First synthesis of two deoxy Lewisx pentaosyl glycosphingolipids

The Lewis^x–Lewis^x interaction has been increasingly studied, using a variety of techniques including nuclear magnetic resonance spectroscopy, mass spectrometry, vesicle adhesion, atomic force microscopy, and surface plasmon resonance spectroscopy. However, the detailed molecular mechanism of these weak, divalent cation dependent interactions remains unclear, and new models are needed to probe the nature of this phenomenon in term of key roles of the different hydroxyl groups on Lewis^x trisaccharide determinant involved in the Lewis^x–Lewis^x interaction. An interesting solution is to synthesize a series of Lewis^x pentaosyl glycosphingolipid derivatives in which one of the eight hydroxyl groups of Lewis^x trisaccharide is replaced by a hydrogen atom, and to test the adhesion induced by interaction of these derivatives, in order to gain insight into the functions played by the hydroxyl groups of the Lewis^x trisaccharide. This article describes the synthesis of 3d-deoxy and 4d-deoxy Lewis^x pentaosyl glycosphingolipids, to be used for study of the Lewis^x–Lewis^x interaction. Botao Fan: Deceased October 22, 2006     

Synthesis and characterization of a sulfated pentasaccharide containing the Lewisx motif

We report the synthesis of a sulfated pentasaccharide containing the Lewis(x) motif used for an NMR study described in Carbohydr. Res. 2003, 338, this issue, see following communication: doi:10.1016/S0008-6215(03)00243-X, using the dibutylstannylene acetal methodology.     

Airway epithelial wound repair: role of carbohydrate sialyl Lewisx

Epithelial repair is a complex cellular and molecular process, the details of which are still not clearly understood. Plasma membrane glycoconjugates can modulate cell function by altering the function of protein and lipids. Sialyl Lewisx (sLex), a fucose-containing tetrasaccharide, decorates membrane-bound and secreted proteins and mediates cell-cell interaction. In the present study we investigated the role of sLex in airway epithelial repair. Using immunohistochemistry, we showed an increased expression of sLex in areas of damaged bronchial epithelium compared with intact regions. Confluent monolayers of airway epithelial cells were mechanically wounded and allowed to close. Wounded monolayers were photographed for wound closure kinetics, fixed for immunocytochemical studies, or subjected to RNA extraction. Examining the expression of different alpha1,3-fucosyltransferases (FucT), enzymes that mediate the final step in the synthesis of sLex, we found that FucT-IV was the common gene expressed in all cell lines and primary airway epithelial cells. We demonstrated an increased expression of sLex over time after mechanical injury. Blocking of sLex with an inhibitory antibody completely prevented epithelial repair. Our data suggest an essential functional role for sLex in epithelial repair. Further studies are necessary to explore the exact mechanism for sLex in mediating cell-cell interaction in bronchial epithelial cells to facilitate epithelial migration and repair.     

Endothelial targeting and enhanced antiinflammatory effects of complement inhibitors possessing sialyl Lewisx moieties

The complement inhibitor soluble complement receptor type 1 (sCR1) and a truncated form of sCR1, sCR1[desLHR-A], have been generated with expression of the selectin-reactive oligosaccharide moiety, sialyl Lewisx (sLex), as N-linked oligosaccharide adducts. These modified proteins, sCR1sLex and sCR1[desLHR-A]sLex, were assessed in the L-selectin- and P-selectin-dependent rat model of lung injury following systemic activation of complement by cobra venom factor and in the L-selectin-, P-selectin-, and E-selectin-dependent model of lung injury following intrapulmonary deposition of IgG immune complexes. In the cobra venom factor model, sCR1sLex and sCR1[desLHR-A]sLex caused substantially greater reductions in neutrophil accumulation and in albumin extravasation in lung when compared with the non-sLex-decorated forms. In this model, increased lung vascular binding of sCR1sLex and sCR1[desLHR-A]sLex occurred in a P-selectin-dependent manner, in contrast to the absence of any increased binding of sCR1 or sCR1[desLHR-A]. In the IgG immune complex model, sCR1[desLHR-A]sLex possessed greater protective effects relative to sCR1[desLHR-A], based on albumin extravasation and neutrophil accumulation. Enhanced protective effects correlated with greater lung vascular binding of sCR1[desLHR-A]sLex as compared with the non-sLex-decorated form. In TNF-alpha-activated HUVEC, substantial in vitro binding occurred with sCR1[desLHR-A]sLex (but not with sCR1[desLHR-A]). This endothelial cell binding was blocked by anti-E-selectin but not by anti-P-selectin. These data suggest that sLex-decorated complement inhibitors have enhanced antiinflammatory effects and appear to have enhanced ability to localize to the activated vascular endothelium.     

Synthesis of fluorinated C-mannopeptides as sialyl Lewisx mimics for E- and P-selectin inhibition

The synthesis of fluorinated C-mannopeptides and their evaluation as E- and P-selectin inhibitors is described. These molecules are difluorinated analogues of CH₂-glycopeptides already reported to act as sLe^x mimics. The α and β anomers of these CF₂-glycopeptides have been prepared, as well as their 1-hydroxy analogues which were present in solution as an equilibrium mixture of α- and β-pyranose and α- and β-furanose forms. These molecules showed inhibitory activities comparable to their CH₂ counterparts with a moderate influence of the pseudo-anomeric center configuration.     

Expression of bisecting type and Lewisx/Lewisy terminated N-glycans on human sperm

Human sperm lack major histocompatibility class I molecules, making them susceptible to lysis by natural killer (NK) cells. Major histocompatibility class I negative tumor cells block NK cell lysis by expressing sufficient amounts of bisecting type N-glycans on their surfaces. Therefore, sperm could employ the same strategy to evade NK cell lysis. The total N-glycans derived from sperm were sequenced using ultrasensitive mass spectrometric and conventional approaches. Three major classes of N-glycans were detected, (i) high mannose, (ii) biantennary bisecting type, and (iii) biantennary, triantennary, and tetraantennary oligosaccharides terminated with Lewisx and Lewisy sequences. Immunostaining of normal sperm showed that glycoproteins bearing Lewisy sequences are localized to the acrosome and not the plasma membrane. In contrast, defective sperm showed distinct surface labeling with anti-Lewisy antibody. The substantial expression of high mannose and complex type N-glycans terminated with Lewisx and Lewisy sequences suggests that sperm glycoproteins are highly decorated with ligands for DC-SIGN. Based on previous studies, the addition of such carbohydrate signals should inhibit antigen-specific responses directed against sperm glycoproteins in both the male and female reproductive systems. Thus, the major N-glycans of human sperm are associated with the inhibition of both innate and adaptive immune responses. These results provide more support for the eutherian fetoembryonic defense system hypothesis that links the expression of carbohydrate functional groups to the protection of gametes and the developing human in utero. This study also highlights the usefulness of glycomic profiling for revealing potential physiological functions of glycans expressed in specific cell types.     

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis^x (fucosylneolactonorpentaosyl ceramide; Le^x), difucosylneolactonorhexaosyl ceramide (dimeric Le^x), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le^x and the complex type of sialyl-Le^x derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le^x glycolipids and sialyl-Le^x were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le^x glycolipid was located on the tip of fine cellular processes. The unique localization of the Le^x glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.     

Selective binding of the scavenger receptor C-type lectin to Lewisx trisaccharide and related glycan ligands

The scavenger receptor C-type lectin (SRCL) is an endothelial receptor that is similar in organization to type A scavenger receptors for modified low density lipoproteins but contains a C-type carbohydrate-recognition domain (CRD). Fragments of the receptor consisting of the entire extracellular domain and the CRD have been expressed and characterized. The extracellular domain is a trimer held together by collagen-like and coiled-coil domains adjacent to the CRD. The amino acid sequence of the CRD is very similar to the CRD of the asialoglycoprotein receptor and other galactose-specific receptors, but SRCL binds selectively to asialo-orosomucoid rather than generally to asialoglycoproteins. Screening of a glycan array and further quantitative binding studies indicate that this selectivity results from high affinity binding to glycans bearing the Lewis(x) trisaccharide. Thus, SRCL shares with the dendritic cell receptor DC-SIGN the ability to bind the Lewis(x) epitope. However, it does so in a fundamentally different way, making a primary binding interaction with the galactose moiety of the glycan rather than the fucose residue. SRCL shares with the asialoglycoprotein receptor the ability to mediate endocytosis and degradation of glycoprotein ligands. These studies suggest that SRCL might be involved in selective clearance of specific desialylated glycoproteins from circulation and/or interaction of cells bearing Lewis(x)-type structures with the vascular endothelium.     

An improved ELISA for the determination of sialyl Lewisx structures on purified glycoconjugates

The membrane carbohydrate antigen, sialyl Lewis x (sLe^x), is involved in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble macromolecules, such as serum glycoproteins, but the precise role of soluble sLe^x in modifying disease processes, or reflecting the pathological changes is still unclear. Although methods were previously reported for the measurement of soluble sLe^x, many of these were not well characterised, measurements were mainly made on mixtures of molecules, and the anti-sLe^x antibodies were used at concentrations that made the assay expensive. In this study an ELISA has been devised that detects sLe^x in purified soluble glycoconjugates using the anti-sLe^x antibody, CSLEX 1. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model substances in developing the procedure. Key steps were washing the antibody/antigen complex with ten times diluted salt solution to prevent dissociation of the complex and the use of bovine serum albumin for blocking non-specific interactions. The assay was shown to be very specific, its precision was in the range 6–12%, and it could detect less than a pmol of sLe^x. It could also distinguish between different densities of sLe^x on the same amount of glycoconjugate. Determination of sLe^x in Hp isolated from small groups of healthy individuals, cancer patients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method, which is an improvement on those previously described, will be useful for determining sLe^x in many different types of soluble glycoconjugate, and used in combination with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLe^x in the future.     

Sialyl Lewisx expression on IgG in rheumatoid arthritis and other arthritic conditions: a preliminary study

Both infiltrating leukocytes and soluble immunoglobulin form aggregates in synovial fluid during the inflammatory process in rheumatoid arthritis (RA). Some of these changes are probably mediated by the adhesion molecule, E-selectin, which increases its expression with disease activity. As glycosylation changes in IgG in RA are well established, the current study was undertaken to measure the expression of the carbohydrate antigen sialyl Lewis x (sLe^x), on IgG in RA. sLe^x is a major ligand for E-selectin. Using a recently developed ELISA, sLe^x expression was determined in IgG isolated from 8 healthy individuals, 20 RA sufferers (10 early and 10 with more long-standing disease) and 20 patients with other rheumatic conditions (osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus). S Le^xexpression on IgG was elevated above the reference range in all but one of the RA patients and this change was highly significant (P < 0.0006). Expression of this antigen on IgG was also significantly different from normal in the other arthritic groups (P < 0.02), but the changes were much less than that observed for RA. In early RA, sLe^x was inversely correlated with parameters used to measure disease activity. This was not observed with the established RA, where there was weak positive association. These preliminary results indicate that a change in sLe^x expression on IgG is an early finding in the development of RA, which may be important in the development of the disease or for predicting its outcome.     

In vivo ligand specificity of E-selectin binding to multivalent sialyl Lewisx N-linked oligosaccharides.

The in vivo specificity for E-selectin binding to a panel of N-linked oligosaccharides containing a clustered array of one to four sialyl Lewisx (SLex; NeuAcalpha2-3Gal[Fucalpha1-3]beta1-4GlcNAc) determinants was studied in mice. Following intraperitoneal dosing with lipopolysaccharide, radioiodinated tyrosinamide N-linked oligosaccharides were dosed i.v. and analyzed for their pharmacokinetics and biodistribution. Specific targeting was determined from the degree of SLex oligosaccharide targeting relative to a sialyl oligosaccharide control. Oligosaccharides targeted the kidney with the greatest selectivity after a 4-h induction period following lipopolysaccharide dosing. Unique pharmacokinetic profiles were identified for SLex biantennary and triantennary oligosaccharides but not for monovalent and tetraantennary SLex oligosaccharides or sialyl oligosaccharide controls. Biodistribution studies established that both SLex biantennary and triantennary oligosaccharides distributed to the kidney with 2-3-fold selectivity over sialyl oligosaccharide controls, whereas monovalent and tetraantennary SLex oligosaccharides failed to mediate specific kidney targeting. Simultaneous dosing of SLex biantennary or triantennary oligosaccharide with a mouse anti-E-selectin monoclonal antibody blocked kidney targeting, whereas co-administration with anti-P-selectin monoclonal antibody did not significantly block kidney targeting. The results suggest that SLex biantennary and triantennary are N-linked oligosaccharide ligands for E-selectin and implicate E-selectin as a bivalent receptor in the murine kidney endothelium.     

Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx

An ultrasound-based molecular imaging technique capable of detecting endothelial cell markers of inflammation may allow early, non-invasive assessment of vascular disease. Clinical application of targeted, acoustically-active microbubbles requires optimization of microbubble-endothelial adhesion strength to maximize image signal-to-noise ratio, as well as the ability to discern the degree of inflammation along a continuum of dysfunction. Accordingly, we hypothesized that adhesion of intercellular adhesion molecule-1 (ICAM-1)-targeted microbubbles is dependent on the degree of endothelial inflammation, and that microbubbles multi-targeted to both ICAM-1 (via anti-ICAM-1 antibodies) and selectins (via sialyl Lewisx) demonstrate greater adhesion strength than microbubbles targeted to either inflammatory marker alone. In a radial flow chamber, microbubbles were perfused across endothelial cells activated with interleukin-1beta to four different levels of inflammation, as assessed by quantitative ICAM-1 expression. ICAM-1-targeted microbubble adhesion strength increased with increasing degree of inflammation, with a relationship that was both positive and linear (r > 0.99). Microbubble adhesion strength was significantly higher for the multi-targeted microbubbles than either of the single-targeted microbubbles. These data thus demonstrate that multi-targeting of contrast microbubbles may offer improved adhesion characteristics, allowing for greater sensitivity to inflammation. Furthermore, the adhesion strength of targeted microbubbles is linearly dependent on the degree of inflammation, suggesting that targeted ultrasound imaging may offer differentiation between various degrees of endothelial dysfunction, and thus detect not only the presence, but also the severity of inflammatory disease processes.     

Cancer-associated quantitative differences in antigen levels of sialosyl Lewisx in stool from patients with colorectal carcinomas

A microenzyme-linked immunosorbent assay system employing monoclonal antibody SNH3 was developed for the detection of sialosyl Lewis^x antigen in stool extracts from 80 patients with colorectal cancer, 13 patients with colorectal non-malignant disorders and 90 normal subjects. Sialosyl Lewis^x antigen was detected in 35% of stool extracts from cancer patients but only in 7.7% and 2.2% of those from non-malignant patients and normal subjects respectively. Hemoglobin in the same stool samples was detected in 37.5% of cancer patients, 15.4% of non-malignant patients and 5.6% of normal subjects. The appearance of sialosyl Lewis^x antigen in stool was not necessary correlated with that of hemoglobin, and overall 61.3% of cancer patients were detected by the combination of the two assays. The combination assay was also impressive in early detection of colorectal cancer (Dukes' A, 52%; Dukes' B, 57.1%). Therefore, the assay for sialosyl Lewis^x antigen in stool would be useful for detecting colorectal cancer.     

FC-2.15, a monoclonal antibody active against human breast cancer, specifically recognizes Lewisx hapten

 FC-2.15 is a murine IgM monoclonal antibody that recognizes breast and colon human carcinomas, chronic myeloid leukemias, Sternberg cells of Hodgkin’s lymphoma and some normal cells, such as peripheral polymorphonuclear granulocytes. It has been previously demonstrated that FC-2.15 recognizes the carbohydrate moiety of different glycoproteins. FC-2.15 is able to mediate the in vitro lysis of Ag-2.15⁺ cells by human complement. In a phase I clinical trial, FC-2.15 induced antitumor responses and reversible neutropenia was its main toxicity. In this work, analysis of epitope specificity has demonstrated that FC-2.15 specifically recognizes terminally exposed Lewis^x trisaccharide but not sialyl-Lewis^x, Lewis^a, trifucosylated Lewis^y, blood-group antigens A and B, globo H and gangliosides. In polymorphonuclear granulocytes (PMN), myeloid leukemic cells and colon carcinoma T84 cells, Lewis^x was found to be almost exclusively N-linked to the protein core, whereas in breast carcinoma MCF-7 cells, Lewis^x appeared to be mostly O-linked. Treatment with neuraminidase increased detection by FC-2.15 in normal PMN, myeloid leukemia cells and T84 cells but not in MCF-7 cells. Received: 20 March 1997 / Accepted: 4 September 1997     

The expression of Lewisx on carcinoembryonic antigen (CEA)-related glycoproteins of normal and inflamed oesophageal squamous mucosa

Carcinoembryonic antigen (CEA)-related antigens were detected histologically in normal and inflamed oesophageal squamous mucosa using polyclonal anti-CEA antisera and monoclonal antibodies recognizing CEA or NCAs (non-specific cross-reacting antigens). Expression was limited to the surface of more mature squames. Immunoblotting of detergent extracts of oesophageal mucosa separated on polyacrylamide gels using polyclonal anti-CEA antisera showed a number of CEA-related proteins, of 195, 145, and 80 kDa. CEA-specific monoclonal antibodies recognized only the 195-kDa glycoprotein. The lower molecular weight species were recognized by anti-NCA antibody DD9 and a CD66 antibody. The carboyhydrate antigen Lewis^x (Le^x, CD15), previously shown to be a marker of mature squames, was present predominantly on a subpopulation of the 195-kDa antigen and was demonstrable on the higher molecular weight component of a doublet recognized by the CEA antibodies. Expression of Le^x carbohydrate antigens in inflamed oesophageal squamous mucosa was shown to be significantly reduced relative to the expression seen in normal tissue. A suprabasal layer of CEA-positive, Le^x-negative cells became apparent in inflamed tissue showing altered glycosylation of the CEA under these conditions. It is postulated that CEA plays a role in maintaining the integrity of the squamous mucosa.     

Conformational dynamics of sialyl Lewisx in aqueous solution and its interaction with selectinE. A study by molecular dynamics

Three dimensional structures of sialyl Lewis(x) (SLe(x)) in aqueous solution and bound to selectinE are described based on an exhaustive conformational analysis and several long molecular dynamics simulations using different glycosidic regions as starting conformations. It appears from this study that when the oligosaccharide is free in solution the NeuNAcalpha(2-3)Gal segment favors glycosidic conformation in three different regions in the (Phi,Psi) plane with propensity of populations in the ratio 1:8:1. Each one of these structures is characteristically stabilized by specific hydrogen bonding interaction between NeuNAc and Gal. On the other hand, the Gal-GlcNAc-Fuc segment can exist in four different conformational states. Based on the topology of SLe(x) we are able to predict that out of all the allowed conformations in solution only two of these structures possess a geometry that would fit without steric clashes into the binding location of selectinE. In both of these binding modes, segment Gal-GlcNAc-Fuc adopts a unique conformation. The only difference between the two SLe(x) conformers that can successfully bind to selectinE is given by two possible regions in glycosidic space in the fragment NeuNAcalpha(2-3)Gal. A large conformational departure from the crystallographic data is observed for two lysine residues at the binding site of selectinE. These two residues play an important role when SLe(x) binds selectinE in aqueous solution. These findings help reconcile the X-ray data, in which these residues appear to be 1 nm away from SLe(x), with recent liquid NMR data reporting couplings between these protein residues and the sugar.     

L-selectin interactions with novel mono- and multisulfated Lewisx sequences in comparison with the potent ligand 3'-sulfated Lewisa.

The cell adhesion molecule L-selectin binds to 3'-sialyl-Lewis (Le)x and -Lea and to 3'-sulfo-Lex and -Lea sequences. The binding to 3'-sialyl-Lex is strongly affected by the presence of 6-O-sulfate as found on oligosaccharides of the counter receptor, GlyCAM-1; 6-O-sulfate on the N-acetylglucosamine (6-sulfation) enhances, whereas 6-O-sulfate on the galactose (6'-sulfation) virtually abolishes binding. To extend knowledge on the specificity of L-selectin, we have investigated interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence. We observe that, also with 3'-sulfo-Lex, the 6-sulfation enhances and 6'-sulfation suppresses L-selectin binding. The 6'-sulfation without 3'-sialyl or 3'-sulfate gives no binding signal with L-selectin. Where the 6-sulfo,3'-sialyl-Lex is on an extended di-N-acetyllactosamine backbone, additional 6-O-sulfates on the inner galactose and inner N-acetylglucosamine do not influence the binding. Although binding to the 6,3'-sulfo-Lex and 6-sulfo, 3'-sialyl-Lex sequences is comparable, the former is a more effective inhibitor of L-selectin binding. This difference is most apparent when L-selectin is in paucivalent form (predominantly di- and tetramer) rather than multivalent. Indeed, as inhibitors of the paucivalent L-selectin, the 3'-sulfo-Lex series are more potent than the corresponding 3'-sialyl-Lex series. Thus, for synthetic strategies to design therapeutic oligosaccharide analogs as antagonists of L-selectin binding, those based on the simpler 3'-sulfo-Lex (and also the 3'-sulfo-Lea) would seem most appropriate.     

Application of homonuclear 3D NMR experiments and 1D analogs to study the conformation of sialyl Lewisx bound to E-selectin

The conformation of the sialyl Lewis ^x tetrasaccharidebound to E-selectin was previously determined from transfer NOE (trNOE)experiments in conjunction with a distance-geometry analysis. However, theorientation of the tetrasaccharide ligand in the binding site of E-selectinis still unknown. It can be predicted that the accurate quantitativeanalysis of all trNOEs, including those originating from spin diffusion, isone key to analyze the orientation of sialyl Lewis^x in thebinding pocket of E-selectin. Therefore, we applied homonuclear 3D NMRexperiments and 1D analogs to obtain trNOEs that could not unambiguously beassigned from previous 2D trNOESY spectra, due to severe resonance-signaloverlap. A 3D TOCSY-trNOESY experiment, a 1D TOCSY-trNOESY experiment, and a1D trNOESY-TOCSY experiment of the sialyl Lewis^x/E-selectincomplex furnished new interglycosidic trNOEs and provided additionalinformation for the interpretation of trNOEs that have been describedbefore. A 2D trROESY spectrum of the sialyl Lewis^x/E-selectincomplex allowed one to identify the amount of spin-diffusion contributionsto trNOEs. Finally, an unambiguous assignment of all trNOEs, and an analysisof spin-diffusion pathways, was obtained, creating a basis for aquantitative analysis of trNOEs in the sialylLewis^x/E-selectin complex.