Insights into the Molecular Basis of Leukocyte Tethering and Rolling Revealed by Structures of P- and E-Selectin Bound to SLeX and PSGL-1

Somers, W.S.; Tang, Jin; Shaw, Gray D.; Camphausen, Raymond T.

doi:10.1016/s0092-8674(00)00138-0

Cited by 698 publications

(784 citation statements)

References 42 publications

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“…Reagents-The chimeric form of P-selectin-IgG Fc (P-selectin) consisting of the lectin, epidermal growth factor, and nine consensus repeat domains for human P-selectin linked to each arm of human IgG1 was a generous gift from Dr. Ray Camphausen of Wyeth External Research (Cambridge, MA) (20). All of the other reagents were purchased from Sigma unless otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

Single Molecule Characterization of P-selectin/Ligand Binding

Hanley

McCarty²,

Jadhav³

et al. 2003

Journal of Biological Chemistry

176

210

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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...

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Section: Methodsmentioning

confidence: 99%

Single Molecule Characterization of P-selectin/Ligand Binding

Hanley

McCarty²,

Jadhav³

et al. 2003

Journal of Biological Chemistry

176

210

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“…Requiring Ca 2ϩ like native PSGL-1 interactions, the 19-aa N terminus has been shown to be sufficient for binding to P-selectin provided that a fucosylated and sialylated oligosaccharide (related to sLe X ) is present at Thr-16 (14). Moreover, the binding affinity is enhanced to varying degrees by sulfation (13,18,19) of three tyrosine residues at positions 5, 7, and 10. Therefore, we tested strengths of P-selectin bonds to two 19-mer polypeptides linked with sLe X at theThr-16 position (13), one fully sulfated at all three tyrosines (called SGP-3) and the other with no tyrosine sulfation (called GP-1), and also to the simple carbohydrate b-sLe X .…”

Section: Tests Of P-selectin-psgl-1 Bonds Under Steady Ramps After a mentioning

confidence: 99%

“…Revealing a mechanical switching between pathways, a quick initial jump to a small force blocks the low-impedance pathway and allows bonds to fail only along the high-impedance pathway, even if then pulled very slowly. Replacing PSGL-1 by variants of its 19-aa N terminus (13) and by the crucial tetrasaccharide sialyl Lewis X (sLe X ) produces significant changes in the impedances to dissociation along the two pathways.…”

mentioning

confidence: 99%

Mechanical switching and coupling between two dissociation pathways in a P-selectin adhesion bond

Evans

Leung

Heinrich

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

306

454

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Many biomolecular bonds exhibit a mechanical strength that increases in proportion to the logarithm of the rate of force application. Consistent with exponential decrease in bond lifetime under rising force, this kinetically limited failure reflects dissociation along a single thermodynamic pathway impeded by a sharp free energy barrier. Using a sensitive force probe to test the leukocyte adhesion bond P-selectin glycoprotein ligand 1 (PSGL-1)-P-selectin, we observed a linear increase of bond strength with each 10-fold increase in the rate of force application from 300 to 30,000 pN͞sec, implying a single pathway for failure. However, the strength and lifetime of PSGL-1-P-selectin bonds dropped anomalously when loaded below 300 pN͞sec, demonstrating unexpectedly faster dissociation and a possible second pathway for failure. Remarkably, if first loaded by a ''jump'' in force to 20 -30 pN, the bonds became strong when subjected to a force ramp as slow as 30 pN͞sec and exhibited the same single-pathway kinetics under all force rates. Applied in this way, a new ''jump͞ramp'' mode of force spectroscopy was used to show that the PSGL-1-P-selectin bond behaves as a mechanochemical switch where force history selects between two dissociation pathways with markedly different properties. Furthermore, replacing PSGL-1 by variants of its 19-aa N terminus and by the crucial tetrasaccharide sialyl Lewis X produces dramatic changes in the failure kinetics, suggesting a structural basis for the two pathways. The two-pathway switch seems to provide a mechanism for the ''catch bond'' response observed recently with PSGL-1-P-selectin bonds subjected to smallconstant forces.N oncovalent interactions among large multidomain proteins underlie most adhesive functions in biology. Well known prototypes are the complexes formed between the selectin family of adhesion receptors, e.g., P-selectin expressed on activated endothelial cells or platelets, and their glycosylated ligands, e.g., the leukocyte mucin P-selectin glycoprotein ligand 1 (PSGL-1). Referred to as ''bonds,'' these interactions transiently interrupt rapid transport of leukocytes in blood flow and enable cells to perform a rolling exploration of vessel walls during the inflammatory response (1, 2). Most of our knowledge about how selectin bonds behave under stress has come from observing the decay in a number of receptor-bearing particles (cells or microspheres) tethered to walls by adhesive ligands in flow chambers. Held under nearly constant ''force clamp'' conditions, particles tethered by ligand͞selectin bonds release at progressively faster rates with increasing shear forces in high flow (3-5) but, at the same time, exhibit an unexpected shear threshold in slow flow below which particles also detach very quickly (6, 7). Recently tested by both flow chamber and atomic force microscope (AFM) techniques in a similar force clamp mode, the lifetimes of PSGL-1-P-selectin attachments were found to first increase with initial application of small forces before crossing over to de...

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“…Each of these two ligands requires sLe x for binding, with the difference that ESL-1 requires sLe x on N-glycans (18), whereas PSGL-1 requires it on O-glycans that carry a core-2 branch (21,24). In addition, PSGL-1 requires sulfation of the tyrosine residues within its N terminus for binding to P-selectin (25)(26)(27)(28)(29). The physiological relevance of PSGL-1 in leukocyte extravasation is well established (23).…”

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confidence: 99%

The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

Huang

Laskowska²,

Vestweber

et al. 2002

Journal of Biological Chemistry

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Fuc-TIV and Fuc-TVII are the two ␣(1, 3)-fucosyltransferases in myeloid cells responsible for the biosynthesis of sialyl Lewis X (sLe x ), the minimal ligand structure for the selectins. We have compared the ability of Fuc-TIV and Fuc-TVII to generate sLe x -like epitopes in transfected Chinese hamster ovary (CHO)-Pro ؊ 5 cells expressing the P-selectin glycoprotein ligand-1 and the core-2 branching enzyme C2GnT. We found that mouse Fuc-TIV and Fuc-TVII can generate similar levels of cell surface sLe x . Surprisingly however, Fuc-TIV-generated sLe x was resistant to proteinase K and trypsin treatment and could be removed from cells by delipidation with chloroform/methanol, whereas 80 -90% of Fuc-TVIIgenerated sLe x was protease-sensitive, and most of it resistant to delipidation. Despite similar levels of sLe x on the cell surface, Fuc-TVII transfectants adhered to immobilized E-selectin-IgG under static and flow conditions better than Fuc-TIV transfectants. Binding was mainly protease sensitive, indicating that glycoproteins were more efficient ligands than glycolipids. In summary, we conclude that the two fucosyltransferases differ in their in vivo specificity for acceptor substrates with Fuc-TVII generating sLe x preferentially on glycoproteins, whereas most of the Fuc-TIV-generated sLe x is found on glycolipids. Interestingly, the non-catalytic portion of Fuc-TIV in a Fuc-TIV/VII chimeric enzyme mediated the specificity for glycolipid substrates.The three known selectins, L-, E-, and P-selectin are cell adhesion molecules that initiate interactions between leukocytes and endothelial cells during leukocyte extravasation (1). The minimal ligand structure for all three selectins is the tetrasaccharide sialyl Lewis X (sLe x ). 1

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Insights into the Molecular Basis of Leukocyte Tethering and Rolling Revealed by Structures of P- and E-Selectin Bound to SLeX and PSGL-1

Cited by 698 publications

References 42 publications

Single Molecule Characterization of P-selectin/Ligand Binding

Single Molecule Characterization of P-selectin/Ligand Binding

Mechanical switching and coupling between two dissociation pathways in a P-selectin adhesion bond

The α(1,3)-Fucosyltransferase Fuc-TIV, but Not Fuc-TVII, Generates Sialyl Lewis X-like Epitopes Preferentially on Glycolipids

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