Structural characterization of the N-glycans of Dictyocaulus viviparus: discovery of the Lewis(x) structure in a nematode

This paper reports the first rigorous evidence for the existence of N-linked oligosaccharides in Dictyocaulus viviparus, an economically important nematode that parasitises cattle. Structural strategies based upon fast atom bombardment mass spectrometry were employed to examine detergent extracts of homogenised adult D.viviparus for their N-glycan content. These revealed that detergent-soluble material is rich in high mannose, truncated and complex-type families of N-linked oligosaccharides. Importantly, the most abundant antennae in the complex-type structures were shown to carry the Lewis(x)epitope (Galbeta1-4(Fucalpha1-3)GlcNAc). Although the Lewis(x)moiety occurs in other helminths such as schistosomes, nematodes have previously been thought to lack this epitope. The Lewis(x)epitopes in D.viviparus are carried on bi-, tri-, and tetraantennary glycans and are therefore candidates for recognition events requiring multivalent ligands. There is compelling evidence from schistosome research that glycoconjugates containing Lewis(x)structures are immunomodulators. We propose that the Lewis(x)-rich glycans identified in this study might similarly be involved in D.viviparus host interactions.     

The expression of free oligosaccharides in human seminal plasma

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis(x) and/or Lewis(y) epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAcbeta1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Galbeta1-4GlcNAc) but not the type 1 sequences (Galbeta1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract.     

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

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

Two squid skin proteoglycans each containing chondroitin sulfates with different sulfation patterns.

Two chondroitin sulfate containing proteoglycans, amounting to approximately 6% of the tissue proteoglycans, were isolated from the skin of the squid. They were almost completely extracted by 4 M guanidine hydrochloride in the presence of proteinase inhibitors, and then they were separated by DEAE-Sephacel chromatography and isolated by further chromatography on Sepharose CL-4B. Each proteoglycan contained two types of chondroitin sulfates that differed in their sulfation patterns. One proteoglycan (molecular mass (M(r)) 5.6 x 10(5)) contained, on the average, four chondroitins (M(r) 8.4 x 10(4)) and five chondroitin sulfates (M(r) 3.4 x 10(4)), whereas the other proteoglycan (M(r) 5.2 x 10(5)) contained three chondroitin sulfates (M(r) 1.1 x 10(5)) and five oversulfated chondroitin sulfates (M(r) 4.3 x 10(4)). The glycosaminoglycans were released from the proteoglycans by treatment with alkaline borohydride, separated from the oligosaccharides by chromatography on Bio-Gel P-30, and isolated by chromatography on DEAE-Sephacel and Sepharose CL-6B. Chondroitin sulfates were degraded by chondroitinase AC to an extent of 70% and consisted of significant amounts of disaccharides sulfated at C-4 of the galactosamine, disulfated disaccharides, and small amounts of nonsulfated disaccharides, as well as disaccharides that bore sulfates at C-6. Oversulfated chondroitin sulfate was degraded by chondroitinase AC to only 40% and contained appreciable amounts of disulfated and trisulfated disaccharides. The glycosaminoglycans also contained neutral monosaccharides; glucose was the predominant neutral sugar. A part of the oligosaccharides of both proteoglycans was of identical structure to that of chondroitin sulfate.     

A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha that mediates rapid clearance of lutropin.

We have identified a receptor in hepatic endothelial and Kupffer cells that binds oligosaccharides terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2-Man alpha (S4GGnM). This receptor can account for the rapid removal of the glycoprotein hormone lutropin, which bears unique Asn-linked oligosaccharides terminating in S4GGnM, from the circulation. Hepatic endothelial cells express 579,000 S4GGnM receptors at their surface and bind lutropin with an apparent Kd of 1.63 x 10(-7) M. Bound ligand is rapidly internalized. Binding does not require divalent cations, is reversed by incubation at pH 5.0 or below, and is inhibited by fucoidin but not by hyaluronate, heparin, chondroitin sulfate, or dextran sulfate. We propose that the S4GGnM-specific receptor represents a major mechanism for clearance of certain sulfated glycoproteins from the blood, including members of the glycoprotein hormone family.     

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

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

Diverse sugar-binding specificities of marine invertebrate C-type lectins

The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized alpha2-6 sialylation but not alpha2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with alpha-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewis(x) and Lewis(a) epitopes.     

Deciphering the Glycan Preference of Bacterial Lectins by Glycan Array and Molecular Docking with Validation by Microcalorimetry and Crystallography

Recent advances in glycobiology revealed the essential role of lectins for deciphering the glycocode by specific recognition of carbohydrates. Integrated multiscale approaches are needed for characterizing lectin specificity: combining on one hand high-throughput analysis by glycan array experiments and systematic molecular docking of oligosaccharide libraries and on the other hand detailed analysis of the lectin/oligosaccharide interaction by x-ray crystallography, microcalorimetry and free energy calculations. The lectins LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria are part of the virulence factors used by the pathogenic bacteria to invade the targeted host. These two lectins are not related but both recognize fucosylated oligosaccharides such as the histo-blood group oligosaccharides of the ABH(O) and Lewis epitopes. The specificities were characterized using semi-quantitative data from glycan array and analyzed by molecular docking with the Glide software. Reliable prediction of protein/oligosaccharide structures could be obtained as validated by existing crystal structures of complexes. Additionally, the crystal structure of BambL/Lewis x was determined at 1.6 Å resolution, which confirms that Lewis x has to adopt a high-energy conformation so as to bind to this lectin. Free energies of binding were calculated using a procedure combining the Glide docking protocol followed by free energy rescoring with the Prime/Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method. The calculated data were in reasonable agreement with experimental free energies of binding obtained by titration microcalorimetry. The established predictive protocol is proposed to rationalize large sets of data such as glycan arrays and to help in lead discovery projects based on such high throughput technology.     

Heparan sulfate and chondroitin sulfate proteoglycans inhibit E-selectin binding to endothelial cells

E-selectin is a cell adhesion molecule involved in the initial rolling and adhesion of leukocytes to the endothelium during inflammation. In addition, in vitro studies have suggested that an interaction between E-selectin and binding sites such as sialyl Lewis X-containing oligosaccharides on endothelial cells may be important for angiogenesis. In order to investigate the binding of E-selectin to endothelial cells, we developed an ELISA assay using chimeric E-selectin-Ig molecules and endothelial cells fixed on poly-L-lysine coated plates. Our results indicate that E-selectin-Ig binds to both bovine capillary endothelial cells and human dermal microvascular endothelial cells in a calcium-dependent and saturable manner. The binding is inhibited markedly by heparin and by syndecan-1 ectodomain, and moderately by chondroitin sulfate, but not by sialyl Lewis X-containing oligosaccharides. These results suggest that heparan sulfate and chondroitin sulfate proteoglycans on endothelial cells are potential ligands for E-selectin.     

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain.     

Solvolytic depolymerization of chondroitin and dermatan sulfates

It is essential to establish a library of glycosaminoglycan oligosaccharides from the chondroitin and dermatan sulfates to investigate their biological functions and structure-activity relationships (SARs). There are several approaches to obtain oligosaccharides using chemical and enzymatic degradation procedures; however, purification of each resulting oligosaccharide is complicated because of the diversity of sulfonation patterns present in these oligosaccharides. We have developed a new method for the solvolytic degradation for chondroitin and dermatan sulfates to obtain an oligosaccharide mixture that can be easily purified into chondro/dermato oligosaccharides for characterization by both ¹H NMR and MALDI-TOFMS. These oligosaccharides have a methyl-esterified uronate residue and a methyl 2-acetamido-2-deoxy-d-galactofuranoside at the nonreducing and reducing ends, respectively. All other internal repeating disaccharide units were desulfonated, but maintained their core carbohydrate structures.     

Glycosylation of dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside as saccharide primers in cells

Syntheses of oligosaccharides expressed on cells are indispensable for the improvement of the functional analyses of the oligosaccharides and their applications. We are developing saccharide primers for synthesizing oligosaccharides using living cells. In this study, dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (GlcNAc-C12) and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside (LacNAc-C12) were examined for their abilities to prime the syntheses of neolacto-series oligosaccharides in HL60 cells. When GlcNAc-C12 was incubated with HL60 cells in serum-free medium for 2 days, 14 kinds of glycosylated products were collected from the culture medium. They were separated by high-performance liquid chromatography. The sequences of the products were determined to be neolacto-series oligosaccharides including Lewis(X), sialyl Lewis(X), polylactosamine, and sialylpolylactosamine by mass spectrometry. GlcNAc-C12 was also glycosylated by B16 cells and gave sialyllactosamine. Furthermore, LacNAc-C12 gave similar glycosylated products to GlcNAc-C12.     

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.     

Experimental autoimmune encephalomyelitis mediated by T lymphocyte lines: genotype of antigen-presenting cells influences immunodominant epitope of basic protein

Lewis rats are susceptible to experimental autoimmune encephalomyelitis (EAE), and their T lymphocytes recognize epitopes in the 68-88 sequence of guinea pig myelin basic protein (BP). BN rats are resistant to EAE, and their T lymphocytes recognize epitopes outside of the 68-88 sequence, probably in the 43-67 portion of BP. To investigate the influence of the genome of antigen-presenting cells (APC) on the dominance of BP epitopes for T lymphocyte lines, we selected anti-BP lines from (Lewis X BN)F1 rats by using the APC of Lewis, BN, or F1 origin. We now report that the F1/Lewis and F1/F1 lines recognized the 68-88 epitopes and were highly encephalitogenic in F1 rats, whereas the F1/BN line recognized the 43-67 epitopes and was only weakly encephalitogenic. Thus, the genotype of the APC can influence the immunologic dominance for T lymphocytes of BP epitopes, and this dominance in turn can influence the expression of disease.     

Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment

Heparin is an excellent inhibitor of P- and L-selectin binding to the carbohydrate determinant, sialyl Lewis(x). As a consequence of its anti-selectin activity, heparin attenuates metastasis and inflammation. Here we show that fucosylated chondroitin sulfate (FucCS), a polysaccharide isolated from sea cucumber composed of a chondroitin sulfate backbone substituted at the 3-position of the beta-D-glucuronic acid residues with 2,4-disulfated alpha-L-fucopyranosyl branches, is a potent inhibitor of P- and L-selectin binding to immobilized sialyl Lewis(x) and LS180 carcinoma cell attachment to immobilized P- and L-selectins. Inhibition occurs in a concentration-dependent manner. Furthermore, FucCS was 4-8-fold more potent than heparin in the inhibition of the P- and L-selectin-sialyl Lewis(x) interactions. No inhibition of E-selectin was observed. FucCS also inhibited lung colonization by adenocarcinoma MC-38 cells in an experimental metastasis model in mice, as well as neutrophil recruitment in two models of inflammation (thioglycollate-induced peritonitis and lipopolysaccharide-induced lung inflammation). Inhibition occurred at a dose that produces no significant change in plasma activated partial thromboplastin time. Removal of the sulfated fucose branches on the FucCS abolished the inhibitory effect in vitro and in vivo. Overall, the results suggest that invertebrate FucCS may be a potential alternative to heparin for blocking metastasis and inflammatory reactions without the undesirable side effects of anticoagulant heparin.