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J. Biol. Chem., Vol. 278, Issue 12, 10556-10561, March 21, 2003
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From the 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
Single Molecule Characterization of P-selectin/Ligand
Binding*,
§,§,,,¶
, and**
Department of Chemical and Biomolecular
Engineering, ** Department of Biomedical Engineering, and
¶ Molecular Biophysics Program, The Johns Hopkins University,
Baltimore, Maryland 21218
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 s1. 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.
*
This work was supported by a Whitaker Foundation grant and
National Science Foundation Grant CTS0210718.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Fig. 1.
§
Both authors contributed equally to this work.
To whom correspondence may be addressed. Dept. of Chemical and
Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles
St., Baltimore, MD 21218. Tel.: 410-516-6290, Fax: 410-516-5510;
E-mail: kkonsta1@jhu.edu (K. K.) or Tel.: 410-516-7006; Fax:
410-516-5510; E-mail: wirtz@jhu.edu (D. W.).
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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