin binding to a novel O-linked, sialylated proteasesensitive 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 Pselectin/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.Cellular adhesion mediated by biological macromolecules and their respective ligands plays an essential role in a number of diverse biological phenomena including inflammation and cancer metastasis. Leukocyte recruitment to sites of infection is regulated by highly specific receptor/ligand interactions that allow leukocytes to first tether and roll on activated endothelium under hydrodynamic shear and then firmly adhere prior to extravazation into the tissue space. These stages are mediated via three distinct classes of adhesion molecules: the selectins, integrins, and immunoglobulins (1-3). Accumulating evidence suggests that tumor cell arrest in the microcirculation is also mediated through receptor/ligand interactions between tumor cells and the vascular endothelium in a manner analogous to leukocyte recruitment (4 -6). Both processes involve highly regulated molecular events that rely on the local circulatory hemodynamics and the mechanical and kinetic properties of participating adhesive molecular groups, which have yet to be characterized at the single molecule level.The involvement of P-selectin is critical within immune system functioning. P-selectin, a cell-surface glycoprotein expressed on activated endothelial cells and platelets, supports leukocyte tethering and rolling in response to inflammatory signals by interac...

Single-molecule binding of CD44 to fibrin versus P-selectin predicts their distinct shear-dependent interactions in cancer

“…We have previously applied force spectroscopy to characterize the interactions between purified selectins immobilized on a cantilever tip and ligands expressed on whole cells (Hanley et al, 2003;Hanley et al, 2004). In view of recent observations showing that CD44v accounts for ~50% of LS174T colon carcinoma binding to P-selectin (Napier et al, 2007;Thomas et al, 2008), we developed a methodology to study binding interactions between purified molecules rather than whole cells.…”

“…2C). To further ensure that most binding events were mediated by a single receptorligand pair, a low probability (Hanley et al, 2003) of binding (15-30%) was achieved by decorating the cantilever and lipid bilayers with dilute fibrin and CD44v concentrations, respectively. The specificity of CD44v-fibrin binding was demonstrated by incubating the CD44v-incorporated lipid vesicles with the function-blocking 1904 Journal of Cell Science 124 (11) anti-CD44 monoclonal antibody Hermes-1, which drastically reduced the frequency of binding events from ~20% down to <5% (Fig.…”

“…4C). The Bell model parameters were validated for both receptor-ligand pairs by performing Monte Carlo simulations of bond rupture (Hanley et al, 2003;Hanley et al, 2004) under constant loading rates (Fig. 4D).…”

“…To test our hypothesis, we used a micro-manipulation method based on single-molecule force spectroscopy (Hanley et al, 2003;Hanley et al, 2004). To this end, P-selectin or fibrin immobilized onto a cantilever tip was brought in contact for prescribed contact durations with either CD44v expressed on immobilized colon carcinoma cells or immunopurified CD44v incorporated into lipid vesicles and layered on a polyethyleneimine (PEI)-cushioned glass slide.…”

The Role of Glycocalyx in Nanocarrier-Cell Adhesion Investigated Using a Thermodynamic Model and Monte Carlo Simulations

“…19,33 This experiment is repeated a number of times to give a rupture force distribution at a given loading rate.…”