Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force.
Carbohydrate--protein bonds interrupt the rapid flow of leukocytes in the circulation by initiation of rolling and tethering at vessel walls. The cell surface carbohydrate ligands are glycosylated proteins like the mucin P-selectin glycoprotein ligand-1 (PSGL-1), which bind ubiquitously to the family of E-, P-, and L-selectin proteins in membranes of leukocytes and endothelium. The current view is that carbohydrate-selectin bonds dissociate a few times per second, and the unbinding rate increases weakly with force. However, such studies have provided little insight into how numerous hydrogen bonds, a Ca(2+) metal ion bond, and other interactions contribute to the mechanical strength of these attachments. Decorating a force probe with very dilute ligands and controlling touch to achieve rare single-bond events, we have varied the unbinding rates of carbohydrate--selectin bonds by detachment with ramps of force/time from 10 to 100,000 pN/sec. Testing PSGL-1, its outer 19 aa (19FT), and sialyl Lewis(X) (sLe(X)) against L-selectin in vitro on glass microspheres and in situ on neutrophils, we found that the unbinding rates followed the same dependence on force and increased by nearly 1,000-fold as rupture forces rose from a few to approximately 200 pN. Plotted on a logarithmic scale of loading rate, the rupture forces reveal two prominent energy barriers along the unbinding pathway. Strengths above 75 pN arise from rapid detachment (<0.01 sec) impeded by an inner barrier that requires a Ca(2+) bond between a single sLe(X) and the lectin domain. Strengths below 75 pN occur under slow detachment (>0.01 sec) impeded by the outer barrier, which appears to involve an array of weak (putatively hydrogen) bonds.
Evans
E
E
Department of Physics, University of British Columbia, Vancouver, BC, Canada V6T 2A6. evans@physics.ubc.ca
Leung
A
A
Hammer
D
D
Simon
S
S
D002118:Calcium / Q000737:chemistry; D005898:Glass / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D019041:L-Selectin / Q000737:chemistry* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry*; D009504:Neutrophils / Q000378:metabolism*; D013057:Spectrum Analysis
D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D019041:L-Selectin; D002118:Calcium