Solution conformation of Lewis a-derived selectin ligands is unaffected by anionic substituents at the 3'- and 6'- positions

The selectins are a family of proteins that mediate leukocytetethering and rolling along the vascular endothelium. E-, P-,and L-selectin recognize various derivatives of the Lewis^a andLewis^x trisaccharides. The distribution of negative chargeson the Lewis^a and Lewis^x oligosaccharides appears to be an importantfactor in their binding by the selectins. Previous work exploringthis electrostatic dependence found that a series of syntheticanionic trisaccharides, 3'-sulfo, 3'-phospho, 6'-sulfo, and3',6'-disulfo Lewis^a. (Glc), exhibited differing selectin inhibitoryefficacies. To explore the possibility that these differencesarise from conformational differences between the sugars, thesolution structures of these trisaccharides were determinedusing NMR and molecular dynamics simulations. Interproton distancesand interglycosidic torsion angles were determined at 37°Cusing NOESY buildup curves and 1D LRJ experiments, respectively.Data from both experiments agreed well with predictions madefrom 2000 picosecond unrestrained molecular dynamics simulations.We found that 3'-sulfation did not alter the core Lewis^a conformation,a finding that reaffirms the results of previous study. In addition,we found that sulfation at the 6' position also leaves the trisaccharideconformation unperturbed. This is significant because the proximityof the 6'-sulfate group to the fucose ring might have alteredthe canonical Lewis^a structure. The disulfate exhibited greaterflexibility than the other derivatives in dynamics simulations,but not so much as to affect NOE and heteronuclear couplingconstant measurements. Taken together, our findings supportthe use of Lewis^a as a template onto which charged groups maybe added without significantly altering the trisaccharide'sstructure. oligosaccharides molecular dynamics simulations NMR sulfated Lewis^a phosphorylated Lewis^a     

Further studies of the binding specificity of the leukocyte adhesion molecule, L-selectin, towards sulphated oligosaccharides--suggestion of a link between the selectin- and the integrin-mediated lymphocyte adhesion systems

This communication is concerned with the binding specficityof the leukocyte-adhesion molecule L-selectin (leukocyte homingreceptor) towards structurally defined sulphated oligosaccharidesof the blood group Le^a and Le^x series, and of the glycolsaminoglycanseries heparin, chondroitin sulphate and keratan sulphate. Therecombinant soluble form of the rat L-selectin (L-selectin-IgGFc chimera) investigated here was shown previously to bind tolipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated atgalactose, such as sulphatides and a mixture of 3-sulphatedLe^a/Le^x type tetrasaccharides isolated from ovarian cystadenoma,as well as to the HNK-1 glycolipid with 3-O sulphated glucuronicacid. In the present study, the L-selectin investigated in bothchromatogram binding and plastic microwell binding experimentsusing neoglycolipids was found to bind to the individual 3-sulphatedLe^a and Le^x sequences (penta-, tetra- and trisaccharides), andwith somewhat lower intensities to their non-fucosylated analogues.Glycosaminoglycan disaccharides of keratan sulphate, heparinand chondroitin sulphate types were also bound by L-selectinin one or both assay systems, leading to the conclusion thatclustered glycosaminoglycan oligosaccharides with 6-O sulphationof N-acetylgalactctosamine, N-acetylglucosamine or glucosamine,4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronicacid, N-sulphation of glucosamine and, to a lesser extent, thenon-sulphated uronic acid-contahing disaccharides, can supportL-selectin adhesion. As inflammatory chemokines (short-rangestimulators of lymphocyte migration which trigger integrin activation)are known to bind to endothelial glycosaminoglycans, we proposethat the binding of the lymphocyte membrane L-selectin to endothelialglycosaminoglycans may provide a link between the selectin-mediatedand integrin-mediated adhesion systems in leukocyte extravasationcascades. The posibility is also raised that lymphocyte L-selectininteractions with glycosaminoglycans may contribute to pathologiesof glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoidarthritis and inflammatory lesions of the cornea. glycosaminoglycans leukocyte adhesion cascades neoglycolipids oligosaccharide presentation sulphated oligosaccharides     

The vascular E-selectin binds to the leukocyte integrins CD11/CD18

Leukocyte adhesion involves at least three molecular familiesof adhesion proteins: the leukocyte integrins CD11/CD18, theintercellular adhesion molecules (ICAMs) and the carbohydrate-bindingL-, E- and P-selectins. The intercellular adhesion moleculesare well-known ligands for the CD11/CD18 integrins. We now showthat E-selectin specifically binds to the sialyl Le^x carbohydrateepitopes of leukocyte integrins. Thus, the different familiesof leukocyte adhesion molecules form an integrated adhesionnetwork. adhesion integrins leukocyte selectin     

Prevention of ischemia-reperfusion lung injury by sulfated Lewisa pentasaccharide

Reignier, Jean, Hassan Sellak, Rémy Lemoine,André Lubineau, Guy Michel Mazmanian, Hélène Detruit,Alain Chapelier, Philippe Hervé, and The Paris-Sud UniversityLung Transplantation Group. Prevention of ischemia-reperfusionlung injury by sulfated Lewis^apentasaccharide. J. Appl. Physiol.82(4): 1058-1063, 1997.Inhibition of polymorphonuclearneutrophil (PMN) adhesion to the pulmonary endothelium attenuatesischemia-reperfusion (I/R) lung injury. We hypothesized that3-sulfated Lewis^a (SuLa), apotent ligand for the selectin adhesion molecules, may have abeneficial effect on I/R lung injury, as measured by the filtrationcoefficient(K_fc),and reduce pulmonary sequestration of PMN as assessed by the lungmyeloperoxidase (MPO) activity. Blood-perfused rat lungs were subjectedto 30 min of perfusion, 60 min of warm ischemia, and 90 min ofreperfusion after treatment with either SuLa (200 µg) or saline.Effects of SuLa on PMN adhesion to cultured human umbilical veinendothelial cells (HUVEC) stimulated with tumor necrosis factor- andcalcium ionophore were also investigated. Compared with preischemiaconditions, I/R induced a significant increase inK_fc,which was attenuated with SuLa (80 ± 8 vs. 30 ± 5%; P < 0.001). SuLa reduced lungMPO and PMN adhesion to stimulated HUVEC. These results indicate thatSuLa reduces I/R-induced lung injury and PMN accumulation in lung. Thisprotective effect might be related to inhibition of PMN adhesion toendothelial cells.

     

Comparison of NMR and molecular modeling results for a rigid and a flexible oligosaccharide

Three-bond heteronuclear coupling constants (³J_CH) are extremelyuseful in describing flexible models for oligosaccharides. Weshow that antiphase methods for measuring ³J_CH in oligosaccharideshave limited reliability but that the coupling constants canbe reliably measured in natural abundance by quantitative J-correlationmethods. Interpretation of ³J_CH data for a pentasaccharide (lacto-N-fuco-pentaose2) from human milk are consistent with a rigid model for theLewis^a trisaccharide epitope but for an antigenic tetrasaccharidefragment from the cell wall polysaccharide of viridans streptococci,³J_CH data imply a considerably more flexible model. NuclearOverhauser effect (NOE) data are reported for a heptasacchariderepeating unit isolated from the cell wall polysaccharide ofStreptococcus gordonii 38. The results for a tetrasaccharidefragment are similar to data reported for the same fragmentin the cell wall polysaccharide from S.mitis 322. This resultimplies a similar conformation for the tetrasaccharide fragmentin the polysaccharide and in the heptasaccharide and also impliesthat anisotropy of motion is not significant in the interpretationof the nuclear Overhauser effects in the polysaccharide. Interpretationof the NOE results for the tetrasaccharide fragment, like the³J_CH data, implies a flexible model with three conformationsin fast exchange. The results of the two experimental techniquesare combined with molecular modeling results including moleculardynamics simulation to provide a clear delineation between flexibleand rigid oligosaccharide epitopes. The blood group Lewis^a trisaccharideantigenic determinant is highly restricted in its motions bysteric interactions while the antigenic tetrasaccharide fragmentof the S.gordonii 38 heptasaccharide is considerably more mobile.We propose that some branched oligosaccharides are relativelyrigid and some are flexible depending on subtle details of thelinkages. oligosaccharide conformation molecular dynamics     

Detection in human blood platelets of sialyl Lewis X gangliosides, potential ligands for CD62 and other selectins

Activated platelets are known to express P-selectin, a lectin-likeadhesion receptor (CD62), through which they bind to sialylLewis X (sLe^x) ligands displayed on the membranes of leukocytes.To determine whether direct platelet-platelet interactions viaP-selectin/sLe^x interactions are also possible, we have examinedthe ganglioside extract of human blood platelets for the presenceof sLe^x ligands. Using the sensitive method of high-performancethin-layer chromatography (HPTLC)-immunostaining with the monoclonalantibody (mAb) CSLEX or with sialidase followed by mAbs MC480or PM81, eight sLe^x bands were demonstrated at R₁ 0.01, 0.03,0.05, 0.06, 0.08, 0.10, 0.14 and 0.21 in the solvent 45:55:10chloroform-methanol-aqueous 0.02% CaCl₂. The sensitivity ofall eight bands to sialidase or endoglycoceramidase confirmedthat they were gangliosides. Comparison of the HPTLC mobilitiesand densities of platelet bands with those from five other humantissues (granulocytes, monoblasts, kidney, aortic endotheliumand erythrocytes) in three different solvents revealed threemajor bands associated with platelets: 3 (R₁ 0.03), 6 (0.08)and 14 (0.21). Platelet bands were demonstrated not to haveresulted from granulocyte contamination. Partial purificationof platelet sLe^x gangliosides by high-performance liquid chromatographyand their reaction with 14 oligosaccharide-specific mAbs (FH4,FH5, LM112-161, LM-181, A5, 1B2, BR55-2, BE2, ES4, MC631, MH04,SH34, P001 and MC813-70) revealed that band 6 is a multifucosylatedneolacto ganglioside and band 14 is a branched, disialo neolactofucoganglioside. Platelet band 3 combined the features of bothbands 6 and 14, and reacted differently than granulocyte band3. These partial structures resemble gangliosides associatedwith adhesion in other cell systems. It is concluded that plateletsexpress tissue-specific sLe^x gangliosides (sLe^x ligands). Thus,it is possible that platelet-platelet binding may be mediatedat least partially through P-selectin/sLe^x interactions, especiallyafter platelet activation. gangliosides HPTLC-immunostaining platelets selectin ligands sLe^x     

Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells

Neutral glycosphingolipids and gangliosides were isolated from3.7 x 10⁹ primary bovine aortic endothelial cells and structurallycharacterized by immunological and chemical methods. Glucosyl-and lactosylceramide were detected as the main neutral glycosphingolipids(28% and 40% of total orcinol stain, respectively); LcOse₃Cerand nLcOse₄Cer were expressed to somewhat minor amounts (16%and 10% of total orcinol stain, respectively), and nLcOse₆Ceroccurred only in trace quantities. No neutral glycosphingolipidsof the ganglio-series (GgOse₃Cer and GgOse₄Cer) and the globo-series(GbOse₄Cer and the Forssman antigen) have been detected; onlytraces of GbOse₃Cer were identified by TLC immunostaining. PositiveCD15 bands obtained by TLC overlay with anti-Galβ1–4(Fucl-3)GlcNAcβ1-Rantibody indicated the presence of lipid bound Lewis^x antigen,whereas the isomeric Lewis^a structure (Galβ1–3(Fuc1–4)GlcNAcβ1-R)was not detectable. G_M3 substituted with Neu5Gc and Neu5Ac ina 2:1 ratio was the major ganglioside comprising about 95% withinthe whole ganglioside fraction. G_M3-structures were furthercharacterized by FAB-MS and GC-MS of the native compounds andtheir permethylated derivatives. C₁₈-sphingosine was the onlylong chain base, whereas variation occurred due to C_24:0,24:1and C₁₆ fatty acids. Terminally 2–3 sialylated neolacto-seriesgangliosides with nL-cOse₄- and nLcOse₆Cer (<5% of totalresordnol stain) were found in almost equal quantities, whereasno 2–6 sialylated counterparts were detected. Fucosylatedgangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis^x,sialyl Lewis^a, and VIM-2 antigen) and sulfoglucuronyl-neolactoseries structures with HNK-1 epitope were not detectable inthe acidic glycosphingoiipid fraction by TLC immunostaining.Gangliotetraose-type gangliosides G_M1 and G_D1a (<1 % of totalresorcinol stain) as well as traces of G_D1b and G_T1b have beendistinctly identified by combined choleragenoid-TLC-immunostainingand previous neur-aminidase treatment.The expression of dominantglycosphingolipids lactosylceramide and G_M3(Neu5Gc) was provedby indirect immunofiuorescence microscopy of cell layers grownin chamber slides, each showing different plasma membrane andsubcellular distribution patterns. The results provide the basisfor investigation of the role of glycosphingolipids as cellsurface antigens of cellular interaction as well as receptorsfor blood components and mac-romolecules of the extracellularmatrix. gangliosides neutral glycosphingolipids antibodies Lewis^x antigen TLC immunostaining     

Probing the carbohydrate recognition domain of E-selectin: The importance of the acid orientation in sLex mimetics

The selectin–leukocyte interaction is the initial event in the early inflammatory cascade. This interplay proceeds via the terminal tetrasaccharide sialyl Lewis^x (sLe^x), present on physiological selectin ligands and E- and P-selectins located on the endothelial surface. Blocking this process is regarded as a promising therapeutic approach for inflammatory diseases where excessive leukocyte efflux is responsible for tissue damage. Selectin antagonists are generally based on sLe^x as lead structure, containing the essential pharmacophores pre-oriented in the bioactive conformation. In this work, we describe a set of competitive sLe^x mimetics possessing the carboxylic acid pharmacophore equipped with additional hydrophobic substituents as neuraminic acid (Neu5Ac) replacements. This small library of antagonists derived from Huisgen-1,3-dipolar cycloadditions allows to further probe the carbohydrate recognition domain of E-selectin.     

Colon carcinoma cell glycolipids,integrins, and other glycoproteins mediate adhesion to HUVECs under flow

This study was undertaken toinvestigate the molecular constituents mediating LS174T colonadenocarcinoma cell adhesion to 4-h TNF--stimulated human umbilicalvein endothelial cells (HUVECs) under flow. At 1 dyn/cm²,~57% of cells rolled and then became firmly adherent, whereas otherscontinuously rolled on endothelium. Initial cell binding was primarilymediated by endothelial E-selectin. By using neuraminidase, glycolipidbiosynthesis inhibitord,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol · HCl,trypsin, and flow cytometry, LS174T cells were shown to express sialylLewis^x (sLe^x)- anddi-sLe^x-decorated, but not sLe^a-decorated,glycolipid and glycoprotein ligands for E-selectin. The cellspreferentially employed sialylated glycoproteins over glycolipids inadhesion as measured by conversion of rolling to firm adhesion,resistance to detachment by increased shear stress, and rollingvelocity. However, a nonsialylated E-selectin counterreceptor alsoexists. Furthermore, LS174T ₂, ₆, and₁ integrins support a minor pathway in adhesion toHUVECs. Finally, tumor cell attachment specifically increases HUVECendocytosis of E-selectin. Altogether, the data indicate the complexityof carcinoma cell-endothelium adhesion via sialylated glycoconjugates,integrins, and their respective counterreceptors.

     

Le(X) and related structures as adhesion molecules.

Summary and conclusion Le^x (13 fucosylated type 2 chain) functions as an adhesion molecule capable of Ca²⁺-mediated homotypic binding. Cells with high surface expression of Le^x therefore exhibit strong self-aggregation (based on Le^x-Le^x interaction) in the presence of Ca²⁺. In this review, I have summarized several lines of supporting data for this concept, and the role of Le^x-Le^x interaction in the process of embryo compaction and autoaggregation of F9 teratocarcinoma cells. In general, cell adhesion events based on Le^x-Le^x interaction may be followed and reinforced by integrin- or Ig receptor-based adhesion systems.SLe^x, the 23 sialosyl derivative of Le^x, and its positional isomer SLe^a, have been identified as the target molecules for selectin-dependent cell adhesion. Adhesion of leukocytes or tumour cells to ECs or platelets, which express E-selectin and P-selectin respectively, is initiated by this process. The target epitopes SLe^x and SLe^a are presented mainly on transmembrane glycoproteins having many clusters of O-linked carbohydrate chains. Therefore, inhibition of O-glycosylation may be effective for blocking selectin-mediated cell adhesion. The abundant presence of Le^x epitope in the central nervous system, and the physiological changes of Le^x expression as described in this monograph, reflect the adhesive properties of this molecule and its sialyosylated and/or fucosylated derivatives.     

The Fate of Sulphate within the Capsular Polysaccharide of the Unicellular Red Alga Rhodella maculata

³⁵SO^2–₄ was used in pulse-chase experiments to study themetabolism of sulphate in the capsular polysaccharides of R.maculata. Radioactivity reached an equilibrium between the chiefintracellular pools during an 8 h pulse and there was no lossof radioactivity from the cells during an extended chase ofup to 14 d, until cultures reached stationary growth phase.Any loss then found was due to dissociation of sulphated capsularmaterial from the cells. The failure of biochemical assays todetect extracellular sulphatase or sulphotransferase activitiessupports the conclusion from pulse-chase experiments that thereis no turnover of sulphate in the capsular polysaccharide ofR. maculata. Key words: sulphated polysaccharides, sulphate turnover, Rhodella     

Human Lewis {alpha}1->3/4fucosyltransferase: specificity of fucose transfer to GlcNAc{beta}1->3Gal{beta}1->4Glc{beta}1->1Cer (LcOse3Cer)

Biosynthesis of the Le^x series of carbohydrate antigens proceedsby fucose transfer in 13-linkage to the penultimate GlcNAc residueof a neolacto-series oligosaccharide acceptor, a reaction catalysedby multiple enzymes expressed in human tissues. Particularlybroad acceptor specificity, including the ability to catalysefucose transfer to both lacto- and neolacto-series acceptorsas well as the precursor Lc₃ structure (where Lc₃ lactotriaosylceramide,is GlcNAcß13Galß14Glcß1Cer), existsfor one human fucosyltransferase form, the Lewis 13/4fucosyltransferase(FucT-III). To determine if fucose transfer to Lc₃may representan alternate early step in Le^xor Le^a antigen biosynthesis withthis enzyme, the chemical structure of the fucosylated Lc₃ reactionproduct formed by the Lewis 13/4fucosyltransferase from Colo205 cells has been defined. Transfer of [¹⁴C]fucose to Lc₃ yieldeda labelled product migrating as a tetrasaccharide on thin layerchromatography plates. This product remained an acceptor forboth ß13- and ß14-galactosyl transfer onthe terminal GlcNAc residue. The product was degraded to a fucosylatedtrisaccharide derivative by bovine kidney ß-N-acetylglucosaminidase.Fast atom bombardment mass spectrometry and methylation analysisconfirmed that the product was composed exclusively of the followingstructure containing a fucose linked to the 3-position of theinternal Glc residue: GlcNAcß13Galß14Glcß11Cer Such a structure does not represent an intermdiate in Le^xorLe^a antigen biosynthesis. Thus, the evidence suggests that Le^xor Le^a antigen synthesis results exclusively from fucosylationof complete core chains. fucosyltransferase lacto-series LcOse₃Cer Lewis antigen transfer specificity     

Incomplete synthesis of the Sd/Cad blood group carbohydrate in gastrointestinal cancer

Cancer-associated changes in cell surface carbohydrates, including incomplete synthesis of normal carbohydrate epitopes, strongly affect malignant and metastatic potential. Here, we report that compensating for the cancer-associated loss of a single glycosyltransferase, β1,4N-acetylgalactosaminyltransferese T2, dramatically decreased cell surface expression of both E-selectin ligands (sialyl Lewis^x and sialyl Lewis^a). This modification was associated with elevated expression of the Sd^a carbohydrate determinant, which is expressed in normal gastrointestinal mucosa and is strikingly downregulated in cancer tissues. Loss of E-selectin ligands resulted in decreased adhesion of cancer cells to activated human endothelial cells in vitro and eventually suppressed metastatic potential in vivo.     

Selectin ligands on human melanoma cells

Twelve established human melanoma lines were screened for surface expression of the carbohydrate antigens Lewis^a (Le^a), sialyl Lewis^a (SLe^a), dimeric sialyl Lewis^a (diSLe^a), sialyl Lewis^X (SLe^X) and dimeric sialyl Lewis^X (diSLe^X). None of the lines expressed SLe^X, but 11/12 were positive for diSLe^X and 7/12 were positive for SLe^a. Although both diSLe^X and SLe^a have been reported to bind to E-selectin, none of the melanoma lines exhibited E-selectin-dependent adhesion to activated human umbilical vein endothelial cells (HUVECs). Three melanoma lines infected with a retroviral vector carrying the cDNA for the human Lewis fucosyltransferase (FucT-III) subsequently expressed SLe^X at their cell surface and exhibited E-selectin-dependent adhesion to activated HUVECs. Treatment of these transduced cells with inhibitors of O-linked or N-linked protein glycosylation significantly inhibited E-selectin-meiiated adhesion, though fluorescence-activated cell sorter analysis indicated no decrease in cell surface expression of SLe^X, SLe^a or diSLe^X. This suggests that the majority of SLe^X/SLe^a-type glycans endogenously procuded by human melanoma cells are not protein-associated and do not mediate E-selectin-dependent adhesion. These results support the hypothesis that E-selectin-dependent adhesion requires presentation of SLe^X-type moieties on appropriate glycoproteins.     

Sulphate release from keratan sulphate and heparan sulphate by bovine N-acetylglucosamine-6-sulphate sulphatase

The functions of sulphated monosaccharides within glycosaminoglycans(GAGs) and glycoproteins are being studied intensely, but progressis hindered by an inability to selectively desulphate glycoconjugates.We recently identified an N-acetylglucosamine-6-sulphate sulphatase(NG6SS) from bovine kidney that can remove sulphate from N-acetylglucosamine-6-sulphate(GlcNAc-6-SO₄) within oligosaccharides and glycoproteins. However,the potential ‘endosulphatase’ activity of the NG6SStoward GAGs is not known. To test for this possibility, [³H]glucosamine-,[³H]galactose- and ³⁵SO_4- labelled keratan sulphate (KS) wereseparately prepared by metabolic radiolabelling of bovine cornea.NG6SS quantitatively removed sulphate from KS without releaseof sugar fragments. The enzyme had a K_m of 4.7 mM toward freeGlcNAc-6-SO₄, but its K_m for commercially available bovine cornealKS was found to be 9.1 µM. Analyses of both KS and heparansulphate after treatment with NG6SS demonstrated significantloss of sulphate from GlcNAc-6-SO₄ in both GAGs. These findingsmay be relevant for future studies aimed at defining the function(s)of GlcNAc-6-SO₄ residues in GAGs and understanding the catabolismof GAGs, especially in regard to sulphatidoses, such as SanfilippoD syndrome in humans, which involves a deficiency of NG6SS activity catabolism endosulphatase glycosaminoglycans sulphation     

Comparison of human and mouse E-selectin binding to Sialyl-Lewis<Superscript>x</Superscript>

Background

During inflammation, leukocytes are captured by the selectin family of adhesion receptors lining blood vessels to facilitate exit from the bloodstream. E-selectin is upregulated on stimulated endothelial cells and binds to several ligands on the surface of leukocytes. Selectin:ligand interactions are mediated in part by the interaction between the lectin domain and Sialyl-Lewis x (sLe^x), a tetrasaccharide common to selectin ligands. There is a high degree of homology between selectins of various species: about 72 and 60 % in the lectin and EGF domains, respectively. In this study, molecular dynamics, docking, and steered molecular dynamics simulations were used to compare the binding and dissociation mechanisms of sLe^x with mouse and human E-selectin. First, a mouse E-selectin homology model was generated using the human E-selectin crystal structure as a template.

Results

Mouse E-selectin was found to have a greater interdomain angle, which has been previously shown to correlate with stronger binding among selectins. sLe^x was docked onto human and mouse E-selectin, and the mouse complex was found to have a higher free energy of binding and a lower dissociation constant, suggesting stronger binding. The mouse complex had higher flexibility in a few key residues. Finally, steered molecular dynamics was used to dissociate the complexes at force loading rates of 2000–5000 pm/ps². The mouse complex took longer to dissociate at every force loading rate and the difference was statistically significant at 3000 pm/ps². When sLe^x-coated microspheres were perfused through microtubes coated with human or mouse E-selectin, the particles rolled more slowly on mouse E-selectin.

Conclusions

Both molecular dynamics simulations and microsphere adhesion experiments show that mouse E-selectin protein binds more strongly to sialyl Lewis x ligand than human E-selectin. This difference was explained by a greater interdomain angle for mouse E-selectin, and greater flexibility in key residues. Future work could introduce similar amino acid substitutions into the human E-selectin sequence to further modulate adhesion behavior.

     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Repression of the Lewis fucosyl transferase by retinoic acid increases apical sialosyl Lewisa secretion in colorectal carcinoma cultures

The rate of polarised secretion of sialosyl Lewis^a(19-9) molecular species (SiaLeams) by SW1116 colorectal carcinoma cells is stimulated at least ninefold by the presence of 3 μM retinoic acid (RA). In order to investigate the intracellular origins of this augmentation, carcinoma cell membranes, membrane subfractions, and media were studied to determine alterations in sialosyl Lewis^a levels, oligosaccharide composition, and core structures accompanying the capacity to increase export of this epitope. We observed a nine- to twentyfold increase in sialosyl Lewis^a epitope levels in a light membrane subfraction from RA-treated cells. Antigenic molecules of < 200,000 Mr on acrylamide gradient gels were concentrated in two doublets in the apparent Mr range 106,000–152,000 on Western blots. Carbohydrates analyses of oligosaccharides from SiaLeams of membrane subfraction and apical media indicated much higher fucose/mannose, fucose/sialic, fucose/sialosyl Lewis^a, fucose/total CHO, and (³H) fucose incorporation in control samples than RA samples. Western blots of samples from membranes subfractions and media indicated that, in contrast to the effect of RA on the sialosyl Lewis^a epitope, RA treatment did not augment cysteine-rich, PDTRP, blood group H-2, blood group A, and EGF receptor-like region epitopes in the media. In addition, Northern blots using the Lewis fucosyl transferase (FTIII) cDNA showed a dramatic diminution of mRNA encoding FTIII but apparently unaltered levels of sialyl transferase (ST4) mRNA. Since subterminal fucosylation of lactosyl termini blocks terminal sialylation, we conclude that one mechanism of sialosyl Lewis^a induction in this culture system is the lower expression of the Lewis fucosyl transferase mRNA. Therefore less subterminal fucosylation of GlcNAc permits the prior sialylation of terminal Galβ1-3 moieties at oligosaccharide termini destined for export from the Golgi.     

Synthesis of a BSA-Lex glycoconjugate and recognition of Lex analogues by the anti-Lex monoclonal antibody SH1: The identification of a non-cross reactive analogue

A Le^x trisaccharide functionalized with a cysteamine arm was prepared and this synthesis provided additional information on the reactivity of N-acetylglucosamine O-4 acceptors when they are glycosylated with trichloroacetimidate donors activated with excess BF₃·OEt₂. In turn, this trisaccharide was conjugated to BSA lysine side chains through a squarate–mediated coupling. This BSA-Le^x glycoconjugate displayed 35 Le^x haptens per BSA molecule. The relative affinity of the anti-Le^x monoclonal antibody SH1 for the Le^x antigen and analogues of Le^x in which the d-glucosamine, l-fucose or d-galactose residues were replaced with d-glucose, l-rhamnose and d-glucose, respectively, was measured by competitive ELISA experiments. While all analogues were weaker inhibitors than the Le^x antigen, only the analogue of Le^x in which the galactose residue was replaced by a glucose unit showed no binding to the SH1 mAb. To confirm that the reduced or loss of recognition of the Le^x analogues by the anti-Le^x mAb SH1 did not result from different conformations adopted by the analogues when compared to the native Le^x antigen, we assessed the conformational behavior of all trisaccharides by a combination of stochastic searches and NMR experiments. Our results showed that, indeed, the analogues adopted the same stacked conformation as that identified for the Le^x antigen. The identification of a trisaccharide analogue that does not cross-react with Le^x but still retains the same conformation as Le^x constitutes the first step to the design of a safe anti-cancer vaccine based on the dimeric Le^x tumor associated carbohydrate antigen.