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.     

Characterization of the O-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)

P-selectin glypoprotein ligand-1, PSGL-1, a specific ligand for P-, E-, and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin and platelet P-selectin- or E-selectin receptor globulin-agarose chromatography. The O-linked oligosaccharides on the ligand were released by mild alkaline sodium borohydride treatment and analyzed by a combination of ion-exchange, size exclusion, lectin, and paper chromatography, together with specific exoglycosidase treatments and chemical modifications. Approximately 91% of the radioactivity released from PSGL-1 was recovered in five O-linked glycans: GalNAc (approximately 4% of the total structures), Gal, 3GalNAc (36%), and Gal, 3GalNAc substituted with one (45%), two (6 %), or three (3%) N-acetyllactosamine repeat units. None of these structures contained fucose, and the majority were substituted with at least one sialic acid. The N-acetyllactosmine-containing structures appeared to be core 2. The remaining 9% of the radioactivity recovered in O-linked oligosaccharides from PSGL-1, eluted in two peaks at 11.8 and 10.2 glucose units, on size-exclusion chromatography. Results from lectin chromatography and chemical and enzymatic degradation experiments suggest that the major portion of the radioactivity in these peaks is associated with sialylated N-acetyllactosamine-type oligosaccharides, substituted with fucose at the penultimate residue in the nonreducing end. Since both sialic acid and fucose reportedly are crucial requirements for selectin binding, these results suggest that only a minor portion, approximately 4.5%, of the O-linked oligosaccharides on PSGL-1 are involved in the interaction with the selectins.     

Cytoplasmic domain of P-selectin glycoprotein ligand-1 facilitates dimerization and export from the endoplasmic reticulum

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.     

重组人P-选凝素及P-选凝素糖蛋白配基-1的表达与鉴定

P-选凝素表达于血管内皮细胞及血小板膜上,它可以与白细胞膜表面的P-选凝素糖蛋白配基-1(P-selectin glyco-protein ligand-1,PSGL-1)相互作用,在炎症过程中介导白细胞的滚动并启动随后的白细胞迁移级联过程.我们构建了重组人野生型可溶性P-选凝素及其钙离子结合位点突变体,同时构建了重组PSGL-1免疫球蛋白融合分子(PSGL-1-Rg),并应用昆虫杆状病毒表达系统在Sf9细胞中表达这些重组蛋白,最后用镍金属螯和柱或Protein A亲和柱予以纯化.结果显示,用该系统表达的P-选凝素或PSGL-1是有活性的,但是P-选凝素的4个钙离子结合位点突变体却没有活性.该研究证明了P-选凝素钙离子结合位点在其与配基相互作用中的重要性.     

Anti-pig antibody adsorption efficacy of {alpha}-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 {beta}1, 6-N-acetylglucosaminyltransferase expression

We have previously described the construction of a P-selectin glycoprotein ligand-1-mouse immunoglobulin Fc fusion protein, which when transiently coexpressed with the porcine alpha1,3 galactosyltransferase in COS cells becomes a very efficient adsorber of xenoreactive, anti-pig antibodies. To relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO-K1, COS, and 293T cells producing this fusion protein have been engineered. On alpha1,3 galactosyltransferase coexpression, high-affinity adsorbers were produced by both COS and 293T cells, whereas an adsorber of lower affinity was derived from CHO-K1 cells. Stable coexpression of a core 2 beta1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased alpha-Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG(2b) produced in an alpha1,3 galactosyl- and core 2 beta1,6 N-acetylglucosaminyltransferase expressing CHO-K1 cell clone revealed a number of structures with carbohydrate sequences consistent with terminal Gal-Gal. In contrast, no O-glycan structures with terminal Gal-Gal were identified on the fusion protein when expressed alone or in combination with the alpha1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of alpha-Gal epitopes on PSGL-1/mIgG(2b) was dependent on the expression of O-linked glycans with core 2 structures and lactosamine extensions. The structural complexity of the terminal Gal-Gal expressing O-glycans with both neutral as well as sialic acid-containing structures is likely to contribute to the high adsorption efficacy.     

Noncovalent association of P-selectin glycoprotein ligand-1 and minimal determinants for binding to P-selectin

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded, homodimeric mucin ( approximately 250 kDa) on leukocytes that binds to P-selectin on platelets and endothelial cells during the initial steps in inflammation. Because it has been proposed that only covalently dimerized PSGL-1 can bind P-selectin, we investigated the factors controlling dimerization of PSGL-1 and re-examined whether covalent dimers are required for binding its P-selectin. Recombinant forms of PSGL-1 were created in which the single extracellular Cys (Cys(320)) was replaced with either Ser (C320S-PSGL-1) or Ala (C320A-PSGL-1). Both recombinants migrated as monomeric species of approximately 120 kDa under both nonreducing and reducing conditions on SDS-polyacrylamide gel electrophoresis. P-selectin bound similarly to cells expressing either wild type or mutated forms of PSGL-1 in both flow cytometric and rolling adhesion assays. Unexpectedly, chemical cross-linking studies revealed that both C320S- and C320A-PSGL-1 noncovalently associate in the plasma membrane and cross-linking generates dimeric species. Chimeric recombinants of PSGL-1 in which the transmembrane domain in PSGL-1 was replaced with the transmembrane domain of CD43 (CD43TMD-PSGL-1) could not be chemically cross-linked, suggesting that residues within the transmembrane domain of PSGL-1 are required for noncovalent association. Cells expressing CD43TMD-PSGL-1 bound P-selectin. To further address the ability of P-selectin to bind monomeric derivatives of PSGL-1, intact HL-60 cells were trypsin-treated, which generated a soluble approximately 25-kDa NH(2)-terminal fragment of PSGL-1 that bound to immobilized P-selectin. Because N-glycosylation of PSGL-1 hinders trypsin cleavage, a recombinant form of PSGL-1 was generated in which all three potential N-glycosylation sites were mutated (DeltaN-PSGL-1). Cells expressing DeltaN-PSGL-1 bound P-selectin, and trypsin treatment of the cells generated NH(2)-terminal monomeric fragments (<10 kDa) of PSGL-1 that bound to P-selectin. These results demonstrate that Cys(320)-dependent dimerization of PSGL-1 is not required for binding to P-selectin and that a small monomeric fragment of PSGL-1 is sufficient for P-selectin recognition.     

Model glycosulfopeptides from P-selectin glycoprotein ligand-1 require tyrosine sulfation and a core 2-branched O-glycan to bind to L-selectin

L-selectin expressed on leukocytes is involved in lymphocyte homing to secondary lymphoid organs and leukocyte recruitment into inflamed tissue. L-selectin binds to the sulfated sialyl Lewis x (6-sulfo-sLex) epitope present on O-glycans of various glycoproteins in high endothelial venules. In addition, L-selectin interacts with the dimeric mucin P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes. PSGL-1 lacks 6-sulfo-sLex but contains sulfated tyrosine residues (Tyr-SO3)at positions 46, 48, and 51 and sLex in a core 2-based O-glycan (C2-O-sLex) on Thr at position 57. The role of tyrosine sulfation and core 2 O-glycans in binding of PSGL-1 to L-selectin is not well defined. Here, we show that L-selectin binds to a glycosulfopeptide (GSP-6) modeled after the extreme N terminus of human PSGL-1, containing three Tyr-SO3 and a nearby Thr modified with C2-O-sLex. Leukocytes roll on immobilized GSP-6 in an L-selectin-dependent manner, and rolling is dependent on Tyr-SO3 and C2-O-sLex on GSP-6. The dissociation constant for binding of L-selectin to GSP-6, as measured by equilibrium gel filtration, is approximately 5 microm. Binding is dependent on Tyr-SO3 residues as well as the sialic acid and fucose residues of C2-O-sLex. Binding to an isomeric glycosulfopeptide containing three Tyr-SO3 residues and a core 1-based O-glycan expressing sLex was reduced by approximately 90%. All three Tyr-SO3 residues of GSP-6 are required for high affinity binding to L-selectin. Low affinity binding to mono- and disulfated GSPs is largely independent of the position of the Tyr-SO3 residues, except for some binding preference for an isomer sulfated on both Tyr-48 and -51. These results demonstrate that L-selectin binds with high affinity to the N-terminal region of PSGL-1 through cooperative interactions with three sulfated tyrosine residues and an appropriately positioned C2-O-sLex O-glycan.     

P-selectin glycoprotein ligand-1 and E-selectin ligand-1 are differentially modified by fucosyltransferases Fuc-TIV and Fuc-TVII in mouse neutrophils

P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are the two major selectin ligands on mouse neutrophils. Transfection experiments demonstrate that each ligand requires alpha1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid alpha1, 3-fucosyltransferases Fuc-TVII or Fuc-TIV to this alpha1, 3-fucosylation are not yet clear. To address this issue, we have used mice deficient in Fuc-TIV and/or Fuc-TVII to examine how these enzymes generate selectin-binding glycoforms of PSGL-1 and ESL-1 in mouse neutrophils. Selectin binding was analyzed by affinity isolation experiments using recombinant, antibody-like forms of the respective endothelial selectins. We observe essentially normal binding of E- or P-selectin to PSGL-1 expressed by Fuc-TIV-deficient neutrophils but find that PSGL-1 expressed by Fuc-TVII-deficient neutrophils is not bound by E- or P-selectin. By contrast, E-selectin binds with normal efficiency to ESL-1 on Fuc-TVII-deficient neutrophils but exhibits an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV-deficient neutrophils. The same specificity with which Fuc-TVII and Fuc-TIV generate selectin-binding forms of PSGL-1 and ESL-1 was found in transfection experiments with CHO-Pro(-)5 cells. In contrast, each fucosyltransferase alone could generate selectin-binding glycoforms of each of the two ligands in CHO-DUKX-B1 cells. Our data imply that in mouse neutrophils and their precursors, Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. This substrate specificity can be mimicked in CHO-Pro(-)5 cells.     

Functional role of P-selectin glycoprotein ligand 1/P-selectin interaction in the generation of tolerogenic dendritic cells

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-beta genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells, which expressed high levels of TGF-beta1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4(+)CD25(-) T cells. Accordingly, we found that DCs from PSGL-1(-/-) mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.     

Partial characterization of the N－linked oligosaccharide structures on Pselectin glycoprotein ligand－1（PSGL－1）

PSGL-1, a specific ligand for P-, E- and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography. N-linked oligosaccharides were released from the purified, denatured ligand molecule by peptide: N-glycosidase F treatment and, following separation by Sephacryl S-200 chromatography, partially characterized using lectin, ion-exchange and size-exclusion chromatography in combination with glycosidase digestions. The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated, multiantennary complex type structures with extended, poly-N-acetyllactosamine containing outer chains. A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans, in addition to the O-glycans on PSGL-1, may be involved in selectin binding.     

Partial characterization of the N-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)

INTroDUCTIONP-, E- and L-selectin are C-type lectins in-volved in the binding of circulating leukocytes tovarious target cellsll, 2]. The interaction(s) be-tween the selectins and the target cells is mediatedby oligosaccharide structures conjugated to specificligand molecules expressed on the taxget cell sur-faces. These ligand molecules may be recognized byone, two or all three of the selectins[1-31. AlthoughaVailable data suggest that the interaction(s) be-tween the selectins and their…     

Impact of carrier stiffness and microtopology on two-dimensional kinetics of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) interactions

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.     

Deficiency of P-selectin or P-selectin glycoprotein ligand-1 leads to accelerated development of glomerulonephritis and increased expression of CC chemokine ligand 2 in lupus-prone mice

The selectins and their ligands mediate leukocyte rolling on endothelial cells, the initial step in the emigration cascade leading to leukocyte infiltration of tissue. These adhesion molecules have been shown to be key promoters of acute leukocyte emigration events; however, their roles in the development of long-term inflammatory responses, including those that occur during chronic inflammatory diseases such as systemic lupus erythematosus, are unclear. To assess participation of P-selectin in such disorders, we studied the progression of systemic lupus erythematosus-like disease in P-selectin-deficient and control MRL/MpJ-Fas(lpr) (Fas(lpr)) mice. Surprisingly, we found that P-selectin deficiency resulted in significantly earlier mortality, characterized by a more rapid development of glomerulonephritis and dermatitis. Expression of CCL2 (MCP-1) was increased in the kidneys of P-selectin mutant mice and in supernatants of LPS-stimulated primary renal endothelial cell cultures from these mice. A closely similar phenotype, including elevated renal expression of CCL2, was also observed in Fas(lpr) mice deficient in the major P-selectin ligand, P-selectin glycoprotein ligand-1. These results indicate that P-selectin and P-selectin glycoprotein ligand-1 are not required for leukocyte infiltration and the development of autoimmune disease in Fas(lpr) mice, but rather expression of these adhesion molecules is important for modulating the progression of glomerulonephritis, possibly through down-regulation of endothelial CCL2 expression.     

Activation of human leukocytes reduces surface P-selectin glycoprotein ligand-1 (PSGL-1, CD162) and adhesion to P-selectin in vitro

P-selectin glycoprotein ligand-1 (PSGL-1), the primary ligand for P-selectin, is constitutively expressed on the surface of circulating leukocytes. The objective of this study was to examine the effect of leukocyte activation on PSGL-1 expression and PSGL-1-mediated leukocyte adhesion to P-selectin. PSGL-1 expression was examined via indirect immunofluorescence and flow cytometry before and after leukocyte stimulation with platelet activating factor (PAF) and PMA. Human neutrophils, monocytes, and eosinophils were all demonstrated to have significant surface expression of PSGL-1 at baseline, which decreased within minutes of exposure to PAF or PMA. PSGL-1 was detected in the supernatants of PAF-activated neutrophils by immunoprecipitation. Along with the expression data, this suggests removal of PSGL-1 from the cell surface. Soluble PSGL-1 was also detected in human bronchoalveolar lavage fluids. Down-regulation of PSGL-1 was inhibited by EDTA. However, inhibitors of L-selectin shedding and other sheddase inhibitors did not affect PSGL-1 release, suggesting that PSGL-1 may be shed by an as yet unidentified sheddase or removed by some other mechanism. Functionally, PSGL-1 down-regulation was associated with decreased neutrophil adhesion to immobilized P-selectin under both static and flow conditions, with the most profound effects seen under flow conditions. Together, these data indicate that PSGL-1 can be removed from the surface of activated leukocytes, and that this decrease in PSGL-1 expression has profound effects on leukocyte binding to P-selectin, especially under conditions of flow.     

Characterization of a sialic acid- and P-selectin glycoprotein ligand-1-independent adhesin activity in the granulocytotropic bacterium Anaplasma phagocytophilum

Anaplasma phagocytophilum, the aetiologic agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that colonizes neutrophils and neutrophil precursors. The granulocytotropic bacterium uses multiple adhesins that cooperatively bind to the N-terminal region of P-selectin glycoprotein ligand-1 (PSGL-1) and to sialyl Lewis x (sLe(x)) expressed on myeloid cell surfaces. Recognition of sLe(x) occurs through interactions with alpha2,3-sialic acid and alpha1,3-fucose. It is unknown whether other bacteria-host cell interactions are involved. In this study, we have enriched for A. phagocytophilum organisms that do not rely on sialic acid for cellular adhesion and entry by maintaining strain NCH-1 in HL-60 cells that are severely undersialylated. The selected bacteria, termed NCH-1A, also exhibit lessened dependencies on PSGL-1 and alpha1,3-fucose. Optimal adhesion and invasion by NCH-1A require interactions with the known determinants (sialic acid, PSGL-1 and alpha1,3-fucose), but none of them is absolutely necessary. NCH-1A binding to sLe(x)-modified PSGL-1 requires recognition of the known determinants in the same manners as other A. phagocytophilum strains. These data suggest that A. phagocytophilum expresses a separate adhesin from those targeting sialic acid, alpha1,3-fucose and the N-terminal region of PSGL-1. We propose that NCH-1A upregulates expression of this adhesin.     

Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1-6 (GlcNAc beta 1-2)Man alpha-R (GlcNAc to Man) beta-4-N-acetylglucosaminyltransferase VI

Hen oviduct membranes were shown to contain high activity of a novel enzyme, UDP-GlcNac:GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-R (GlcNAc to Man) beta 4-GlcNAc-transferase VI. The enzyme was shown to transfer GlcNAc in beta 1-4 linkage to the D-mannose residue of GlcNAc beta 1-6 (GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or methyl. Radioactive enzyme products were purified by several chromatographic steps, including high performance liquid chromatography, and structures were determined by proton nmr, fast atom bombardment-mass spectrometry, and methylation analysis to be GlcNAc beta 1-6 ([14C]GlcNAc beta 1-4) (GlcNAc beta 1-2) Man alpha-R. The enzyme is stimulated by Triton X-100 and has optimum activity at a relatively high MnCl2 concentration of about 100 mM; Co2+, Mg2+, and Ca2+ could partially substitute for Mn2+. A tissue survey demonstrated high GlcNAc-transferase VI activity in hen oviduct and lower activity in chicken liver and colon, duck colon, and turkey intestine. No activity was found in mammalian tissues. Hen oviduct membranes cannot act on GlcNAc beta 1-6Man alpha-R but have a beta 4-GlcNAc-transferase activity that converts GlcNAc beta 1-2Man alpha-R to GlcNAc beta 1-4(GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or 1-6Man beta methyl. The latter activity is probably due to GlcNAc-transferase IV which preferentially adds GlcNAc in beta 1-4 linkage to the Man alpha 1-3 arm of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn core structure of asparagine-linked glycans. The minimum structural requirement for a substrate of beta 4-GlcNAc-transferase VI is therefore the trisaccharide GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-; this trisaccharide is found on the Man alpha 6 arm of many branched complex asparagine-linked oligosaccharides. The data suggest that GlcNAc-transferase VI acts after the synthesis of the GlcNAc beta 1-2Man alpha 1-3-, GlcNAc beta 1-2Man alpha 1-6-, and GlcNAc beta 1-6 Man alpha 1-6-branches by GlcNAc-transferases I, II, and V, respectively, and is responsible for the synthesis of branched oligosaccharides containing the GlcNAc beta 1-6(GlcNAc beta 1-4)(GlcNAc beta 1-2)Man alpha 1-6Man beta moiety.     

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.     

Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by beta 1-3- and beta 1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma

The N-acetylglucosaminyltransferases probably involved in the biosynthesis in vitro of Ii core glycosphingolipids have been solubilized from a membrane preparation of mouse lymphoma P-1798 and partially characterized. The detergent-extracted membrane supernatant contains both beta 1-3- and beta 1-6-N-acetylglucosaminyltransferase activities that transfer [3H]GlcNAc from UDP-[3H]GlcNAc to the terminal galactose of neolactotetraosylceramide (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; nLcOse4ceramide), to form the Ii core structures. The linkage of [3H]N-acetylglucosamine incorporated into the terminal galactose of nLcOse4Cer was determined from identification of 2,4,6-tri-O-methyl[3H]galactose and 2,3,4-tri-O-methyl[3H]galactose after hydrolysis of the permethylated enzymatic products, GlcNAc beta-[3H]Gal-GlcNAc-Gal-Glc-ceramide. In addition to the presence of beta-N-acetylglucosaminyltransferases, we have detected a galactosyltransferase activity in this soluble supernatant fraction that catalyzes the transfer of [14C]galactose from UDP-[14C]galactose to lactotriaosylceramide (GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; LcOse3ceramide) to form nLcOse4ceramide, the acceptor in the N-acetylglucosaminyltransferase-catalyzed reaction.     

CEA is the major PHA-L-reactive glycoprotein in colon carcinoma cell lines and tumors: relationship between K-ras activation and beta1-6 branching of N-linked carbohydrate on CEA

Previously we have shown that a positive correlation existed between the presence of beta1-6 branching of N-linked carbohydrate (detected as PHA-L reactivity) and the level of Ras activation in colon carcinoma cell lines. In these cell lines the major PHA-L-reactive species was found to be 180 kDa. Here we identified this species to be carcinoembryonic antigen (CEA) by demonstrating that: (a) CEA immunoreactivity and PHA-L reactivity colocalized on blots of crude cellular membranes from these cell lines, and that (b) immunoprecipitation of CEA resulted in quantitative coprecipitation of PHA-L reactivity at 180 kDa. Metabolic labeling of cell line HTB39 with [(3)H]mannose revealed that CEA was the predominantly labeled glycoprotein. This indicated that CEA was the major PHA-L-reactive species due its high level of expression. The amount of PHA-L reactivity present on CEA, expressed as the PHA-L/CEA ratio, was found to vary between cell lines. This ratio was found to correlate closely with the level of Ras activation in these cells. In cellular membrane isolated from primary colon carcinoma, the major PHA-L-reactive species was also 180 kDa. This reactivity colocalized with CEA immunoreactivity, indicating that the major beta1-6-branching glycoprotein in membranes from primary colon carcinoma was CEA. Similar to that seen in cell lines, the amount of PHA-L reactivity on CEA in human tumor samples varied, suggesting that a similar paradigm of Ras-induced expression of beta1-6 branching may occur in human colon carcinoma.     

Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties

The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80-104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55-69 and aa 80-104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody-assisted epitope analysis of N-acetylgalactosaminidase-treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate-binding domain of gC-1.