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Leukocyte adhesion to P-selectin on activated platelets and endothelial cells induces shedding of the P-selectin ectodomain into the circulation. Plasma soluble P-selectin (sP-selectin) is elevated threefold to fourfold in patients with cardiovascular disease. Circulating sP-selectin is thought to trigger signaling in leukocytes that directly contributes to inflammation and thrombosis. However, sP-selectin likely circulates as a monomer, and in vitro studies suggest that sP-selectin must dimerize to induce signaling in leukocytes. To address this discrepancy, we expressed the entire ectodomain of mouse P-selectin as a monomer (sP-selectin) or as a disulfide-linked dimer fused to the Fc portion of mouse immunoglobulin G (sP-selectin-Fc). Dimeric sP-selectin-Fc, but not monomeric sP-selectin, triggered integrin-dependent adhesion of mouse leukocytes in vitro. Antibody-induced oligomerization of sP-selectin or sP-selectin-Fc was required to trigger formation of neutrophil extracellular traps. Injecting sP-selectin-Fc, but not sP-selectin, into mice augmented integrin-dependent adhesion of neutrophils in venules, generated tissue factor-bearing microparticles, shortened plasma-clotting times, and increased thrombus frequency in the inferior vena cava. Furthermore, transgenic mice that overexpressed monomeric sP-selectin did not exhibit increased inflammation or thrombosis. We conclude that elevated plasma sP-selectin is a consequence rather than a cause of cardiovascular disease., (© 2017 by The American Society of Hematology.)

Selectin interactions with fucosylated glycan ligands mediate leukocyte rolling in the vasculature under shear forces. Crystal structures of P- and E-selectin suggest a two-state model in which ligand binding to the lectin domain closes loop 83-89 around the Ca 2+ coordination site, enabling Glu-88 to engage Ca 2+ and fucose. This triggers further allostery that opens the lectin/EGF domain hinge. The model posits that force accelerates transition from the bent (low affinity) to the extended (high affinity) state. However, transition intermediates have not been described, and the role of Glu-88 in force-assisted allostery has not been examined. Here we report the structure of the lectin and EGF domains of L-selectin bound to a fucose mimetic; that is, a terminal mannose on an N -glycan attached to a symmetry-related molecule. The structure is a transition intermediate where loop 83-89 closes to engage Ca 2+ and mannose without triggering allostery that opens the lectin/EGF domain hinge. We used three complementary assays to compare ligand binding to WT selectins and to E88D selectins that replaced Glu-88 with Asp. Soluble P-selectinE88D bound with an ∼9-fold lower affinity to PSGL-1, a physiological ligand, due to faster dissociation. Adhesion frequency experiments with a biomembrane force probe could not detect interactions of P-selectinE88D with PSGL-1. Cells expressing transmembrane P-selectinE88D or L-selectinE88D detached from immobilized ligands immediately after initiating flow. Cells expressing E-selectinE88D rolled but detached faster. Our data support a two-state model for selectins in which Glu-88 must engage ligand to trigger allostery that stabilizes the high affinity state under force., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion.

Interactions of selectins with cell surface glycoconjugates mediate the first step of the adhesion and signaling cascade that recruits circulating leukocytes to sites of infection or injury. P-selectin dimerizes on the surface of endothelial cells and forms dimeric bonds with P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric sialomucin on leukocytes. It is not known whether leukocyte L-selectin or endothelial cell E-selectin are monomeric or oligomeric. Here we used the micropipette technique to analyze two-dimensional binding of monomeric or dimeric L- and E-selectin with monomeric or dimeric PSGL-1. Adhesion frequency analysis demonstrated that E-selectin on human aortic endothelial cells supported dimeric interactions with dimeric PSGL-1 and monomeric interactions with monomeric PSGL-1. In contrast, L-selectin on human neutrophils supported monomeric interactions with dimeric or monomeric PSGL-1. Our work provides a new method to analyze oligomeric cross-junctional molecular binding at the interface of two interacting cells.

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)β(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)β(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)β(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)β(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)β(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)β(2)-mediated arrest.

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation, reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization, and the cytoplasmic domain propagates signals that activate β(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in "ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly, they have 10-fold less PSGL-1 on their surfaces than WT leukocytes. Using glycosidases, proteases, Western blotting, confocal microscopy, cell-surface cross-linking, FRET, and pulse-chase metabolic labeling, we demonstrate that deleting the cytoplasmic domain impaired dimerization and delayed export of PSGL-1 from the endoplasmic reticulum (ER), markedly increasing a monomeric precursor in the ER and decreasing mature PSGL-1 on the cell surface. A monomeric full-length PSGL-1 made by substituting the transmembrane domain with that of CD43 exited the ER normally, revealing that dimerization was not required for ER export. Thus, the transmembrane and cytoplasmic domains cooperate to promote dimerization of PSGL-1. Furthermore, the cytoplasmic domain provides a key signal to export precursors of PSGL-1 from the ER to the Golgi apparatus en route to the cell surface.

In inflamed venules, neutrophils rolling on E-selectin induce integrin alpha(L)beta(2)-dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-gamma (FcRgamma), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential E-selectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raft-dependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, E-selectin-mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce alpha(L)beta(2)-dependent slow rolling.

In inflamed venules, leukocytes use P-selectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin alphaLbeta2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on P-selectin, and to signal through spleen tyrosine kinase (Syk). We generated "DeltaCD" mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. DeltaCD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on DeltaCD neutrophils. At matched PSGL-1 densities, both DeltaCD and wild-type neutrophils rolled similarly on P-selectin. However, DeltaCD neutrophils rolling on P-selectin did not trigger Syk-dependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate beta2 integrins to slow rolling on ICAM-1.

Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-selectin glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-selectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-selectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-selectin, L-selectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-selectin than P-selectin.

Flow-enhanced cell adhesion is a counterintuitive phenomenon that has been observed in several biological systems. Flow augments L-selectin-dependent adhesion by increasing the initial tethering of leukocytes to vascular surfaces and by strengthening their subsequent rolling interactions. Tethering or rolling might be influenced by physical factors that affect the formation or dissociation of selectin-ligand bonds. We recently demonstrated that flow enhanced rolling of L-selectin-bearing microspheres or neutrophils on P-selectin glycoprotein ligand-1 by force decreased bond dissociation. Here, we show that flow augmented tethering of these microspheres or cells to P-selectin glycoprotein ligand-1 by three transport mechanisms that increased bond formation: sliding of the sphere bottom on the surface, Brownian motion, and molecular diffusion. These results elucidate the mechanisms for flow-enhanced tethering through L-selectin.

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.

Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin-PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin-bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin-dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.

Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.

Carcinoembryonic antigen (CEA) is an oncofoetal cell surface glycoprotein that serves as an important tumour marker for colorectal and some other carcinomas. Its immunoglobulin-like structure places CEA within the immunoglobulin superfamily. CEA functions in several biological roles including homotypic and heterotypic (with other CEA family members) cell adhesion. Cell-cell interaction can be modulated by different factors, e.g., post-translational modifications such as glycosylation. The purpose of this study was to examine whether changes in carbohydrate composition of CEA oligosaccharides can influence homotypic (CEA-CEA) interactions. In order to modulate glycosylation of CEA we used two different glycosylation mutants of Chinese hamster ovary (CHO) cells, Lec2 and Lec8. Lec2 cells should produce CEA with nonsialylated N-glycans, while Lec8 cells should yield more truncated sugar structures than Lec2. Parental CHO (Pro5) cells and the glycosylation deficient mutants were stably transfected with CEA cDNA. All three CEA glycoforms, tested in a solid-phase cell adhesion assay, showed an ability to mediate CEA-dependent cell adhesion, and no qualitative differences in the adhesion between the glycoforms were observed. Thus, it may be assumed that carbohydrates do not play a role in homotypic adhesion, and the interactions between CEA molecules depend solely on the polypeptide structure.