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.     

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.     

An activated L-selectin mutant with conserved equilibrium binding properties but enhanced ligand recognition under shear flow

Selectins mediate the initial tethering and rolling of leukocytes on vessel walls. Adhesion by selectins is a function of both ligand recognition at equilibrium and mechanical properties of the selectin-ligand bond under applied force. We describe an EGF domain mutant of L-selectin with profoundly augmented adhesiveness over that of native L-selectin but conserved ligand specificity. This mutant, termed LPL, was derived by a substitution of the EGF-like domain of L-selectin with the homologous domain from P-selectin. The mutant bound soluble carbohydrate L-selectin ligand with affinity comparable with that of native L-selectin but interacted with all surface-bound ligands much more readily than native L-selectin, in particular under elevated shear flow. Tethers mediated by both native and mutant L-selectin exhibited similar lifetimes under a range of shear stresses, but the rate of bond formation by the mutant was at least 10-fold higher than that of native L-selectin toward distinct L-selectin ligands. Enhanced rate of bond formation by the mutant was associated with profoundly stronger rolling interactions and reduced dependence of rolling on a threshold of shear stress. This is the first demonstration that the EGF domain can modulate the binding of the lectin domain of a selectin to surface-immobilized ligands under shear flow without affecting the equilibrium properties of the selectin toward soluble ligands.     

Selectins: cell surface lectins which mediate the binding of leukocytes to endothelial cells.

The selectins are the most recently identified family of cell adhesion molecules. The three known members of this family (L-, E- and P-selectin) mediate the binding of leukocytes to endothelial cells and are involved in the homing of lymphocytes to lymph nodes, as well as the extravasation of neutrophilic granulocytes into inflamed tissues. The lectin character of these cell adhesion molecules (CAMs) makes the selectin protein family unique among all known CAM families. The review will summarize present knowledge about the structural organization, the ligands identified (carbohydrates and glycoproteins) and the different regulation mechanisms of the cell surface activity of the three selectins.     

炎症反应中选择素粘附分子与配体间相互作用的研究

在炎症反应中,白细胞在内皮细胞上滚动由选择素分子与其配体分子相互作用所导致,选择素分子有3种,P选择素分子（P—selectin）、E选择素分子（E—selectin）、L选择素分子（L—selectin）,选择素分子与其对应的P-选择素糖蛋白配体-1（PSGL-1）的相互作用起着重要的作用。用等离子共振、流动腔、原子力显微镜等技术能定量分析选择素分子与其配体分子相互作用的动力学反应。     

Affinity and kinetics of sialyl Lewis-X and core-2 based oligosaccharides binding to L- and P-selectin

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLe(X)) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLe(X) and sialyl Lewis-a (sLe(a)) were approximately 5-7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLe(X)-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(GalNAcbeta1,3)GalNAcalpha-OMe was identified which blocked L- and P-selectin binding at 30-100-fold lower doses than sLe(X). (iv) Surface plasmon resonance experiments determined that an sLe(X) analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K(D) of approximately 111.4 microM. The measured on- and off-rates were high (k(off) > 3 s(-)(1), k(on) > 27,000 M(-)(1) s(-)(1)). Similar binding kinetics are expected for sLe(X)-selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.     

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

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.     

Molecular mapping of functional domains of the leukocyte receptor for endothelium, LAM-1

The human lymphocyte homing receptor LAM-1, like its murine counterpart MEL-14, functions as a mammalian lectin, and mediates the binding of leukocytes to specialized high endothelial cells in lymphoid organs (HEV). LAM-1 is a member of a new family of cell adhesion molecules, termed selectins or LEC-CAMs, which also includes ELAM-1 and PAD-GEM (GMP-140/CD62). To localize the regions of LAM-1 that are involved in cell adhesion, we developed chimeric selectins, in which various domains of PAD-GEM were substituted into LAM-1, and used these chimeric proteins to define the domain requirements for carbohydrate binding, and to localize the regions recognized by several mAb which inhibit the adhesion of lymphocytes to lymph node HEV. The binding of PPME or fucoidin, soluble complex carbohydrates that specifically define the lectin activity of LAM-1 and MEL-14, required only the lectin domain of LAM-1. The LAM1-1, LAM1-3, and LAM1-6 mAb each strongly inhibit the binding of lymphocytes to HEV in the in vitro frozen section assay, and defined three independent epitopes on LAM-1. Blocking of PPME or fucoidin binding by LAM1-3 indicated that this site is identical, or in close proximity, to the carbohydrate binding site, and analysis of the binding of LAM1-3 to chimeric selectins showed that the epitope detected by LAM1-3 is located within the lectin domain. Although the LAM1-6 epitope is also located in the lectin domain, LAM1-6 did not affect the binding of PPME or fucoidin. The LAM1-1 epitope was located in, or required, the EGF domain, and, importantly, binding of LAM1-1 significantly enhanced the binding of both PPME and fucoidin. These results suggest that adhesion mediated by LAM-1 may involve cooperativity between functionally and spatially distinct sites, and support previous data suggesting a role for the EGF domain of LAM-1 in lymphocyte adhesion to HEV.     

Characterization of cDNA and genomic sequences encoding rabbit ELAM-1: conservation of structure and functional interactions with leukocytes.

Endothelial leukocyte adhesion molecule-1 (ELAM-1) is a cytokine-inducible endothelial cell surface glycoprotein involved in the adherence of neutrophils. ELAM-1 belongs to the selectin family of cell-surface molecules characterized by the general structure of an amino-terminal lectin domain followed by an epidermal growth factor domain, a variable number of complement regulatory elements, a single transmembrane sequence, and a short cytoplasmic tail. To study the in vivo regulation and expression of ELAM-1, we have isolated a complementary DNA (cDNA) clone encoding the rabbit homolog of human ELAM-1. The nucleotide sequence of the rabbit cDNA as well as its deduced amino acid sequence display extensive conservation compared to the human sequences. Rabbit ELAM-1 contains the characteristic protein domain organization of the selectin gene family and shares 74% amino acid identity with its human counterpart. However, rabbit ELAM-1 contains five complement regulatory elements whereas the human protein has six of these elements. Characterization of the genomic sequence encoding rabbit ELAM-1 indicated that individual extracellular protein domains are encoded by distinct exons. The genomic organization of rabbit ELAM-1 parallels that found for the human ELAM-1 gene and is similar to the pattern observed for other selectin family members (GMP-140, Lam-1), consistent with the hypothesis that the selectins evolved by duplication and rearrangement of individual exons. COS cells transiently expressing the rabbit ELAM-1 cDNA mediate the adhesion of rabbit and human polymorphonuclear leukocytes and are recognized by antibodies prepared against the human protein. Our results suggest that the specificity of molecular interaction between ELAM-1 and its ligand is highly conserved.     

P-selectin glycoprotein ligand-1 decameric repeats regulate selectin-dependent rolling under flow conditions

P-selectin glycoprotein ligand-1 (PSGL-1) interacts with selectins to support leukocyte rolling along vascular wall. L- and P-selectin bind to N-terminal tyrosine sulfate residues and to core-2 O-glycans attached to Thr-57, whereas tyrosine sulfation is not required for E-selectin binding. PSGL-1 extracellular domain contains decameric repeats, which extend L- and P-selectin binding sites far above the plasma membrane. We hypothesized that decamers may play a role in regulating PSGL-1 interactions with selectins. Chinese hamster ovary cells expressing wild-type PSGL-1 or PSGL-1 molecules exhibiting deletion or substitution of decamers with the tandem repeats of platelet glycoprotein Ibalpha were compared in their ability to roll on selectins and to bind soluble L- or P-selectin. Deletion of decamers abrogated soluble L-selectin binding and cell rolling on L-selectin, whereas their substitution partially reversed these diminutions. P-selectin-dependent interactions with PSGL-1 were less affected by decamer deletion. Videomicroscopy analysis showed that decamers are required to stabilize L-selectin-dependent rolling. Importantly, adhesion assays performed on recombinant decamers demonstrated that they directly bind to E-selectin and promote slow rolling. Our results indicate that the role of decamers is to extend PSGL-1 N terminus far above the cell surface to support and stabilize leukocyte rolling on L- or P-selectin. In addition, they function as a cell adhesion receptor, which supports approximately 80% of E-selectin-dependent rolling.     

The neutrophil selectin LECAM-1 presents carbohydrate ligands to the vascular selectins ELAM-1 and GMP-140.

LECAM-1 (leukocyte-endothelial cell adhesion molecule 1), the lymphocyte lectin ("selectin") homing receptor for peripheral lymph nodes (PLNs), participates in the earliest interactions of polymorphonuclear neutrophilic leukocytes (PMNs) with inflamed venules. Here, we present evidence that LECAM-1 mediates this function through a novel mechanism--presentation of oligosaccharide ligands to the inducible vascular selectins endothelial-leukocyte adhesion molecule (ELAM-1) and granule membrane protein 140 (GMP-140). PMN, but not lymphocyte, LECAM-1 is modified with the vascular selectin ligand sialyl Lewis x (sLex) and specifically binds ELAM-1-transfected cells. Although only a small fraction of total cell surface sLex, LECAM-1-associated sLex appears to play a prominent role in PMN interactions with cell-associated ELAM-1 and GMP-140, as anti-LECAM-1 monoclonal antibodies or selective removal of cell surface LECAM-1 inhibits PMN binding to vascular selectin transfectants by up to 70%. The enhanced function of LECAM-1-associated sLex may reflect the striking concentration, shown here, of LECAM-1 on PMN surface microvilli, the site of initial cellular contact.     

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

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

Mucin 16 is a functional selectin ligand on pancreatic cancer cells

Selectins promote metastasis by mediating specific interactions between selectin ligands on tumor cells and selectin-expressing host cells in the microvasculature. Using affinity chromatography in conjunction with tandem mass spectrometry and bioinformatics tools, we identified mucin 16 (MUC16) as a novel selectin ligand expressed by metastatic pancreatic cancer cells. While up-regulated in many pancreatic cancers, the biological function of sialofucosylated MUC16 has yet to be fully elucidated. To address this, we employed blot rolling and cell-free flow-based adhesion assays using MUC16 immunopurified from pancreatic cancer cells and found that it efficiently binds E- and L- but not P-selectin. The selectin-binding determinants are sialofucosylated structures displayed on O- and N-linked glycans. Silencing MUC16 expression by RNAi markedly reduces pancreatic cancer cell binding to E- and L-selectin under flow. These findings provide a novel integrated perspective on the enhanced metastatic potential associated with MUC16 overexpression and the role of selectins in metastasis.     

Interactions through L-selectin between leukocytes and adherent leukocytes nucleate rolling adhesions on selectins and VCAM-1 in shear flow

We demonstrate an additional step and a positive feedback loop in leukocyte accumulation on inflamed endothelium. Leukocytes in shear flow bind to adherent leukocytes through L-selectin/ligand interactions and subsequently bind downstream and roll on inflamed endothelium, purified E-selectin, P-selectin, L-selectin, VCAM-1, or peripheral node addressin. Thus adherent leukocytes nucleate formation of strings of rolling cells and synergistically enhance leukocyte accumulation. Neutrophils, monocytes, and activated T cell lines, but not peripheral blood T lymphocytes, tether to each other through L-selectin. L- selectin is not involved in direct binding to either E- or P-selectin and is not a major counterreceptor of endothelial selectins. Leukocyte- leukocyte tethers are more tolerant to high shear than direct tethers to endothelial selectins and, like other L-selectin-mediated interactions, require a shear threshold. Synergism between leukocyte- leukocyte and leukocyte-endothelial interactions introduces novel regulatory mechanisms in recruitment of leukocytes in inflammation.     

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

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.     

How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X.

Domains homologous to the epidermal growth factor (EGF) are important building blocks for extracellular proteins. Proteins containing these domains have been shown to function in such diverse biological processes as blood coagulation, complement activation, and the developmental determination of embryonic cell fates. Many of these proteins require calcium for their biological function. In the case of coagulation factors IX and X and anticoagulants proteins C and S, calcium has been found to bind to the EGF-like domains. We have now determined the three-dimensional structure of the calcium-bound form of the NH2-terminal EGF-like domain in coagulation factor X by two-dimensional NMR and simulated folding. Ligands to the calcium ion are the two backbone carbonyls in Gly-47 and Gly-64, as well as the side chains in Gln-49, erythro-beta-hydroxyaspartic acid (Hya) 63, and possibly Asp-46. The conserved Asp-48 is not a ligand in our present structures. The remaining ligands are assumed to be solvent molecules or, in the intact protein, ligands from neighboring domains. Other proteins interacting in a calcium-dependent manner may also contribute ligands. A comparison with the calcium-free form shows that calcium binding induces strictly local structural changes in the domain. Residues corresponding to the side chain ligands in factor X are conserved in many other proteins, such as the integral membrane protein TAN-1 of human lymphocytes and its developmentally important homolog, Notch, in Drosophila. Calcium binding to EGF-like domains may be crucial for numerous protein-protein interactions involving EGF-like domains in coagulation factors, plasma proteins, and membrane proteins. Therefore, there is reason to believe that this novel calcium site plays an important role in the biochemistry of extracellular proteins.     

Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx into inflammatory sites in vivo

The selectins are cell adhesion molecules whose carbohydrate-bindingdomain (C-type lectin) is thought to be involved in leukocyteadhesion to activated vascular endothelium in the inflammatoryprocess. A series of peptides, based on a conserved region (⁴⁸YYWIGIRK⁵⁵-NH₂)of the lectin domain of E-, L- and P-selectins, were analysedfor their ability to block selectin-mediated cell adhesion invitro, and neutrophil infiltration into sites of inflammationin vivo. The peptides inhibited the adhesion of myeloid cellsto recombinant forms of E- and P-selectin. The adhesion of myeloidcells to human endothelial cells, stimulated to express E-selectin,was also inhibited by the peptides. Finally, the peptides blockedthe adhesion of lymphocytes, expressing L-selectin, to highendothelial venules in lymph nodes which contain the ligandfor L-selectin. A clear structure-activity relationship wasestablished when peptides of different amino acid chain lengthswere tested in these assays. Peptides lacking tyrosine residues(e.g. WIGIR-NH₂) at their amino terminus were poor inhibitorsof selectin-mediated cell adhesion in vitro. The peptides thatwere found to be inhibitors of cell adhesion in vitro were alsofound to inhibit (up to 70%) neutrophil infiltration into sitesof inflammation in a thioglycollate-induced peritonitis mousemodel system. They also significantly reduced (>50%) themigration of neutrophils into cytokine-treated skin. These resultsstrongly suggest that compounds based on these tyrosine-containing,selectin-derived peptides could be used as anti-inflammatorytherapeutic agents. inflammation neutrophils peptides selectins     

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.     

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.     

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.