Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.     

Sialyl Lewis(x)-mediated, PSGL-1-independent rolling adhesion on P-selectin

Selectin-mediated cell adhesion is an essential component of the inflammatory response. In an attempt to unambiguously identify molecular features of ligands that are necessary to support rolling adhesion on P-selectin, we have used a reconstituted ("cell-free") system in which ligand-coated beads are perfused over soluble P-selectin surfaces. We find that beads coated with the saccharides sialyl Lewis(x) (sLe(x)), sialyl Lewis(a) (sLe(a)), and sulfated Lewis(x) (HSO(3)Le(x) support rolling adhesion on P-selectin surfaces. Although it has been suggested that glycosylation and sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) is required for high-affinity binding and rolling on P-selectin, our findings indicate that sulfation of N-terminal tyrosine residues is not required for binding or rolling. However, beads coated with a tyrosine-sulfated, sLe(x)-modified, PSGL-1-Fc chimera support slower rolling on P-selectin than beads coated with sLe(x) alone, suggesting that sulfation improves rolling adhesion by modulating binding to P-selectin. In addition, we find it is not necessary that P-selectin carbohydrate ligands be multivalent for robust rolling to occur. Our results demonstrate that beads coated with monovalent sLe(x), exhibiting a more sparse distribution of carbohydrate than a similar amount of the multivalent form, are sufficient to yield rolling adhesion. The relative abilities of various ligands to support rolling on P-selectin are quantitatively examined among themselves and in comparison to human neutrophils. Using stop-time distributions, rolling dynamics at video frame rate resolution, and the average and variance of the rolling velocity, we find that P-selectin ligands display the following quantitative trend, in order of decreasing ability to support rolling adhesion on P-selectin: PSGL-1-Fc > sLe(a) approximately sLe(x) > HSO(3)Le(x).     

Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1-P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1-EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1-selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1-EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes.     

P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin

Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P- selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P- selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein- dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL- 1 must interact with P-selectin in order for neutrophils to roll on P- selectin at physiological shear stresses.     

Structurally distinct requirements for binding of P-selectin glycoprotein ligand-1 and sialyl Lewis x to Anaplasma phagocytophilum and P-selectin

Colonization of neutrophils by the bacterium Anaplasma phagocytophilum causes the disease human granulocytic ehrlichiosis. The pathogen also infects mice, its natural host. Like binding of P-selectin, binding of A. phagocytophilum to human neutrophils requires expression of P-selectin glycoprotein ligand-1 (PSGL-1) and alpha1-3-fucosyltransferases that construct the glycan determinant sialyl Lewis x (sLex). Binding of A. phagocytophilum to murine neutrophils, however, requires expression of alpha1-3-fucosyltransferases but not PSGL-1. To further characterize the molecular features that A. phagocytophilum recognizes, we measured bacterial binding to microspheres bearing specific glycoconjugates or to cells expressing human PSGL-1 and particular glycosyltransferases. Like P-selectin, A. phagocytophilum bound to purified human PSGL-1 and to glycopeptides modeled after the N terminus of human PSGL-1 that presented sLex on an O-glycan. Unlike P-selectin, A. phagocytophilum bound to glycopeptides that contained sLex but lacked tyrosine sulfation or a specific core-2 orientation of sLex on the O-glycan. A. phagocytophilum bound only to glycopeptides that contained a short amino acid sequence found in the N-terminal region of human but not murine PSGL-1. Unlike P-selectin, A. phagocytophilum bound to cells expressing PSGL-1 in cooperation with sLex on both N-and O-glycans. Moreover, bacteria bound to microspheres coupled independently with glycopeptide lacking sLex and with sLex lacking peptide. These results demonstrate that, unlike P-selectin, A. phagocytophilum binds cooperatively to a nonsulfated N-terminal peptide in human PSGL-1 and to sLex expressed on PSGL-1 or other glycoproteins. Distinct bacterial adhesins may mediate these cooperative interactions.     

Distinct sulfation requirements of selectins disclosed using cells that support rolling mediated by all three selectins under shear flow. L-selectin prefers carbohydrate 6-sulfation totyrosine sulfation,whereas p-selectin does not

l- and P-selectin are known to require sulfation in their ligand molecules. We investigated the significance of carbohydrate 6-sulfation and tyrosine sulfation in selectin-mediated cell adhesion. COS-7 cells were genetically engineered to express P-selectin glycoprotein ligand-1 (PSGL-1) or its mutant in various combinations with 6-O-sulfotransferase (6-Sul-T) and/or alpha1-->3fucosyltransferase VII (Fuc-T VII). The cells transfected with PSGL-1, 6-Sul-T, and Fuc-T VII cDNAs supported rolling mediated by all three selectins and provided the best experimental system so far to estimate kinetic parameters in selectin-mediated cell adhesion for all three selectins using the identical rolling substrate and to compare the ligand specificity of each selectin. L-selectin-mediated rolling was drastically impaired if the cells lacked carbohydrate 6-sulfation elaborated by 6-Sul-T, but not affected when PSGL-1 was replaced with a mutant lacking three tyrosine residues at its NH(2) terminus. L-selectin-mediated adhesion was also hardly affected by mocarhagin treatment of the cells, which cleaved a short peptide containing sulfated tyrosine residues from PSGL-1. In contrast, P-selectin-mediated rolling was abolished when PSGL-1 was either mutated or cleaved by mocarhagin at its NH(2) terminus, whereas the cells expressing PSGL-1 and Fuc-T VII but not 6-Sul-T showed only a modest decrease in P-selectin-mediated adhesion. These results indicate that L-selectin prefers carbohydrate 6-sulfation much more than tyrosine sulfation, whereas P-selectin favors tyrosine sulfation in the PSGL-1 molecule far more than carbohydrate 6-sulfation. E-selectin-mediated adhesion was sulfation-independent requiring only Fuc-T VII, and thus the three members of the selectin family have distinct requirements for ligand sulfation.     

Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets

Background: P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate.     

P-selectin mediates metastatic progression through binding to sulfatides on tumor cells

Hematogenous carcinoma metastasis is associated with tumor cell emboli formation, which is now known to be facilitated by selectins. P-selectin-mediated interactions of platelets with cancer cells are based mostly on mucin- and glycosaminoglycan-type selectin ligands. We previously showed that mouse colon carcinoma cells (MC-38) carry P-selectin ligands of nonmucin origin, which were not identified. Here we show that P-selectin ligands recognized on MC-38 cells are sulfated glycolipids, thereby facilitating experimental metastasis in a syngeneic mouse model. Metabolic inhibition of sulfation by incubation of cells with sodium chlorate almost completely abrogated P-selectin binding. Metabolic labeling of MC-38 cells with (35)S sulfate revealed only a single band as detected by high-performance thin layer chromatography analysis of a total lipid extract. Matrix-assisted laser desorption/ionization tandem time-of-flight/time-of-flight analysis (MALDI-TOF-TOF) analysis of the purified sulfate-containing lipid fraction identified the selectin ligand to be a sulfated galactosylceramide SM4 (HSO(3)-3Galbeta-1Cer). Modulation of glycolipid biosynthesis in MC-38 cells altered P-selectin binding, thereby confirming sulfoglycolipids to be major P-selectin ligands. In addition, P-selectin was also found to recognize lactosylceramide sulfate SM3 (HSO(3)-3Galbeta-4Glcbeta-1Cer) and gangliotriaosylceramide sulfate SM2 [GalNAcbeta-4(HSO(3)-3)Galbeta-4Glcbeta-1Cer] in human hepatoma cells. Finally, the enzymatic removal of sulfation from the cell surface of MC-38 cells resulted in decreased P-selectin binding and led to attenuation of metastasis. Thus, SM4 sulfatide serves as a native ligand for P-selectin contributing to cell-cell interactions and to facilitation of metastasis.     

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.     

Tyrosine sulfation enhances but is not required for PSGL-1 rolling adhesion on P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a large (240 kDa) glycoprotein found on the surface of nearly all leukocytes. The mature molecule is decorated with multiple N- and O-linked glycans and displays copies of the tetrasaccharide sialyl-Lewis(x) (sLe(X)), as well as a cluster of three tyrosine sulfate (tyr-SO(3)) groups near the N-terminus of the processed protein. Previous studies have suggested that PSGL-1 needs to be tyrosine-sulfated, in addition to glycosylated with sLe(X), to successfully interact with P-selectin. To better understand how biochemical features of the PSGL-1 ligand are related to its adhesion phenotype, we have measured the dynamics of adhesion under flow of a series of well-defined PSGL-1 variants that differ in their biochemical modification, to both P- and E-selectin-coated substrates. These variants are distinct PSGL-1 peptides: one that possesses sLe(X) in conjunction with three N-terminal tyr-SO(3) groups (SGP3), one that possesses sLe(X) without tyrosine sulfation (GP1), and one that lacks sLe(X) but has three N-terminal tyr-SO(3) groups (SP3). Although all peptides expressing sLe(X), tyr-SO(3), or both supported some form of rolling adhesion on P-selectin, only peptides expressing sLe(X) groups showed rolling adhesion on E-selectin. On P-selectin, the PSGL-1 peptides demonstrated a decreasing strength of adhesion in the following order: SGP3 > GP1 > SP3. Robust, rolling adhesion on P-selectin was mediated by the GP1 peptide, despite its lack of tyrosine sulfation. However, the addition of tyrosine sulfation to glycosylated peptides (SGP3) creates a super ligand for P-selectin that supports slower rolling adhesion at all shear rates and supports rolling adhesion at much higher shear rates. Tyrosine sulfation has no similar effect on PSGL-1 rolling on E-selectin. Such functional distinctions in rolling dynamics are uniquely realized with a cell-free system, which permits precise, unambiguous identification of the functional activity of adhesive ligands. These findings are consistent with structural and functional characterizations of the interactions between these peptides and E- and P-selectin published recently.     

Inhibition of protein tyrosine phosphatases suppresses P-selectin exocytosis in activated human platelets

P-selectin (CD62P), a cell adhesion molecule for most leukocytes, is stored in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Upon thrombogenic and inflammatory challenges, P-selectin is rapidly expressed, by exocytosis, on activated platelets and stimulated endothelial cells. However, little is known for the molecular mechanisms governing the regulation of the rapid mobilization of P-selectin in these cells. Here we show that phenylarsine oxide (PAO) and diamide (both were inhibitors for protein tyrosine phosphatases), but not genistein (an inhibitor for protein tyrosine kinases), adenosine, wortmannin and LY294002 (all were inhibitors for phosphatidylinositol 3- and 4-kinases), could inhibit P-selectin exocytosis on activated platelets and could abolish the P-selectin mediated aggregation of activated platelets to neutrophils. However, PAO did not attenuate the P-selectin mediated adhesion of human promyeloid HL-60 cells on the stimulated endothelial cells under flow conditions. Further, PAO had no detectable effects on the exocytosis of P-selectin in the stimulated endothelial cells. These results indicate that protein tyrosine phosphatases are necessary for P-selectin exocytosis on the activated platelets, but not on the stimulated endothelial cells, and suggest that inhibitors for protein tyrosine phosphatases may be potentially valuable for treatment of platelet/leukocyte aggregation.     

Molecular determinants of the prothrombogenic phenotype assumed by inflamed colonic venules

Although platelets have been implicated in the pathogenesis of human inflammatory bowel diseases, little is known about the magnitude of platelet accumulation in the inflamed bowel, what regulates this process, and its relevance to the overall inflammatory response. In this study, intravital video microscopy was used to monitor the trafficking of platelets and leukocytes and vascular permeability in colonic venules during the development of colonic inflammation induced by 3% dextran sodium sulfate (DSS). Blocking antibodies directed against different adhesion molecules as well as P-selectin-deficient mice were used to define the adhesive determinants of DSS-induced platelet recruitment. DSS induced an accumulation of adherent platelets that was temporally correlated with the appearance of adherent leukocytes and with disease severity. Platelet adhesion and, to a lesser extent, leukocyte adhesion were attenuated by immunoblockade of P-selectin and its ligand P-selectin glycoprotein ligand-1 (PSGL-1), with contributions from both platelet- and endothelial cell-associated P-selectin. DSS induced a rapid and sustained increase in vascular permeability that was greatly attenuated in P-selectin-deficient mice. P-selectin bone marrow chimeras revealed that both endothelial cell- and platelet-associated P-selectin contribute to the P-selectin expression detected in the inflamed colonic microvasculature, with endothelial P-selectin making a larger contribution. Our findings indicate that colonic inflammation is associated with the induction of a prothrombogenic phenotype in the colonic microcirculation, with P-selectin and its ligand PSGL-1 playing a major role in the recruitment of platelets.     

Bending rigidities of cell surface molecules P-selectin and PSGL-1

P-selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets. P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin expressed on leukocytes. The interaction of P-selectin and PSGL-1 mediates leukocyte tethering to and rolling on the vascular surface, which are initiating events in inflammatory and thrombotic processes. In the hemodynamic environment of the circulation, P-selectin and PSGL-1 are subject to a wide range of forces, which can cause deformation. For P-selectin/PSGL-1 interaction to be physically possible, these molecules may need to project above much of the glycocalyx layers of the respective cell surfaces, suggesting that they are either longer than the thickness of glycocalyx or better able to support compression than the glycocalyx. As such, the mechanical properties of these molecules and their functional implications merit investigation. Here we report determination of the bending rigidities of P-selectin and PSGL-1 by analyzing their thermally excited curvature fluctuations, whose values are of the order of magnitude of 100 pN nm².     

Time-dependent platelet-vessel wall interactions induced by intestinal ischemia-reperfusion

Platelets roll and adhere in venules exposed to ischemia-reperfusion (I/R). This platelet-endothelial adhesion may influence leukocyte trafficking because platelet depletion decreases I/R-induced leukocyte emigration. The objectives of this study were 1) to assess the time course of platelet adhesion in the small bowel after I/R and 2) to determine the roles of endothelial and/or platelet P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) in this adhesion. The adhesion of fluorescently labeled platelets was monitored by intravital microscopy in postcapillary venules exposed to 45 min of ischemia and up to 8 h of reperfusion. Peak platelet adhesion was observed at 4 h of reperfusion. To assess the contributions of platelet and endothelial cell P-selectin, platelets from P-selectin-deficient and wild-type mice were infused into wild-type and P-selectin-deficient mice, respectively. Platelets deficient in P-selectin exhibited low levels of adhesion comparable to that in sham-treated animals. In the absence of endothelial P-selectin, platelet adhesion was reduced by 65%. Treatment with a blocking antibody against PSGL-1 reduced adhesion by 57%. These results indicate that I/R induces a time-dependent platelet-endothelial adhesion response in postcapillary venules via a mechanism that involves PSGL-1 and both platelet and endothelial P-selectin, with platelet P-selectin playing a greater role.     

Mechanisms of platelet and leukocyte recruitment in experimental colitis

Both leukocytes and platelets accumulate in the colonic microvasculature during experimental colitis, leading to microvascular dysfunction and tissue injury. The objective of this study was to determine whether the recruitment of leukocytes and platelets in inflamed colonic venules are codependent processes. The rolling and adherence of leukocytes and platelets in colonic venules of mice with dextran sodium sulfate (DSS)-induced colitis were monitored by intravital videomicroscopy. DSS elicited an increased recruitment of both rolling and adherent leukocytes and platelets. DSS-colitic mice rendered thrombocytopenic with anti-platelet serum exhibited profound reductions in leukocyte adhesion. Neutropenia, induced with anti-neutrophil serum, significantly reduced the adhesion of leukocytes and the accumulation of platelet-leukocyte aggregates while greatly enhancing the number of platelets that roll and adhere directly to venular endothelial cells. The enhanced platelet adhesion associated with neutropenia was mediated by platelet P-selectin interactions with endothelial cell P-selectin glycoprotein ligand (PSGL-1). DSS colitis was also associated with an increased expression of PSGL-1 in the colonic vasculature. These findings indicate that the recruitment of leukocytes and platelets in inflamed colonic venules are co-dependent processes.     

P-selectin Cross-links PSGL-1 and Enhances Neutrophil Adhesion to Fibrinogen and ICAM-1 in a Src Kinase-Dependent,but GPCR-Independent Mechanism

Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab’)2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of αMβ2, but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (<0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by Pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.     

P-selectin must extend a sufficient length from the plasma membrane to mediate rolling of neutrophils

Under physiological shear stress, neutrophils roll on P-selectin on activated endothelial cells or platelets through interactions with P- selectin glycoprotein ligand-1 (PSGL-1). Both P-selectin and PSGL-1 are extended molecules. Human P-selectin contains an NH2-terminal lectin domain, an EGF domain, nine consensus repeats (CRs), a transmembrane domain, and a cytoplasmic tail. To determine whether the length of P- selectin affected its interactions with PSGL-1, we examined the adhesion of neutrophils to CHO cells expressing membrane-anchored P- selectin constructs in which various numbers of CRs were deleted. Under static conditions, neutrophils attached equivalently to wild-type P- selectin and to constructs containing from 2-6 CRs. Under shear stress, neutrophils attached equivalently to wild-type and 6 CR P-selectin and nearly as well to 5 CR P-selectin. However, fewer neutrophils attached to the 4 CR construct, and those that did attach rolled faster and were more readily detached by increasing shear stress. Flowing neutrophils failed to attach to the 3 CR and 2 CR constructs. Neutrophils attached and rolled more efficiently on 4 CR P-selectin expressed on glycosylation-defective Lec8 CHO cells, which have less glycocalyx. We conclude that P-selectin must project its lectin domain well above the membrane to mediate optimal attachment of neutrophils under shear forces. The length of P-selectin may: (a) facilitate interactions with PSGL-1 on flowing neutrophils, and (b) increase the intermembrane distance where specific bonds form, minimizing contacts between the glycocalyces that result in cell-cell repulsion.     

P-Selectin Glycoprotein Ligand-1 Forms Dimeric Interactions with E-Selectin but Monomeric Interactions with L-Selectin on Cell Surfaces

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.     

Binding of glycosulfopeptides to P-selectin requires stereospecific contributions of individual tyrosine sulfate and sugar residues

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin on leukocytes that binds to selectins. P-selectin binds to an N-terminal region of PSGL-1 that requires sulfation of at least one of three clustered tyrosines (TyrSO(3)) and an adjacent core-2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). We synthesized glycosulfopeptides (GSPs) modeled after this region of PSGL-1 to explore the roles of individual TyrSO(3) residues, the placement of C2-O-sLe(x) relative to TyrSO(3), the relative contributions of fucose and sialic acid on C2-O-sLe(x), and the function of the peptide sequence for binding to P-selectin. Binding of GSPs to P-selectin was measured by affinity chromatography and equilibrium gel filtration. 2-GSP-6, which has C2-O-sLe(x) at Thr-57 and TyrSO(3) at residues 46, 48, and 51, bound to P-selectin with high affinity (K(d) approximately 650 nm), whereas an isomeric trisulfated GSP containing C2-O-sLe(x) at Thr-44 bound much less well. Non-sulfated glycopeptide (2-GP-6) containing C2-O-sLe(x) at Thr-57 bound to P-selectin with approximately 40-fold lower affinity (K(d) approximately 25 microm). Proteolysis of 2-GP-6 abolished detectable binding of the residual C2-O-sLe(x)-Thr to P-selectin, demonstrating that the peptide backbone contributes to binding. Monosulfated and disulfated GSPs bound significantly better than non-sulfated 2-GP-6, but sulfation of Tyr-48 enhanced affinity (K(d) approximately 6 microm) more than sulfation of Tyr-46 or Tyr-51. 2-GSP-6 lacking sialic acid bound to P-selectin at approximately 10% that of the level of the parent 2-GSP-6, whereas 2-GSP-6 lacking fucose did not detectably bind; thus, fucose contributes more than sialic acid to binding. Reducing NaCl from 150 to 50 mm markedly enhanced binding of 2-GSP-6 to P-selectin (K(d) approximately 75 nm), demonstrating the charge dependence of the interaction. These results reveal a stereospecific interaction of P-selectin with PSGL-1 that includes distinct contributions of each of the three TyrSO(3) residues, adjacent peptide determinants, and fucose/sialic acid on an optimally positioned core-2 O-glycan.