Single molecule characterization of P-selectin/ligand binding

William Hanley, Owen McCarty, Sameer Jadhav, Yiider Tseng, Denis Wirtz, K Konstantopoulos

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)10556-10561
Number of pages6
JournalJournal of Biological Chemistry
Volume278
Issue number12
DOIs
StatePublished - Mar 21 2003

Fingerprint

P-Selectin
Ligands
Molecules
Carcinoma
Colon
Tensile Strength
Rupture
Blood
Tensile strength
Adhesiveness
Vascular Endothelium
Substrates
Post Translational Protein Processing
Platelets
Adhesives
Neutrophils
Peptide Hydrolases
Blood Platelets
Spectroscopy
Neoplasm Metastasis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Single molecule characterization of P-selectin/ligand binding. / Hanley, William; McCarty, Owen; Jadhav, Sameer; Tseng, Yiider; Wirtz, Denis; Konstantopoulos, K.

In: Journal of Biological Chemistry, Vol. 278, No. 12, 21.03.2003, p. 10556-10561.

Research output: Contribution to journalArticle

Hanley, William ; McCarty, Owen ; Jadhav, Sameer ; Tseng, Yiider ; Wirtz, Denis ; Konstantopoulos, K. / Single molecule characterization of P-selectin/ligand binding. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 12. pp. 10556-10561.
@article{8f850892db8b4d738301434789a37e22,
title = "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 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.",
author = "William Hanley and Owen McCarty and Sameer Jadhav and Yiider Tseng and Denis Wirtz and K Konstantopoulos",
year = "2003",
month = "3",
day = "21",
doi = "10.1074/jbc.M213233200",
language = "English (US)",
volume = "278",
pages = "10556--10561",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "12",

}

TY - JOUR

T1 - Single molecule characterization of P-selectin/ligand binding

AU - Hanley, William

AU - McCarty, Owen

AU - Jadhav, Sameer

AU - Tseng, Yiider

AU - Wirtz, Denis

AU - Konstantopoulos, K

PY - 2003/3/21

Y1 - 2003/3/21

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0013198341&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0013198341&partnerID=8YFLogxK

U2 - 10.1074/jbc.M213233200

DO - 10.1074/jbc.M213233200

M3 - Article

C2 - 12522146

AN - SCOPUS:0013198341

VL - 278

SP - 10556

EP - 10561

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 12

ER -