Specific Expression of a Complex Sialyl Lewis X Antigen on High Endothelial Venules of Human Lymph Nodes: Possible Candidate for L-Selectin Ligand

Sawada, Mikiko; Takada, Ayako; Ohwaki, Ichiro; Takahashi, Naofumi; Tateno, Hiroo; Sakamoto, Junichi; Kannagi, Reiji

doi:10.1006/bbrc.1993.1629

Cited by 63 publications

(47 citation statements)

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“…Monoclonal Antibodies Directed against Sialyl Lewis X-related Carbohydrate Determinants-The anti-sialyl Lewis X antibodies 2H5 (established against a mixture of complex sialyl Lewis X-active determinants prepared from human cancer cells) and 2F3 (established against the synthetic sialyl Lewis X glycolipid) were prepared as described previously (11,16). The antibody HECA-452 was a kind gift from Dr. T. K. Kishimoto (Department of Pathology, Stanford University School of Medicine, Stanford, CA) (17).…”

Section: Methodsmentioning

confidence: 99%

“…However, most of the known anti-sialyl Lewis X antibodies as well as anti-sialyl Lewis A antibodies did not react efficiently to HEV endothelial cells. In an earlier report, we showed that one of the anti-sialyl Lewis X antibodies (2H5) specifically detected a sialyl Lewis X-like determinant on human HEV, and the antigenic determinant served as a ligand for L-selectin since the antibody inhibited the binding of lymphoid cells to human HEV endothelial cells in Stamper-Woodruff assays (11). We suggested that the antigenic determinant detected by the antibody was closely related, but not exactly identical, to genuine sialyl Lewis X and called it a complex-type sialyl Lewis X determinant, which was defined by the 2H5 antibody at that time (11).…”

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confidence: 99%

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Identification of a Major Carbohydrate Capping Group of the L-selectin Ligand on High Endothelial Venules in Human Lymph Nodes as 6-Sulfo Sialyl Lewis X

Mitsuoka¹,

Sawada‐Kasugai²,

Ando‐Furui³

et al. 1998

Journal of Biological Chemistry

243

211

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We investigated the molecular species of sulfated sialyl Lewis X determinants, the putative L-selectin ligand, expressed on high endothelial venules (HEV) in human lymph nodes. Comparison of the reactivity pattern of HEV with the reactivity of the pure 6-sulfo, 6-sulfo, or 6,6-bissulfo sialyl Lewis X determinant with hitherto known anti-sialyl Lewis X antibodies strongly suggested 6-sulfo sialyl Lewis X to be the best candidate for the major sulfated sialyl Lewis X determinant on HEV, followed by 6,6-bissulfo sialyl Lewis X, whereas 6-sulfo sialyl Lewis X was unlikely. We newly generated monoclonal antibodies (mAbs) G152 and G72 directed against 6-sulfo sialyl Lewis X, which intensely labeled HEV in immunohistochemical examination and inhibited binding of recombinant L-selectin-IgG to HEV, suggesting that the determinant serves as a ligand for L-selectin. To test the concomitant expression of 6,6-bissulfo sialyl Lewis X, specific mAbs (G2706, G27011, G27037, and G27039) were generated, but all antibodies failed to react to HEV. Next, we established mAbs (AG97 and AG273) directed against 6-sulfo Lewis X, the asialo form of 6-sulfo sialyl Lewis X. The antibodies were not reactive to untreated HEV, but strongly reacted to sialidasetreated HEV. This indicated the predominance of the sialylated form of 6-sulfo sialyl Lewis X and minimal expression of its asialo form, corroborating that it was synthesized by fucosyltransferase VII, the isoenzyme that preferentially produces the sialylated form of the determinant.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of a Major Carbohydrate Capping Group of the L-selectin Ligand on High Endothelial Venules in Human Lymph Nodes as 6-Sulfo Sialyl Lewis X

Mitsuoka¹,

Sawada‐Kasugai²,

Ando‐Furui³

et al. 1998

Journal of Biological Chemistry

243

211

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“…6B). To confirm the presence of sLe x on the extended core 1 O-glycan, enzyme-linked immunosorbent assays were performed using 2H5, a monoclonal antibody that recognizes the sLe x determinant (46). 2H5 bound to immobilized GP-6 and GP-6Ј, but not to the control glycopeptide GP-2, which lacks sLe x (data not shown).…”

Section: Synthesis Of Glyco(sulfo)peptides-mentioning

confidence: 99%

A Novel Glycosulfopeptide Binds to P-selectin and Inhibits Leukocyte Adhesion to P-selectin

Leppänen¹,

Mehta²,

Ouyang³

et al. 1999

Journal of Biological Chemistry

199

213

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P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO 3 ) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe x ) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO 3 or C2-O-sLe x do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6, which contains sLe x on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K d of ϳ350 nM. GSP-6 (<5 M) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe x (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.The interactions between selectins and their carbohydratebased ligands initiate adhesion of leukocytes to the vascular wall during inflammation. Although L-, E-, and P-selectin can bind a simple glycan containing sialyl Lewis x (sLe x ) 1 (NeuAc␣233Gal␤134[Fuc␣133]GlcNAc␤13 R) in a Ca 2ϩ -dependent manner, each selectin binds with higher affinity to a limited number of macromolecular ligands expressing sialylated and fucosylated glycans (1-4). P-selectin, which is expressed by activated platelets and endothelial cells, demonstrates the most discriminating ligand specificity of any selectin. It interacts predominantly with a disulfide-bonded dimeric mucin on leukocytes termed P-selectin glycoprotein ligand-1 (PSGL-1) (subunit mass ϳ120 kDa) (5).Each 120-kDa subunit of human PSGL-1 contains numerous sialic acids and approximately 70 extracellular Ser and Thr residues, which are potential sites for O-glycosylation, plus three potential sites for N-glycosylation (6, 7) (Fig. 1). These features suggested that the large amount of carbohydrate on the mucin might promote high avidity binding to P-selectin. However, indirect evidence suggests that the extreme N-terminal extracellular region of mature PSGL-1, which begins at residue 42, is important for high affinity binding to P-selectin (reviewed in Ref. 3). Specifically, tyrosine sulfate residues and O-glycans within that region have been considered essential for binding (Fig. 1). A monoclonal antibody directed to a peptide ep...

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“…The anti-CD15s antibody CSLEX1 (12) was obtained from Becton Dickinson Immunocytometry Systems (San Jose, California, USA); KM93 (13) from Kamiya Biomedical (Tukwila, Washington, USA); and 2H5 (14) and 2F3 (15) from PharMingen (San Diego, California, USA). VIM-2 (16) was obtained from BioSource International (Camarillo, California, USA).…”

Section: Hge Infection Cultivation and Antibody Blocking Studiesmentioning

confidence: 99%

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand

et al. 1999

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Human granulocytic ehrlichiosis (HGE) is a newly recognized tickborne infection of humans (1-3). HGE is a febrile illness characterized by reductions in blood leukocyte and platelet counts and the presence of colonies of infecting organisms (morulae) proliferating within cytoplasmic vacuoles of circulating neutrophils. No other organism is known to replicate within human granulocytes. Our laboratory recently isolated and propagated the causative agent of this disease using the human promyelocytic leukemia cell line HL60 (4). In addition, we found that the agent can grow in primary bone marrow granulocytic and monocytic precursors, blood neutrophils, and nonactivated monocytes (5).The clinical and in vitro tropism of this obligate intracellular parasite for myelomonocytic cells suggested that specific cell-surface molecules are required for adhesion, cell signaling, and entry into a permissive intracellular compartment. Of interest, sialyl Lewis x ([NeuAcα(2− 3)GalΒ1−4(Fucα1-3)GlcNAc, or CD15s]), which serves as a ligand to initially tether leukocytes to endothelial cell Eselectin (6, 7), is richly expressed on both HL60 cells and on those bone marrow and blood cells permissive for the HGE agent (8-11), where it modifies a variety of lipid and protein molecules. We hypothesized that cell-surface sialylated Lewis x might be important for infection by HGE. Methods HGE infection, cultivation, and antibody blocking studies.A cell-free bacterial inoculum of HGE was prepared from fully infected HL60 cells, propagated as described (4). The infected cells, at a density of 10 6 per ml, were passed through a 25-gauge needle three times, and cellular debris were removed by centrifugation at 700 g for 5 min. The supernatant was centrifuged at 1,236 g for 5 min, and the resultant bacterial pellet was resuspended in 10 µl supernatant per ml of the original culture. Counting in a Petroff-Hausser chamber revealed ∼10 7 organisms per 10 µl. Infections were carried out by coincubating 10 µl of this cell-free HGE preparation with 10 6 cells for a multiplicity of infection of ∼10 organisms per cell used in all experiments. For studies to determine whether bacterial binding requires calcium, both HL60 cells and the bacterial inoculum were separately incubated in HBSS with or without calcium for 20-70 min before coincubation, as described later here. For blocking studies, 10 µg (or the quantity specified) of the monoclonal antibody (MAB) tested (or control diluent or murine IgM alone) was added to 1 ml of 10 6 HL60 cells (or bone marrow progenitors, peripheral blood monocytes, or granulocytes, prepared as described; ref. 5) in medium and incubated at room temperature for 1 h. Blocking studies were performed six times with similar results. As noted, antibody blocking studies were also performed at 4°C or 37°C. Cells were centrifuged at 200 g for 5 min, 900 µl of supernatant saved, the cells resuspended in the 100 µl of medium remaining, and 10 µl of the HGE bacteria added and then coincubated for 15 min at room temperature. The cells w...

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Specific Expression of a Complex Sialyl Lewis X Antigen on High Endothelial Venules of Human Lymph Nodes: Possible Candidate for L-Selectin Ligand

Cited by 63 publications

References 0 publications

Identification of a Major Carbohydrate Capping Group of the L-selectin Ligand on High Endothelial Venules in Human Lymph Nodes as 6-Sulfo Sialyl Lewis X

Identification of a Major Carbohydrate Capping Group of the L-selectin Ligand on High Endothelial Venules in Human Lymph Nodes as 6-Sulfo Sialyl Lewis X

A Novel Glycosulfopeptide Binds to P-selectin and Inhibits Leukocyte Adhesion to P-selectin

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand

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