Core saccharide dependence of sialyl Lewis X biosynthesis

The sialyl-Lewis X (SLe^x) determinant is important in leukocyte extravasation, metastasis and bacterial adhesion. The role of the protein, N-glycan and O-glycan core structures for the biosynthesis of SLe^x in vivo by fucosyltransferases (FucTs) is not known. Immunoglobulin G (IgG) Fc fusion proteins of α₁-acid glycoprotein (AGP), P-selectin glycoprotein ligand-1 (PSGL-1) or CD43 were used to probe the specificity of FucT-III-VII expressed alone in 293T and COS cells or together with O-glycan core enzymes in Chinese hamster ovary (CHO)-K1 cells. Western blotting with the monoclonal antibodies CSLEX and KM93 showed that FucT-III and V-VII produced SLe^x on core 2 in CHO cells. Only FucT-V, -VI and, with low activity, -VII worked on core 3 on CD43/IgG, but no SLe^x was detected with CSLEX on PSGL-1/IgG with core 3. KM93 stained SLe^x on core 2, but was not reactive with SLe^x on core 3. FucT-III, V-VII made SLe^x on N-glycans of AGP/IgG in CHO, but not in COS and 293T cells, even though the same FucTs could make SLe^x on CD43/IgG and PSGL-1/IgG in these cells. Our results define the specificities of FucT-III-VII in SLe^x biosynthesis on O-glycans with different core structures and the fine specificity of the widely used anti-SLe^x monoclonal antibody, KM93.     

Synthesis of a highly hydrophobic dimeric Lewis X containing glycolipid: A model for the study of homotypic carbohydrate--carbohydrate interaction

This paper describes the synthesis of an octasaccharidic glycolipid 1 based on a stereo- and regioselective glycosylation between a Lewis X trisaccharidic donor and a pentasaccharidic acceptor. A highly hydrophobic lipid moiety of a new type was selected, making the compound 1 a good candidate for the study of the interaction of Lewis X functionalised vesicles.     

Dynamics of sialyl Lewis(a) in aqueous solution and prediction of the structure of the sialyl Lewis(a)-SelectinE complex

In this article we investigate all possible three-dimensional structures for sialyl Lewis^a (SLe^a) in aqueous solution and we predict without a priori experimental information its conformation when bound to SelectinE by using a combination of long molecular dynamics (MD) simulations. Based on 10 ns MD studies, three structures differing in glycosidic conformations are proposed for SLe^a in aqueous solution. Based on a 4 ns MD study of the SLe^a-SelectinE complex with initial structures derived from our prediction tools, we find that, fucose and N-acetyl neuraminic acid are in close contact with SelectinE and therefore expect interactions of the protein with these two sugar rings to be significantly more important than in the case of galactose and N-acetyl glucosamine. Our predictions indicate that the N-acetyl glucosamine of SLe^a is positioned primarily in the aqueous phase. In order to be able to interact with SLe^a the side chains of amino acid residues Lys99 and Lys111 in SelectinE appear to undergo large conformational changes when contrasted with the positions of these residues in the X-ray crystal structure. Furthermore, amino acid residues Arg97, Glu98 and Lys99 are acting as a holding arm to position the NeuNAc of SLe^a in the binding pocket.     

Chemoenzymatic synthesis of L-galactosylated dimeric sialyl Lewis X structures employing alpha-1,3-fucosyltransferase V

L-Galactosylated dimeric sialyl Lewis X (SLeX) has been prepared employing a combination of chemical and enzymatic synthetic methods. GDP-L-galactose has been chemically synthesised. Enzymatic transfer of L-galactose onto the acceptor (Sia-alpha2,3-Gal-beta1,4-GlcNAc-beta1,3/6)2-Man-alpha1-OMe was achieved using the human alpha-1,3-fucosyltransferase V.     

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLex and sLea respectively) decorated ligands. Endothelial cells have been shown to express sLex epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLex on sialylated N-acetyllactosamine via the action of α(1,3)fucosyltransferase(s), endothelial cells can also degrade sLex to Lewis x through the action of α(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLex, which facilitates their adhesion to endothelial E- and P-selectin. This revised version was published online in November 2006 with corrections to the Cover Date.     

Glycomic mapping of pseudomucinous human ovarian cyst glycoproteins: identification of Lewis and sialyl Lewis glycotopes

Expression of sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) on cell-surface glycoproteins endows cells with the ability to adhere to E-, P-, and L-selectins present on endothelia, platelets, or leukocytes. Special arrangements of these glycotopes in cancers are thought to play a key role in metastasis. Previous studies have mostly described membrane-bound sLe(x) and sLe(a) activities. In this report, the major O-glycans of the secreted human ovarian cyst sialoglycoproteins from a Le(a+) nonsecretor individual (human ovarian cyst sample 350) were characterized by MS/MS analyses and immuno-/lectin-chemical assays. The results showed that HOC 350 carries a large number of epitopes for sLe(x), sLe(a), and Le(a) reactive antibodies. Advanced MS/MS sequencing coupled with mild periodate oxidation and exoglycosidase digestions further revealed that the O-glycans from HOC 350 are mostly of core 1 and 2 structures, extended and branched on the 3-arm with both type I and type II chains, complete with variable degrees of terminal sialylation and/or fucosylation to yield the sLe(x) or sLe(a) epitopes. Thus, the underlying core and peripheral backbone structures are similar to that of a previously proposed composite structural model for nonsialylated human ovarian cysts O-glycans, but with some notable distinguishing structural features in addition to sialylation.     

IgG antibodies to Lewis type 2 antigens in serum of H. pylori-infected and noninfected blood donors of different Lewis(a,b) blood-group phenotype

Individuals of the Le(b+)/secretor phenotype revealed a stronger natural immune response to Le(x) and Le(y) epitopes irrespective of Helicobacter pylori serologic status. In contrast, H. pylori-infected Le(b-) type individuals showed a significantly higher proportion of strong responders to Le(x) antigen compared with the H. pylori-uninfected subgroup. The data suggest that the immune response to Lewis type 2 determinants is related to both the H. pylori serologic status and the Le(a,b) phenotype of the host.     

Visualization of sialyl Lewis(X) glycosphingolipid microdomains in model membranes as selectin recognition motifs using a fluorescence label

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the cellular immune response. For efficient recognition, the relevant carbohydrate epitope sialyl Lewis(X) (sLe(X); alpha-Neup5Ac-(2-->3)-beta-Galp-(1-->4)-[alpha-Fucp-(1-->3)]GlcpNAc) has to be arranged in clusters. We describe the synthesis of the sLe(X)-glycosphingolipid (sLe(X)-GSL) with a NBD fluorescence label in the tail region, which allows the direct visualization of sLe(X)-GSL microdomains to very low concentrations (0.01mol%) in various planar phosphocholine matrices by fluorescence microscopy. Cell rolling experiments of E-selectin expressing cells along these membranes confirmed that the fluorescence analog behaves similar to the naturally occuring sLe(X)-GSL. This is direct evidence for recent hypotheses on multivalent sLe(X) binding as molecular basis for selectin-mediated cell rolling.     

Characterization of a diagnostic Fab fragment binding trimeric Lewis X

Lewis X trisaccharides normally function as essential cell–cell interaction mediators. However, oligomers of Lewis X trisaccharides expressed by the parasite Schistosoma mansoni seem to be related to its evasion of the immune response of its human host. Here we show that monoclonal antibody 54‐5C10‐A, which is used to diagnose schistosomiasis in humans, interacts with oligomers of at least three Lewis X trisaccharides, but not with monomeric Lewis X. We describe the sequence and the 2.5 Å crystal structure of its Fab fragment and infer a possible mode of binding of the polymeric Lewis X from docking studies. Our studies indicate a radically different mode of binding compared to Fab 291‐2G3‐A, which is specific for monomeric Lewis X, thus providing a structural explanation of the diagnostic success of 54‐5C10‐A. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Investigation of glycosylating properties of 1-deoxy-1-ethoxysulfonyl-hept-2-ulopyranosyl derivatives. Synthesis of a new sulfonic acid mimetic of the sialyl Lewis X tetrasaccharide

Glycosylation reactions of the ethylthio, bromo and chloro derivatives of 1-deoxy-1-ethoxysulfonyl-hept-2-ulopyranose were studied applying different acceptors under different conditions. Elimination side-reactions affording exo- and endoglycals occured in all cases, however, with different proportions. Glycosyl chloride donor was applied to glycosylate a trisaccharide acceptor obtaining a new sulfonic acid mimetic of the sialyl Lewis X tetrasaccharide in high yield.     

Synthesis and biological evaluation of a sialyl Lewis X mimic with significantly improved E-selectin inhibition

The synthesis of the highly potent E-selectin inhibitor 5 is described. Sialyl Lewis X mimic 5 was rationally designed by combining two previously disclosed beneficial sLe(x) modifications in a single molecule. The compound was found to be 30-fold more potent than sLe(x) in a static, cell-free equilibrium assay. Furthermore, compound 5 was highly active (IC50 = 10 microM) in a dynamic non-equilibrium assay in which sLe(x) did not inhibit neutrophil rolling at up to 1000 microM.     

NMR study on the hydroxy protons of the Lewis X and Lewis Y oligosaccharides

The ¹H NMR chemical shifts and NOEs of hydroxy protons in Lewis X trisaccharide, β-d-Galp-(1 → 4)[α-l-Fucp-(1 → 3)]-β-d-GlcpNAc, and Lewis Y tetrasaccharide, α-l-Fucp-(1 → 2)-β-d-Galp-(1 → 4)[α-l-Fucp-(1 → 3)]-β-d-GlcpNAc, were obtained for 85% H₂O/15% (CD₃)₂CO solutions. The OH-4 signal of Galp in Lewis X and OH-3, OH-4 signals of Galp, and OH-2 signal of Fucp linked to Galp in Lewis Y had chemical shifts which indicate reduced hydration due to their proximity to the hydrophobic face of the Fucp unit linked to GlcpNAc. The inter-residue NOEs involving the exchangeable NH and OH protons confirmed the stacking interaction between the Fucp linked to the GlcpNAc and the Galp residues in Lewis X and Lewis Y.     

New derivatives of reducing oligosaccharides and their use in enzymatic reactions: efficient synthesis of sialyl Lewis a and sialyl dimeric Lewis x glycoconjugates

The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbonylaminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyaldi tol oligosaccharide derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars. Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene treatment, and protein coupling was carried out.     

Chemoenzymatic synthesis of the sialyl Lewis X glycan and its derivatives

A combination of recombinant FKP and α-(1→3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5′-diphosphate-β-l-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.     

Synthesis of a dimeric Lewis X hexasaccharide derivative corresponding to a tumor-associated glycolipid

The dimeric Lewis X hexasaccharide p-trifluoroacetamidophenylethyl O-beta-D-galactopyranosyl-(1----4)-O-[alpha-L-fucopyranosyl-(1----3)]-O- (2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----3)-O-beta-D-galactopyrano syl- (1----4)-O-[alpha-L-fucopyranosyl-(1----3)]-2-acetamido-2-deoxy-beta-D- glucopyranoside (14), which is a derivative of a tumor-associated glycolipid, was synthesized from thioglycoside intermediates. A protected disaccharide was used as a key-intermediate for synthesis of the p-nitrophenylethyl glycoside of suitably protected O-beta-D-Galp-(1----4)-O-beta-D-GlcpN-(1----3)-O-beta-D-Galp-(1--- -4)-beta-D- GlcpN, which, after selective deblocking, was di-L-fucosylated and deprotected to give 14.     

Chemical preparation of sialyl Lewis x using an enzymatically synthesized sialoside building block

The sialyl Lewis x tetrasaccharide with a propylamine aglycon was assembled by chemoselective glycosylation from a p-tolyl thioglycosyl donor obtained from an enzymatically synthesized sialodisaccharide. Combining the advantages of highly efficient enzymatic synthesis of sialoside building blocks, and diverse chemical glycosylation, this chemoenzymatic approach is practical for obtaining complex sialosides and their analogues.