scholarly journals Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells.

1994 ◽  
Vol 125 (2) ◽  
pp. 471-481 ◽  
Author(s):  
M Lenter ◽  
A Levinovitz ◽  
S Isenmann ◽  
D Vestweber
Keyword(s):  
Sialic Acid ◽  
Cell Adhesion Molecules ◽  
Recombinant Antibody ◽  
Myeloid Cells ◽  
Cell Binding ◽  
Hl60 Cells ◽  
Selectin Ligands ◽  
Mature Mouse ◽  
Selectin Ligand ◽  
Selectin Binding

E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150-kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160-kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)-dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N-glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N-linked carbohydrates and is common for both endothelial selectins.

The Nature of the P-Selectin Ligands on Sickle Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 363-363
Author(s):  
Stephen H. Embury ◽  
Christine E. Baran ◽  
Colleen A. Hefner ◽  
Christi K. Seto ◽  
Neil M. Matsui
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Transferrin Receptor ◽  
Cell Disease ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Selectin Binding ◽  
Binding Determinants

Abstract Elucidation of the adhesive interactions that effect microvascular occlusion in sickle cell disease both increases our understanding of the pathophysiology of vasoocclusion and identifies molecular targets for the development of therapeutic interventions. Work from our laboratory has established that sickle RBC adhere to P-selectin on thrombin-activated endothelial cells and to immobilized, recombinant P-selectin in vitro (Matsui et al. Blood 98:1955, 2001) and that this adhesion can be inhibited by agents that block P-selectin (Matsui et al. Blood 100:3790, 2002). Based on these findings, we established that sickle RBC adherence to endothelial P-selectin has a substantial influence on microvascular blood flow in vivo and that blocking P-selectin enhances microvascular flow (Embury et al. Blood In Press). We reasoned that characterization of the cognate ligands for P-selectin ligand on sickle RBC could identify additional targets for therapeutic intervention. We had determined that that sickle RBC did not express the P-selectin ligand, P-selectin glycoprotein-1, but that membrane sialic acid is required for sickle RBC binding to P-selectin. Here we describe further characterization of the P-selectin binding determinants on sickle RBC membranes. We assessed the expression of sialyl Lewis X (sLeX) on sickle RBC using flow cytometry and the importance of sLeX expression to the rolling adhesion of sickle RBC to P-selectin in vitro. Using the monoclonal antibodies (mAb) HECA-452 and CSLEX-1 in flow cytometry we detected significant expression of sLeX on sickle RBC (p < 0.003 and p < 0.02, respectively) but not on non-sickle RBC (p < 0.07 and p < 0.3, respectively). Treatment of sickle RBC with sialidase caused a partial, dose dependant reduction of the level of detectable sLeX and of rolling adhesion to immobilized P-selectin (approximately 40% and 85%, respectively), which correlated positively. To assess the possible selective contribution of reticulocytes as a subset of higher sLeX expressing sickle RBC we employed dual label flow cytometry to determine whether sLeX and the transferrin receptor (CD71) are co-expressed. Using mAb YDJ1.2.2 for the transferrin receptor as a reticulocyte marker and CSLEX-1 showed that sLeX was expressed both on sickle reticulocytes and on older sickle RBC. Treatment of sickle RBC with O-sialoglycoprotein endopeptidase, which cleaves sialylated O-glycans, also reduced both their sLeX expression and rolling adhesion on P-selectin (approximately 30% and 65%, respectively). Treatment of sickle RBC with N-glycosidase F did not reduce sLeX or adhesion levels, trypsin treatment produced inconsistent effects, and phosphatidylinositol-specific phospholipase C caused a significant decrease in adhesion but not a significant reduction in sLex expression. These findings suggest that sickle RBC possess more than one type of glycoprotein as a ligand for P-selectin. We also used a solid-phase binding assay to detect a significant level of P-selectin binding to membrane lipids extracted from sickle RBC. Thus, the P-selectin binding determinants on sickle RBC include sialic acid, sLeX, O-linked glycans, PI-linked glycoproteins, and glycolipids. Each of these P-selectin ligands represents a potential target of new adhesion blocking drugs for the treatment of sickle cell disease.

scholarly journals Differential L-Selectin Binding Activities of Human Hematopoietic Cell L-Selectin Ligands, HCELL and PSGL-1

2001 ◽  
Vol 276 (50) ◽  
pp. 47623-47631 ◽  
Author(s):  
Charles J. Dimitroff ◽  
Jack Y. Lee ◽  
Kenneth S. Schor ◽  
Brenda M. Sandmaier ◽  
Robert Sackstein
Keyword(s):  
Flow Chamber ◽  
Hematopoietic Cell ◽  
Shear Stresses ◽  
Parallel Plate Flow Chamber ◽  
Hematopoietic Stem ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Structural Biochemistry ◽  
Adhesive Interactions ◽  
Selectin Binding

Expression of L-selectin on human hematopoietic cells (HC) is associated with a higher proliferative activity and a more rapid engraftment after hematopoietic stem cell transplantation. Two L-selectin ligands are expressed on human HCs, P-selectin glycoprotein ligand-1 (PSGL-1) and a specialized glycoform of CD44 (hematopoietic cell E- and L-selectin ligand, HCELL). Although the structural biochemistry of HCELL and PSGL-1 is well characterized, the relative capacity of these molecules to mediate L-selectin-dependent adhesion has not been explored. In this study, we examined under shear stress conditions L-selectin-dependent leukocyte adhesive interactions mediated by HCELL and PSGL-1, both as naturally expressed on human HC membranes and as purified molecules. By utilizing both Stamper-Woodruff and parallel-plate flow chamber assays, we found that HCELL displayed a 5-fold greater capacity to support L-selectin-dependent leukocyte adherence across a broad range of shear stresses compared with that of PSGL-1. Moreover, L-selectin-mediated leukocyte binding to immunopurified HCELL was consistently >5-fold higher than leukocyte binding to equivalent amounts of PSGL-1. Taken together, these data indicate that HCELL is a more avid L-selectin ligand than PSGL-1 and may be the preferential mediator of L-selectin-dependent adhesive interactions among human HCs in the bone marrow.

scholarly journals Podocalyxin-like protein is an E-/L-selectin ligand on colon carcinoma cells: comparative biochemical properties of selectin ligands in host and tumor cells

2009 ◽  
Vol 296 (3) ◽  
pp. C505-C513 ◽  
Author(s):  
Susan N. Thomas ◽  
Ronald L. Schnaar ◽  
Konstantinos Konstantopoulos
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Colon Carcinoma ◽  
Carcinoma Cells ◽  
Host Cells ◽  
High Endothelial Venules ◽  
Colon Carcinoma Cells ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Selectin Binding

Selectins facilitate metastasis and tumor cell arrest in the microvasculature by mediating binding of selectin-expressing host cells to ligands on tumor cells. We recently identified CD44 variant isoforms as functional P-, but not E-/L-, selectin ligands on colon carcinoma cells. Furthermore, a ∼180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify podocalyxin-like protein (PCLP) as an alternative selectin ligand. Blot rolling and cell-free flow-based adhesion assays disclose that PCLP on LS174T colon carcinoma cells possesses E-/L-, but not P-, selectin binding activity. The selectin-binding determinants on LS174T PCLP are non-MECA-79-reactive sialofucosylated structures displayed on O-linked glycans, distinct from the MECA-79-reactive O-glycans on PCLP expressed by high endothelial venules, which is an L-selectin ligand. PCLP on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than PCLP on wild-type cells, suggesting that PCLP functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactivity on PCLP from CD44-knockdown cells correlates with the increased avidity of PCLP for E- but not L-selectin. The novel finding that PCLP is an E-/L-selectin ligand on carcinoma cells offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell PCLP overexpression and the role of selectins in metastasis.

PSGL-1 participates in E-selectin–mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and α4 integrin

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2060-2067 ◽  
Author(s):  
Yoshio Katayama ◽  
Andrés Hidalgo ◽  
Barbara C. Furie ◽  
Dietmar Vestweber ◽  
Bruce Furie ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Activity ◽  
Hematopoietic Progenitor Cell ◽  
Drastic Reduction ◽  
Selectin Ligands ◽  
Exact Function ◽  
Stem Cell Activity ◽  
Selectin Ligand ◽  
Selectin Binding

Abstract The nature and exact function of selectin ligands involved in hematopoietic progenitor cell (HPC) homing to the bone marrow (BM) are unclear. Using murine progenitor homing assays in lethally irradiated recipients, we found that the P-selectin glycoprotein ligand-1 (PSGL-1) plays a partial role in HPC homing to the BM (a reduction of about 35% when the P-selectin binding region is blocked). Blockade of both PSGL-1 and α4 integrin did not further enhance the effect of anti-α4 integrin (a reduction of about 55%). We suspected that E-selectin ligands might contribute to the remaining homing activity. To test this hypothesis, HPC homing assays were carried out in E-selectin–deficient recipients and revealed a profound alteration in HPC homing when E-selectin and α4 integrin were inactivated (&gt; 90% reduction). Competitive assays to test homing of long-term repopulating stem cells revealed a drastic reduction (&gt; 99%) of the homed stem cell activity when both α4 integrin and E-selectin functions were absent. Further homing studies with PSGL-1–deficient HPCs pretreated with anti-α4 integrin antibody revealed that PSGL-1 contributes to approximately 60% of E-selectin ligand–mediated homing activity. Our results thus underscore a major difference between mature myeloid cells and immature stem/progenitor cells in that E-selectin ligands cooperate with α4 integrin rather than P-selectin ligands.

scholarly journals Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.

1992 ◽  
Vol 118 (2) ◽  
pp. 445-456 ◽  
Author(s):  
K L Moore ◽  
N L Stults ◽  
S Diaz ◽  
D F Smith ◽  
R D Cummings ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Myeloid Cells ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Similar Molecular Weight ◽  
Cell Extracts ◽  
Activated Platelets ◽  
Selectin Ligands ◽  
Reducing Conditions ◽  
Selectin Binding

P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.

scholarly journals Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

Blood ◽  
2011 ◽  
Vol 118 (7) ◽  
pp. 1774-1783 ◽  
Author(s):  
Jasmeen S. Merzaban ◽  
Monica M. Burdick ◽  
S. Zeineb Gadhoum ◽  
Nilesh M. Dagia ◽  
Julia T. Chu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Human Cells ◽  
Glycoprotein E ◽  
Hematopoietic Stem ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Mouse Cells ◽  
Selectin Binding ◽  
Human And Mouse ◽  
Marrow Cells

Abstract Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin−/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved by Western blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand-1 glycoform “CLA,” and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform “HCELL.” E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL's contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures.

scholarly journals P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells.

1996 ◽  
Vol 135 (2) ◽  
pp. 523-531 ◽  
Author(s):  
O Spertini ◽  
A S Cordey ◽  
N Monai ◽  
L Giuffrè ◽  
M Schapira
Keyword(s):  
Critical Role ◽  
Myeloid Cells ◽  
Chimeric Protein ◽  
Amino Acid Residues ◽  
Hematopoietic Progenitors ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Selectin Ligand ◽  
Selectin Binding

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L-selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino-terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L-selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.

scholarly journals Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

2017 ◽  
Vol 1 (27) ◽  
pp. 2799-2816 ◽  
Author(s):  
Dina B. AbuSamra ◽  
Fajr A. Aleisa ◽  
Asma S. Al-Amoodi ◽  
Heba M. Jalal Ahmed ◽  
Chee Jia Chin ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Selectin Ligands ◽  
Key Points ◽  
Selectin Ligand ◽  
Selectin Binding

Key Points Human HSPCs expressing CD34 exhibit E-selectin binding activity, whereas those lacking CD34 do not. CD34 is a unique E- and P-selectin ligand on human HSPCs that binds with kinetics comparable to other known selectin ligands.

scholarly journals The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells.

1996 ◽  
Vol 133 (4) ◽  
pp. 911-920 ◽  
Author(s):  
R N Knibbs ◽  
R A Craig ◽  
S Natsuka ◽  
A Chang ◽  
M Cameron ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Jurkat Cells ◽  
Lewis X ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Ligand Synthesis ◽  
T Lymphoblasts

Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E-selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related sLe(x)related epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state levels FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equal or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT-VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.

scholarly journals Coordinated roles of ST3Gal-VI and ST3Gal-IV sialyltransferases in the synthesis of selectin ligands

Blood ◽  
2012 ◽  
Vol 120 (5) ◽  
pp. 1015-1026 ◽  
Author(s):  
Won Ho Yang ◽  
Claudia Nussbaum ◽  
Prabhjit K. Grewal ◽  
Jamey D. Marth ◽  
Markus Sperandio
Keyword(s):  
Secretory Pathway ◽  
Ex Vivo ◽  
Flow Chamber ◽  
Leukocyte Rolling ◽  
High Endothelial Venules ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Selectin Binding ◽  
Secondary Lymphoid Organs ◽  
Deficient Mice

AbstractBinding of selectins to their glycan ligands is a prerequisite for successful leukocyte trafficking. During synthesis and transport through the secretory pathway, selectin ligands are constructed with the participation of one or more sialyltransferases of the ST3Gal subfamily. Previous studies established that ST3Gal-IV only partially contributes to selectin ligand formation, indicating that other ST3Gal-sialyltransferases are involved. By generating and analyzing St3gal6-null mice and St3gal4/St3gal6 double-deficient mice, in the present study, we found that binding of E- and P-selectin to neutrophils and L-selectin binding to lymph node high endothelial venules is reduced in the absence of ST3Gal-VI and to a greater extent in double-deficient mice. In an ex vivo flow chamber assay, P- and E-selectin–dependent leukocyte rolling was mildly reduced in St3gal6-null mice and more severely in double-deficient mice. In inflamed cremaster muscle venules of St3gal6-null mice, we found impaired P-selectin–dependent, but not E-selectin–dependent leukocyte rolling, whereas in double-deficient mice, E-selectin–dependent rolling was almost completely absent. Furthermore, neutrophil recruitment into the inflamed peritoneal cavity and lymphocyte homing to secondary lymphoid organs were impaired in St3gal6-null mice and more severely in double-deficient mice. The results of the present study demonstrate the coordinated participation of both ST3Gal-VI and ST3Gal-IV in the synthesis of functional selectin ligands.

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