Endoglycan,a member of the CD34 family,functions as an L-selectin ligand through modification with tyrosine sulfation and sialyl Lewis x

During lymphocyte homing to secondary lymphoid organs and instances of inflammatory trafficking, the rolling of leukocytes on vascular endothelium is mediated by transient interactions between L-selectin on leukocytes and several carbohydrate-modified ligands on the endothelium. Most L-selectin ligands such as CD34 and podocalyxin present sulfated carbohydrate structures (6-sulfated sialyl Lewis x or 6-sulfo-sLex) as a recognition determinant within their heavily glycosylated mucin domains. We recently identified endoglycan as a new member of the CD34 family. We report here that endoglycan, like the two other members of this family (CD34 and podocalyxin) can function as a L-selectin ligand. However, endoglycan employs a different binding mechanism, interacting with L-selectin through sulfation on two tyrosine residues and O-linked sLex structures that are presented within its highly acidic amino-terminal region. Our analysis establishes striking parallels with PSGL-1, a leukocyte ligand that interacts with all three selectins, mediating leukocyte-endothelial, leukocyte-leukocyte, and platelet-leukocyte interactions. Since the distribution of endoglycan includes hematopoietic precursors and leukocyte subpopulations, in addition to endothelial cells, our findings suggest several potential settings for endoglycan-mediated adhesion events.     

P- and E-selectins recognize sialyl 6-sulfo Lewis X, the recently identified L-selectin ligand

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.     

P- and E-selectin use common sites for carbohydrate ligand recognition and cell adhesion

The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis(x) (sLe(x); sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E- and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-linked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate recognition and cell adhesion (Erbe, D. V., B. A. Wolitzky, L. G. Presta, C. R. Norton, R. J. Ramos, D. K. Burns, R. M. Rumberger, B. N. N. Rao, C. Foxall, B. K. Brandley, and L. A. Lasky. 1992. J. Cell Biol. 119:215-227). Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2'6-linked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E- and P- selectin lectin domains appears to mediate carbohydrate recognition and cell adhesion.     

Inhibition of L-selectin-mediated leukocyte rolling by synthetic glycoprotein mimics

Synthetic carbohydrate and glycoprotein mimics displaying sulfated saccharide residues have been assayed for their L-selectin inhibitory properties under static and flow conditions. Polymers displaying the L-selectin recognition epitopes 3',6-disulfo Lewis x(Glc) (3-O-SO3-Galbeta1alpha4(Fucalpha1alpha3)-6-O-SO3-Glcbeta+ ++-OR) and 3',6'-disulfo Lewis x(Glc) (3, 6-di-O-SO3-Galbeta1alpha4(Fucalpha1alpha3)Glcbeta-OR) both inhibit L-selectin binding to heparin under static, cell-free binding conditions with similar efficacies. Under conditions of shear flow, however, only the polymer displaying 3',6-disulfo Lewis x(Glc) inhibits the rolling of L-selectin-transfected cells on the glycoprotein ligand GlyCAM-1. Although it has been shown to more effective than sialyl Lewis x at blocking the L-selectin-GlyCAM-1 interaction in static binding studies, the corresponding monomer had no effect in the dynamic assay. These data indicate that multivalent ligands are far more effective inhibitors of L-selectin-mediated rolling than their monovalent counterparts and that the inhibitory activities are dependent on the specific sulfation pattern of the recognition epitope. Importantly, our results indicate the L-selectin specificity for one ligand over another found in static, cell-free binding assays is not necessarily retained under the conditions of shear flow. The results suggest that monovalent or polyvalent carbohydrate or glycoprotein mimetics that inhibit selectin binding in static assays may not block the more physiologically relevant process of selectin-mediated rolling.     

ELAM-1--dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA.

Adhesion of circulating leukocytes to the vascular endothelium during inflammation is mediated in part by their interaction with the endothelial-leukocyte adhesion molecule ELAM-1. ELAM-1, a member of the LEC-CAM family of cell adhesion molecules, expresses an N-terminal carbohydrate recognition domain (CRD) homologous to various calcium-dependent mammalian lectins. However, the contribution of the CRD to cell adhesion and its carbohydrate binding specificity have not been elucidated. This study demonstrates that transfection of a human fucosyltransferase cDNA into nonmyeloid cell lines confers ELAM-1--dependent endothelial adhesion. Binding activity correlates with de novo cell surface expression of the sialylated Lewis x tetrasaccharide, whose biosynthesis is determined by the transfected fucosyltransferase cDNA. We propose that specific alpha(1,3)fucosyltransferases regulate cell adhesion to ELAM-1 by modulating cell surface expression of one or more alpha(2,3)sialylated, alpha(1,3)fucosylated lactosaminoglycans represented by the sialyl Lewis x carbohydrate determinant.     

Polymerized liposome assemblies: bifunctional macromolecular selectin inhibitors mimicking physiological selectin ligands

Monomeric sialyl Lewis(X) (sLe(x)) and sLe(x)-like oligosaccharides are minimal structures capable of supporting selectin binding in vitro. However, their weak binding interactions do not correlate with the high-affinity binding interactions witnessed in vivo. The polyvalent display of carbohydrate groups found on cell surface glycoprotein structures may contribute to the enhanced binding strength of selectin-mediated adhesion. Detailed biochemical analyses of physiological selectin ligands have revealed a complicated composition of molecules that bind to the selectins in vivo and suggest that there are other requirements for tight binding beyond simple carbohydrate multimerization. In an effort to mimic the high-affinity binding, polyvalent scaffolds that contain multicomponent displays of selectin-binding ligands have been synthesized. Here, we demonstrate that the presentation of additional anionic functional groups in the form of sulfate esters, on a polymerized liposome surface containing a multimeric array of sLe(x)-like oligosaccharides, generates a highly potent, bifunctional macromolecular assembly. This assembly inhibits L-, E-, and P-selectin binding to GlyCAM-1, a physiological ligand better than sLe(x)-like liposomes without additional anionic charge. These multivalent arrays are 4 orders of magnitude better than the monovalent carbohydrate. Liposomes displaying 3'-sulfo Lewis(X)-like oligosaccharides, on the other hand, show slight loss of binding with introduction of additional anionic functional groups for E- and P-selectin and negligible change for L-selectin. The ability to rapidly and systematically vary the composition of these assemblies is a distinguishing feature of this methodology and may be applied to the study of other systems where composite binding determinants are important for high-affinity binding.     

Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin.

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs by interacting with HEV ligands. These ligands consist of a complex of sialomucins, candidates for which are glycosylation- dependent cell adhesion molecule 1 (GlyCAM-1), CD34, and podocalyxin. The ligands must be sialylated, fucosylated, and sulfated for optimal recognition by L-selectin. Our previous structural characterization of GlyCAM-1 has demonstrated two sulfation modifications, Gal-6-sulfate and GlcNAc-6-sulfate in the context of sialyl Lewis x. We now report the cloning of a Gal-6-sulfotransferase and a GlcNAc-6-sulfotransferase, which can modify GlyCAM-1 and CD34. The Gal-6-sulfotransferase shows a wide tissue distribution. In contrast, the GlcNAc-6-sulfotransferase is highly restricted to HEVs, as revealed by Northern analysis and in situ hybridization. Expression of either enzyme in Chinese hamster ovary cells, along with CD34 and fucosyltransferase VII, results in ligand activity, as detected by binding of an L-selectin/IgM chimera. When coexpressed, the two sulfotransferases synergize to produce strongly enhanced chimera binding.     

Biosynthesis of L-selectin ligands: sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases

The leukocyte adhesion molecule L-selectin mediates lymphocyte homing to secondary lymphoid organs and to certain sites of inflammation. The cognate ligands for L-selectin possess the unusual sulfated tetrasaccharide epitope 6-sulfo sialyl Lewis x (Siaalpha2-->3Galbeta1-->4[Fucalpha1-->3][SO(3)-->6]GlcNAc). Sulfation of GlcNAc within sialyl Lewis x is a crucial modification for L-selectin binding, and thus, the underlying sulfotransferase may be a key modulator of lymphocyte trafficking. Four recently discovered GlcNAc-6-sulfotransferases are the first candidate contributors to the biosynthesis of 6-sulfo sLex in the context of L-selectin ligands. Here we report the in vitro activity of the four GlcNAc-6-sulfotransferases on a panel of synthetic oligosaccharide substrates that comprise structural motifs derived from sialyl Lewis x. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). Surprisingly, for three of the enzymes, significant activity was observed with sialylated LacNAc, and two of the enzymes were capable of detectable sulfation of GlcNAc in the context of sialyl Lewis x. On the basis of these results, we propose possible pathways for 6-sulfo sialyl Lewis x biosynthesis and suggest that sulfation may be an early committed step.     

The selectin GMP-140 binds to sialylated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells

Granule membrane protein-140 (GMP-140) is an inducible receptor for myeloid leukocytes on activated platelets and endothelium. Like other selectins, GMP-140 recognizes specific oligosaccharide ligands. However, prior data on the nature of these ligands are contradictory. We investigated the structural features required for ligand interaction with GMP-140 using purified GMP-140, cells naturally expressing specific oligosaccharides, and cells expressing cloned glycosyltransferases. Like the related selectin endothelial leukocyte adhesion molecule-1 (ELAM-1), GMP-140 recognizes alpha(2-3)sialylated, alpha(1-3)fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells, including the sequence Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNac beta-R (sialyl Lewis x). Recognition requires sialic acid, because cells expressing large amounts of Lewis x, but not sialyl Lewis x, do not interact with GMP-140. Although sialyl Lewis x is expressed by both myeloid HL-60 cells and CHO cells transfected with an alpha 1-3/4 fucosyltransferase, GMP-140 binds with significantly higher affinity to HL-60 cells. Thus, the sialyl Lewis x tetrasaccharide may require additional structural modifications or specific presentations in order for leukocytes in flowing blood to interact rapidly and with high affinity to GMP-140 on activated platelets or endothelium.     

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.     

Sialyl Lewis(x)/E-selectin-mediated rolling in a cell-free system.

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.     

Selectin/glycoconjugate binding assays for the identification and optimization of selectin antagonists.

In this study we describe ELISA-type P- and L-selectin binding assays for the analysis of selectin antagonists. A biotinylated polyacrylamide-type glycoconjugate containing sialyl Lewis A (sLe(a)-polymer) is utilized as a synthetic ligand for both selectins analogous to the E-selectin assay we have developed recently. Following precomplexation of sLe(a)-polymer with streptavidin-peroxidase, the complex is added to microtiter plates coated with the recombinant selectins. Binding of sLe(a)-polymer to the immobilized selectins is measured by the peroxidase reaction. SLe(a)-polymer was found to bind to P- and L-selectin in a cation-dependent manner. The interaction of the polymer was blocked by neutralizing anti-P- and anti-L-selectin antibody, respectively. The reference compounds heparin and fucoidan inhibited in both assays. Sialyl Lewis X (sLe(x)) blocked binding to L-selectin by 46% at 3 mM, whereas no inhibition was observed in the P-selectin assay up to 3 mM. Control polymers containing sialic acid or beta-d-glucose instead of sLe(a) weakly bound or failed to bind to the selectins. Both assays are rapid to perform and of low variability. The P-selectin assay was successfully employed to identify and optimize novel carbohydrate-based P-selectin antagonists. The P-, L-, and E-selectin assays were used to determine the fine selectivity of several sLe(x)-related selectin antagonists. These studies together suggest that sLe(a)-polymer-based selectin assays are well suited for primary screening and the characterization of selectin antagonists.     

L-selectin ligands expressed by human leukocytes are HECA-452 antibody-defined carbohydrate epitopes preferentially displayed by P-selectin glycoprotein ligand-1.

Leukocytes express L-selectin ligands critical for leukocyte-leukocyte interactions at sites of inflammation. The predominant leukocyte L-selectin ligand is P-selectin glycoprotein ligand-1 (PSGL-1), which displays appropriate sialyl Lewis x (sLex)-like carbohydrate determinants for L-selectin recognition. Among the sLex-like determinants expressed by human leukocytes is a unique carbohydrate epitope defined by the HECA-452 mAb. The HECA-452 Ag is a critical component of L-selectin ligands expressed by vascular endothelial cells. However, HECA-452 Ag expression on human leukocyte L-selectin ligands has not been assessed. In this study, the HECA-452 mAb blocked 88-99% of neutrophil rolling on, or attachment to, adherent cells expressing L-selectin in multiple experimental systems. A function-blocking anti-PSGL-1 mAb also inhibited L-selectin binding to neutrophils by 89-98%. In addition, the HECA-452 and anti-PSGL-1 mAbs blocked the majority of P-selectin binding to neutrophils. Western blot analysis revealed that PSGL-1 immunoprecipitated from neutrophils displayed HECA-452 mAb-reactive determinants and that PSGL-1 was the predominant scaffold for HECA-452 Ag display. Leukocyte L-selectin ligands also contained sulfated determinants since culturing ligand-bearing cells with NaClO3 abrogated L-selectin binding. Consistent with this, human neutrophils expressed mRNA encoding five different sulfotransferases associated with the generation of selectin ligands: CHST1, CHST2, CHST3, TPST1, and HEC-GlcNAc6ST. Therefore, the HECA-452-defined carbohydrate determinant displayed on PSGL-1 represented the predominant L-selectin and P-selectin ligand expressed by neutrophils.     

Regulation of PSGL-1 interactions with L-selectin, P-selectin, and E-selectin: role of human fucosyltransferase-IV and -VII

P-selectin glycoprotein ligand-1 (PSGL-1) interactions with selectins regulate leukocyte migration in inflammatory lesions. In mice, selectin ligand activity regulating leukocyte recruitment and lymphocyte homing into lymph nodes results from the sum of unequal contributions of fucosyltransferase (FucT)-IV and FucT-VII, with FucT-VII playing a predominant role. Here we have examined the role of human FucT-IV and -VII in conferring L-selectin, P-selectin, and E-selectin binding activities to PSGL-1. Lewis x (Le(x)) carbohydrate was generated at the CHO(dhfr)(-) cell surface by FucT-IV expression, whereas sialyl Le(x) (sLe(x)) was synthesized by FucT-VII. Both human FucT-IV and -VII had the ability to generate carbohydrate ligands that support L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a major role. Cooperation was observed between FucT-IV and -VII in recruiting L-, P-, or E-selectin-expressing cells on PSGL-1 and in regulating cell rolling velocity and stability. Additional rolling adhesion assays were performed to assess the role of Thr-57-linked core-2 O-glycans in supporting L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1. These studies confirmed that core-2 O-glycans attached to Thr-57 play a critical role in supporting L- and P-selectin-dependent rolling and revealed that additional binding sites support >75% of E-selectin-mediated rolling. The observations presented here indicate that human FucT-IV and -VII both contribute and cooperate in regulating L-selectin-, P-selectin-, and E-selectin-dependent rolling on PSGL-1, with FucT-VII playing a predominant role in conferring selectin binding activity to PSGL-1.     

Selectins and glycosyltransferases in leukocyte rolling in vivo

Leukocyte rolling is an important step for the successful recruitment of leukocytes into tissue and occurs predominantly in inflamed microvessels and in high endothelial venules of secondary lymphoid organs. Leukocyte rolling is mediated by a group of C-type lectins, termed selectins. Three different selectins have been identified - P-, E- and L-selectin - which recognize and bind to crucial carbohydrate determinants on selectin ligands. Among selectin ligands, P-selectin glycoprotein ligand-1 is the main inflammatory selectin ligand, showing binding to all three selectins under in vivo conditions. Functional relevant selectin ligands expressed on high endothelial venules of lymphoid tissue are less clearly defined at the protein level. However, high endothelial venule-expressed selectin ligands were instrumental in uncovering the crucial role of post-translational modifications for selectin ligand activity. Several glycosyltransferases, such as core 2 beta1,6-N-acetylglucosaminyltransferase-I, beta1,4-galactosyltransferases, alpha1,3-fucosyltransferases and alpha2,3-sialyltransferases have been described to participate in the synthesis of core 2 decorated O-glycan structures carrying the tetrasaccharide sialyl Lewis X, a carbohydrate determinant on selectin ligands with binding activity to all three selectins. In addition, modifications, such as carbohydrate or tyrosine sulfation, were also found to contribute to the synthesis of functional selectin ligands.     

Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion [published erratum appears in J Cell Biol 1993 Feb;120(4):1071]

E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLe(x)). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe(x) glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe(x) binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.     

Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewis(x) (sLe(x)) in selectin-dependent mo-DC binding. Our data reveal that sLe(x) is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLe(x)-dependent binding of mo-DC to selectins was further substantiated by using sLe(x) free sugar and anti-sLe(x) antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLe(x) expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.