N-acetylneuraminic Acid Residues Research Articles

Binding of lectins to novel migration promoters on cardiac mesenchymal cells in the chick.

Chicken serum promotes migration of cardiac mesenchymal cells of chick embryos in vitro. In the present study, migration promotion of unknown migration promoters in chicken serum was examined by using lectins. Cardiac mesenchymal cells of the conotruncal and atrioventricular cushions were cultured on collagen type-I gel with medium including chicken serum. A concentration (100 micrograms/ml) of Concanavalin A (Con A), peanut agglutinin (PNA), pisum sativum agglutinin (PSA), soybean agglutinin (SBA) or wheat germ agglutinin (WGA) was added to the medium. Con A, PSA, and WGA inhibited migration, while PNA and SBA did not affect migration of cardiac mesenchymal cells. WGA inhibited migration in a concentration dependent manner. Preincubation of WGA with specific binding monosaccharides (alpha-D-N-acetylglucosamine and alpha-D-N-acetylneuraminic acid) clearly reduced the inhibition ability of WGA, while preincubation of Con A and PSA with alpha-D-mannose and alpha-D-glucose did not. On the other hand, Con A-binding proteins, eluted from a Con A affinity column with the buffer including alpha-D-mannose and alpha-D-glucose, promoted migration of cardiac mesenchymal cells, as did WGA-binding proteins. These proteins promoted migration in a concentration dependent manner. Western blotting showed that PSA bound the subunits of collagen type-I, but ConA and WGA did not. In migration inhibition assays by monosaccharides, only N-acetylneuraminic acid inhibited migration of cardiac mesenchymal cells. These results suggested that chicken serum contains novel migration promoters for chick cardiac mesenchymal cells. The promoters are proposed to have the terminal N-acetylglucosamine, N-acetylneuraminic acid, and glucose and/or mannose residues.

Sialic Acid Specificity of Myelin-associated Glycoprotein Binding

Myelin-associated glycoprotein (MAG), a nervous system cell adhesion molecule, is an I-type lectin that binds to sialylated glycoconjugates, including gangliosides bearing characteristic structural determinants (Yang, L. J.-S., Zeller, C. B., Shaper, N. L., Kiso, M., Hasegawa, A., Shapiro, R. E., and Schnaar, R. L. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 814-818). Two cell adhesion systems, COS-1 monkey kidney fibroblasts transiently transfected to express MAG and Chinese hamster ovary (CHO) cells stably transfected to express MAG, were used to probe the structural specificity of MAG-ganglioside binding. Both cell types bound to the same gangliosides: GQ1b alpha (IV3NeuAc,III6NeuAc,II3(NeuAc)2Gg4Cer) > GT1b = GD1a > GM3 > GM1, GD1b, and GQ1b (the latter do not support adhesion). Binding was enhanced by pretreatment of MAG-expressing cells with neuraminidase. MAG-expressing Chinese hamster ovary cells bound directly to gangliosides resolved on thin layer chromatograms, allowing detection of MAG binding species in a mixture. The simplest ganglioside ligand for MAG was GM3 bearing N-acetylneuraminic acid, whereas GM3 bearing N-glycolylneuraminic acid did not support adhesion. Chemical modifications of N-acetylneuraminic acid residues (on GD1a) abrogated MAG binding. Mild periodate oxidation of sialic acids to their corresponding seven-carbon (or eight-carbon) sialic acid aldehydes abolished MAG binding, as did further conversion to the corresponding primary alcohols. Eliminating the anionic charge by ethyl esterification, amidation, or reduction also abolished MAG-mediated cell adhesion. These data demonstrate that MAG-ganglioside binding is highly specific and defines key carbohydrate structural determinants for MAG-mediated cell adhesion to gangliosides.

Dural microvessels: molecular properties of their luminal anionic sites

(1) Neurogenic inflammation has been implicated in the pathogenesis of the vascular headaches of migraine and cluster headaches. (2) Durai blood vessels are both pain-sensitive and show neurogenic plasma extravasation. (3) Endothelial cell (EC) surface anionic sites appear to be a determinant of vascular permeability. We therefore examined the anionic sites of durai EC to determine whether they are different from those of pial and parenchymal vessels. Luminal anionic sites of rat optic nerve EC were labelled with cationic colloidal gold (CCG) and cationic ferritin (CF) and examined by electron microscopy. Employing a battery of enzymes, the effects of digestion of ultrathin sections on subsequent labelling with CCG was quantified using image analysis software. In addition, a gold-labelled lectin, wheat-germ agglutinin (WGA), was employed to locate specific saccharide residues. Of the enzymes with a narrow specificity, only neuraminidase substantially reduced CCG binding. Of the proteolytic enzymes, papain was most effective in reducing labelling. These results show that the luminal EC anionic sites are chiefly composed of sialoglycoproteins. The labelling with biotinylated WGA-streptavidin gold was similar to that with CCG without enzyme digestion. This suggests that WGA is binding to N-acetylneuraminic (sialic) acid residues and not to the neutral N-acetylglucosamine (since CCG would not label uncharged molecules). These results do not differ significantly from those for pial and parenchymal EC. It is therefore likely that factors other than anionic site molecular composition account for the susceptibility of dural vessels to neurogenic plasma extravasation. The relevance of these observations in an experimental animal model to the human clinical condition remains to be determined.

Dural microvessels: molecular properties of their luminal anionic sites

(1) Neurogenic inflammation has been implicated in the pathogenesis of the vascular headaches of migraine and cluster headaches. (2) Durai blood vessels are both pain-sensitive and show neurogenic plasma extravasation. (3) Endothelial cell (EC) surface anionic sites appear to be a determinant of vascular permeability. We therefore examined the anionic sites of durai EC to determine whether they are different from those of pial and parenchymal vessels. Luminal anionic sites of rat optic nerve EC were labelled with cationic colloidal gold (CCG) and cationic ferritin (CF) and examined by electron microscopy. Employing a battery of enzymes, the effects of digestion of ultrathin sections on subsequent labelling with CCG was quantified using image analysis software. In addition, a gold-labelled lectin, wheat-germ agglutinin (WGA), was employed to locate specific saccharide residues. Of the enzymes with a narrow specificity, only neuraminidase substantially reduced CCG binding. Of the proteolytic enzymes, papain was most effective in reducing labelling. These results show that the luminal EC anionic sites are chiefly composed of sialoglycoproteins. The labelling with biotinylated WGA-streptavidin gold was similar to that with CCG without enzyme digestion. This suggests that WGA is binding to N-acetylneuraminic (sialic) acid residues and not to the neutral N-acetylglucosamine (since CCG would not label uncharged molecules). These results do not differ significantly from those for pial and parenchymal EC. It is therefore likely that factors other than anionic site molecular composition account for the susceptibility of dural vessels to neurogenic plasma extravasation. The relevance of these observations in an experimental animal model to the human clinical condition remains to be determined.

Synthesis of N-thioacetyl gangliosides GM 1 and GM 3

Reaction of de- N-acetyl gangliosides GM 1 ( 1) and GM 3 ( 2b) with methyl dithioacetate ( 3) afforded N-thioacetyl GM 1 ( 1c) and N-thioacetyl GM 3 ( 2c) with the oxygen of the acetamido group of N-acetylneuraminic acid residue replaced by sulfur.

Identification of rat serotonin 5-HT2C receptors as glycoproteins containing N-linked oligosaccharides.

Antibodies against a portion of the rat 5-HT2C receptor third intracellular loop were generated and used to identify receptors solubilized from cell lines and rat brain. Western blots of CHAPS-soluble proteins were probed with affinity-purified anti-2C antibodies. The specificity of anti-2C was demonstrated with extracts prepared from NIH/3T3 fibroblasts which stably express functional rat 5-HT2C or 5-HT2A receptors. Extracts from the 5-HT2C cell line, but not the 5-HT2A cell line, contained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid plexus extracts with masses of 51 kDa and 58-62 kDa. The major 58-62 kDa and minor 51 kDa proteins were not detected in extracts prepared from the hippocampus, striatum, or frontal cortex using the same amount of CHAPS-soluble protein. These results are consistent with previous studies demonstrating that 5-HT2C receptor binding sites and mRNA are most abundant in choroid plexus. The association of asparagine-linked (N-linked) oligosaccharides with the receptors was examined next. The 5-HT2C receptor cell line (3T3/2C) was grown in the presence of tunicamycin to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts prepared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically remove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroid plexus. Neuraminidase, which cleaves sialic acid (N-acetylneuraminic acid) residues from glycoproteins, reduced the mass of the 51 and 58-62 kDa proteins from the choroid plexus to 50 and 54-58 kDa. In contrast, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affected by treatment with neuraminidase. These results demonstrate that 5-HT2C receptors contain N-linked sugars and suggest that sialic acid residues associate with 5-HT2C receptors in the choroid plexus. The oligosaccharide moieties, which contribute up to approximately 30% of the relative mass as judged by SDS-polyacrylamide gel electrophoresis, may impart functional properties to 5-HT2C receptors.

Synthetic Studies on Sialoglycoconjugates 73: Synthesis of KDN-α-(2→6)-lactotetraosylceramide and KDN-α-(2→6)-neolactotetraosylceramide

Abstract Analogs of sialyl-α(2→6)-lactotetraosylceramide and sialyl-α(2→6)-neolactotetraosylceramide, in which the N-acetylneuraminic acid residue is replaced by a 3-deoxy-d-glycero-d-glacto-2-nonulopyranosylonic acid (KDN) unit, have been synthesized. Methyl O-(methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-d-glycero-α-d-galacto-2-nonulopyranosylonate)-(2→6)-2,4-di-O-benzoyl-3-O-benzyl-1-thio-β-d-galactopyranoside (6) was prepared from the phenyl β-thioglycoside derivative 1 of KDN and 2-(trimethylsilyl)ethyl 3-O-benzyl-β-d-galactopyranoside (2) in four steps. Each coupling of 2-(trimethylsilyl)ethyl O-(2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranosyl)-(1→3′)-per-O-benzyl-β-lactoside (7) or 2-(trimethylsilyl)ethyl O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-d-glucopyranosyl)-(1→3′)-per-O-benzyl-β-d-lactoside (8), with 6 gave the pentasaccharides 9 and 13 in good yields. Compounds 9 and 13 were converted into the corresponding α-trichloroacetimidates 12 and 16 which on glycosylation with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol (17), gave the β-glycosides 18 and 21, respectively. Finally, 18 and 21 were transformed, via selective reduction of the azido group, condensation with octadecanoic acid, O-deacylation, and saponification of the methyl ester group, into the target compounds 20 and 23, respectively.

Heterogeneity of the bovine κ-casein caseinomacropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry

Microheterogeneity occurs in the population of caseinomacropeptides (residues 106–169 of κ-casein) due to variation in the extent and type of oligosaccharide linked to this phosphoglycopeptide. Although caseinomacropeptide A variant (CMP A) was poorly resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) with spectrophotometric detection, it could be analysed with on-line electrospray-ionization mass spectrometry (ESI-MS). From the already established O-linked glycan chains at least fourteen glycosylated forms of CMP A were identified, besides the non-glycosylated and multiphosphorylated (1, 2 or 3 phosphate groups) peptides, giving a maximum number of eighteen known forms. Major subcomponents in CMP A are disialylated species. A maximum of three out of the five potential glycosylation sites were found to be substituted with carbohydrate chains in the most highly glycosylated forms, which may contain up to six N-acetylneuraminic acid residues per molecule. A minor form, diphosphorylated with one disialylated chain, was also detected. From these results, it was shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for the analysis of complex mixtures.

Abnormal expression and glycosylation of the large and small isoforms of myelin‐associated glycoprotein in dysmyelinating quaking mutants

The relative expression of large (L) and small (S) isoforms of the myelin-associated glycoprotein (MAG) and their glycosylation were compared in developing spinal cord of quaking and control mice. Using antisera specific for L- and S-MAG, respectively, it was shown that S-MAG is the principal isoform in quaking mice at all ages between 13 and 72 days, although L-MAG was just detectable by western blotting at the early ages. Both L- and S-MAG have higher apparent molecular weights in quaking mice than in controls. Experiments involving lectin binding and glycosidase treatment demonstrated that the higher molecular weight of MAG in the quaking mutant was due to a higher content of N-acetylneuraminic acid residues linked alpha 2-3 to galactose as well as to more branching of oligosaccharide moieties indicated by a higher content of subterminal galactose residues. The total sialic acid measured by HPAE-chromatography in purified quaking MAG was 40% higher than in control MAG. By contrast, quaking MAG contained less of the adhesion-related, HNK-1 carbohydrate epitope. Another difference was that a lower molecular weight form of MAG with predominantly high mannose oligosaccharides was prominent in young quaking mice, but not in controls. The abnormalities of MAG expression related to splicing of its mRNA and glycosylation may contribute to the myelin pathology in quaking mutants.

Microheterogeneity of Erythropoietin Carbohydrate Structure

The microheterogeneity of the carbohydrate structures on recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary cells has been evaluated by electrospray ionization (ESI) mass spectrometry (MS) of glycopeptide fragments. The microheterogeneity is largely associated with the presence or absence of terminal N-acetylneuraminic acid (Neu5Ac) residues, varying amounts of O-acetylation of the Neu5Ac residues, and the presence or absence of N-acetyllactosamine extensions. The N-linked carbohydrate structures were structurally diverse; 52 different N-linked oligosaccharide structures were identified. Consistent structural assignments could be made from data obtained using different proteolytic digests, ESI solvent systems (aqueous/methanol systems with acetic or formic acid), and on-line or off-line LC/MS analysis. All glycosylation sites exhibited some level of O-acetylation of Neu5Ac residues. Interestingly, glycosylation site asparagine-83 exhibits mono-O-acetyl and di-O-acetyl Neu5Ac residues, while the other sites, asparagine-24, asparagine-38, and serine-126, exhibit mainly mono-O-acetyl Neu5Ac derivatization. This difference in O-acetylation may be site specific or due to sample handling of labile structures. However, mild base treatment of rHuEPO with NaOH on ice removed the O-acetyl groups associated with a given carbohydrate structure, without adversely affecting the underlying oligosaccharide structure, resulting in a simplified mass spectra. Nuclear magnetic resonance spectroscopy of Neu5Ac residues released by neuraminidase treatment of total rHuEPO indicated that Neu5,9Ac2 residues were present. Additional resonances were also observed that were consistent with other Neu5Ac O-acetyl linkages; these O-acetyl resonances could be removed by mild base hydrolysis of rHuEPO.

Oligosaccharide behavior of complex natural glycosphingolipids in multicomponent model membranes.

Wideline 2H NMR of model membranes was used to consider the molecular consequences of factors often suggested as modulators of complex glycosphingolipid oligosaccharide arrangement and motional characteristics at cell surfaces. GM1, asialo-GM1, and globoside were studied as examples of plasma membrane recognition sites. The experimental approach involved substitution of deuterons (D) for protons at specific locations within the carbohydrate chains. Deuterated glycolipids were then dispersed at 7-10 mol% in unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine. Factors tested for their significance to carbohydrate chain conformation and dynamics included glycolipid natural alkyl and acyl chain variability, membrane fluidity, and the presence of cholesterol and a charged sugar residue (neuraminic acid). Effects of Ca2+ and membrane-associated protein were briefly considered. Two distinct strategies were employed in substituting deuterons for selected protons of carbohydrate residues. Neither approach necessitated alteration of the glycolipid natural fatty acid composition. (i) Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of GM1 and asialo-GM1, and on the terminal N-acetylgalactosamine (GalNAc) residue of globoside, were replaced with deuterium (producing -CDHOH) by an enzymatic oxidation/reduction cycle. This represents the first application of such an approach to deuteration of complex neutral glycolipids. Spectral results were compared to those obtained for the similarly-deuterated monoglycosyl lipid, galactosylceramide (GalCer), with natural fatty acid composition. Efficacy of this labeling method may in principle be influenced by structural variations within a given glycolipid family. Also, asymmetric rotation of the deuterated group made it less attractive than the second method for relating spectral features to receptor geometry. (ii) A general synthetic, nonenzymatic method was investigated for replacing amino sugar N-acetyl groups with deuterated acetate (-COCD3). The acetate group of the GalNAc residue of globoside, GM1, and asialo-GM1, as well as that on neuraminic acid in GM1, was replaced with -COCD3. This second method afforded better signal-to-noise--an important consideration for 2H NMR. The NMR technique employed had the potential for detecting changes of as little as 10% in oligosaccharide orientation or motional order. Each glycolipid demonstrated clear evidence of preferred average oligosaccharide conformations in all (fluid) membrane environments examined. The most striking observation was that, in fluid matrices, conformation and motional order of the complex oligosaccharide chains were only modestly influenced by factors tested, including natural variation in the glycolipid hydrocarbon chains, membrane fluidity, temperature, and the presence of cholesterol or the N-acetylneuraminic acid (NeuAc) residue on GM1.(ABSTRACT TRUNCATED AT 400 WORDS)

Optic nerve microvessels: a partial molecular definition of cell surface anionic sites

Incorporated in the luminal glycocalyx of vascular endothelia (EC) are negatively charged microdomains (anionic sites). These sites are considered functionally important (a) in their interaction with circulating blood constituents, and (b) as a determinant of vascular permeability. The molecular composition of these EC sites, described for a number of tissues, has demonstrated a heterogeneity dependent on their anatomical location. Luminal anionic sites have not been characterized for EC of optic nerve. Optic nerves were removed from Sprague-Dawley rats previously fixed by vascular perfusion. EC anionic sites were labelled with the probes cationic colloidal gold (CCG) and cationic ferritin (CF), using the pre- and post-embedding techniques, and examined by electron microscopy. The effects of enzyme digestion of ultrathin sections on subsequent CCG labelling were determined using a battery of enzymes in association with the post-embedding technique. CCG labelling was quantified following each enzyme treatment using image analysis software. The biotinylated lectin wheat germ agglutinin (WGA) with streptavidin gold was also used to localize specific monosaccharide residues. The luminal front of intraneural EC showed a uniform labelling with CCG and CF which was greater than on the abluminal surface. Extracellular matrix components and basal laminae were moderately labelled. Digestion of tissue sections with heparitinase and trypsin had no significant effect on subsequent CCG labelling. Proteinase K was less effective than papain but both produced a significant reduction. Neuraminidase almost completely eliminated labelling. CCG binding to the luminal plasma membrane of optic nerve EC can be significantly reduced with proteolytic and glycolytic enzymes. The results demonstrate that sialoglycoproteins principally constitute these luminal EC anionic sites. Biotinylated WGA-streptavidin gold, which detects both N-acetylneuraminic (sialic) acid and N-acetylglucosamine, gave a similar pattern of labelling to CCG alone on the luminal versus abluminal EC fronts. These findings suggest that WGA is binding predominantly to N-acetylneuraminic acid residues since CCG would not label the neutral (uncharged) N-acetylglucosamine.

Carbohydrate Structures of β‐Trace Protein from Human Cerebrospinal Fluid: Evidence for “Brain‐Type”N‐Glycosylation

The carbohydrate structures of beta-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N-glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N-acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N-acetylglucosamine residues of nearly all structures were found to be alpha 1,6-fucosylated. Peripheral fucose (about 20% alpha 1,3-linked to N-acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N-acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N-acetylglucosamine only were present. At least 20 different N-glycans were identified. Analysis of the site-specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N-acetylglucosamine, as well as terminal N-acetylglucosamine and galactose residues, and significant amounts of N-acetylneuraminic acid in alpha 2,3 linkage), this protein can be classified as "brain-type" glycosylated.

Tyrosine Derivatization and Preparative Purification of the Sialyl and Asialyl N-Linked Oligosaccharides from Porcine Fibrinogen

The N-linked oligosaccharides from porcine fibrinogen were purified following their release from glycopeptides using N-glycosidase F. In separate experiments, both sialyl and asialyl oligosaccharides were prepared from 5 g of fibrinogen. The reducing oligosaccharides were reacted with ammonium bicarbonate to form oligosaccharide-glycosylamines and then derivatized with tert-butoxycarbonyl-L-tyrosine N-hydroxysuccinimidyl ester. Tyrosinamide-oligosaccharides were purified first by gel filtration chromatography and then by reverse-phase HPLC and the products were characterized by proton NMR and fast atom bombardment-MS. Porcine fibrinogen was found to have predominantly a single asialyl biantennary oligosaccharide containing a fucose linked α1-6 to GlcNAc 1. The oligosaccharide possesses two sialylation patterns with a major form (70%) having a single N-acetyl neuraminic acid (NeuAc) residue linked α2-6 to galactose on only one antenna and a minor form (30%) possessing two NeuAc residues linked α2-6 to both terminal galactose residues. In addition to developing an isolation procedure and establishing the structures of porcine fibrinogen oligosaccharides, this study improves on the tyrosine derivatization technique as a general approach to isolate structurally diverse N-linked oligosaccharides from glycoproteins.

Synthesis of KDN-lactotetraosylceramide, KDN-neolactotetraosylceramide, and KDN-Lewis X ganglioside

Analogues of sialyl-lactotetraosylceramide, sialyl-neolactotetraosylceramide, and sialyl Lewis X ganglioside, in which the N-acetylneuraminic acid residue is replaced by a 3-deoxy- d- glycero- d- galacto-2-nonulopyranosonic acid (KDN) unit, have been synthesized. Methyl O-(methyl 4,5,7,8,9-penta- O-acetyl-3-deoxy- d- glycero-α- d- galacto-2-nonulopyranosylonate)-(2 → 3)-2,4,6-tri- O-benzoyl-1-thio-β- d-galactopyranoside ( 4) was prepared from 2-(trimethylsilyl)ethyl O-(methyl 4,5,7,8,9-penta- O-acetyl-3-deoxy- d- glycero-α- d- galacto-2-nonulopyranosylonate)-(2 → 3)-6- O-benzoyl-β- d-galactopyranoside, via O-benzoylation, replacement of the 2-(trimethylsilyl)ethyl group by acetyl, and introduction of the methylthio group with trimethyl(methylthio)silane. Glycosylation of 2-(trimethylsilyl)ethyl O-(2-acetamido-4, 6- O-benzylidene-2-deoxy-β- d-glucopyranosyl)-(1 → 3)- O-(2,4,6-tri- O-benzyl-β- d-galactopyranosyl)-(1 → 4)-2,3,6-tri- O-benzyl-β- d-glucopyranoside ( 5) or of 2-(trimethylsilyl)ethyl O-(2-acetamido-3,6-di- O-benzyl-2-deoxy-β- d-glucopyranosyl)-(1 → 3)- O-(2,4,6-tri- O-benzyl-β- d-galactopyranosyl)-(1 → 4)-2,3,6-tri- O-benzyl-β- d-glucopyranoside, prepared from 5 via O-benzylation and reductive opening of the benzylidene acetal ring, with 4 as a donor gave the corresponding pentasaccharides 9 and 13 in good yields. In the same way, 4 was reacted with 2-(trimethylsilyl)ethyl O-(2,3,4-tri- O-benzyl-α- l-fucopyranosyl)-(1 → 3)- O-(2-acetamido-6- O-benzyl-2-deoxy-β- d-glucopyranosyl)-(1 → 3)- O-(2,4,6-tri- O-benzyl-β- d-galactopyranosyl)-(1 → 4)-2,3,6-tri- O-benzyl-β- d-glucopyranoside to yield the hexasaccharide 17. These three oligosaccharides 9, 13, and 17 were converted via reductive removal of the benzyl groups and benzylidene group, O-acetylation, removal of the 2-(trimethylsilyl)ethyl group, and subsequent reaction with trichloroacetonitrile, into the corresponding trichloroacetimidates 12, 16, and 20, respectively. Glycosylation of (2 S,3 R,4 E)-2-azido-3- O-benzoyl-4-octadecene-1,3-diol with 12, 16, and 20 in the presence of boron trifluoride etherate afforded the expected β-glycosides, which were transformed via selective reduction of the azido group, coupling with octadecanoic acid, O-deacylation, and de-esterification, into the target gangliosides in high yields.

Molecular cloning of a murine N-acetylgalactosamine transferase cDNA that determines expression of the T lymphocyte-specific CT oligosaccharide differentiation antigen.

Two monoclonal antibodies termed CT1 and CT2 define a cell surface oligosaccharide molecule expressed on restricted populations of murine lymphocytes. This oligosaccharide structure is largely associated with the extracellular domain of the CD45 family of tyrosine phosphatases, molecules required for lymphocytes to proliferate in response to antigen stimulation. Previous work has shown that the oligosaccharide structure recognized by the CT antibodies is identical, at least in part, to that of the human Sda blood group, a structure formed through enzymatic addition of N-acetylgalactosamine in beta 1,4-linkage to a sub-terminal galactose substituted with an alpha 2,3-linked N-acetylneuraminic acid residue. We have used a mammalian transient expression cloning approach to isolate a murine cDNA that determines expression of an oligosaccharide structure recognized by the CT antibodies as well as human anti-Sda serum. The nucleotide sequence predicts a 510-amino acid type II transmembrane protein characteristic of other mammalian glycosyltransferases. Enzymatic characterization of the protein expressed by this cDNA demonstrates that it encodes a beta 1,4-N-acetylgalactosamine transferase activity that can add to the low molecular weight acceptor 3'-sialyl-N-acetyllactosamine, to form the nonreducing terminal tetrasaccharide Sda blood group structure. This cDNA shares 51% nucleotide sequence identity with a cDNA encoding the human GM2/GD2 synthase, particularly throughout the regions encoding their putative catalytic domains. Southern blot analysis demonstrates that these two cDNA's represent distinct loci in the murine genome. The CT-GalNAc transferase cDNA isolated here represents a tool with which to define the role(s) of lymphocyte cell surface CT determinants, and may facilitate the isolation of the human Sda blood group locus through cross-hybridization approaches.

Subcellular characterization of glycoproteins in the principal cells of human gallbladder. A lectin cytochemical study.

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide side-chains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(alpha 2-3)galactose(beta 1-3)N-acetylgalactosamine, N-acetylneuraminic acid(alpha 2-3)galactose(beta 1-4)N-acetylglucosamine and galactose(beta 1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.

Lipopolysaccharides of Campylobacter jejuni serotype O:19: structures of core oligosaccharide regions from the serostrain and two bacterial isolates from patients with the Guillain-Barré syndrome.

Lipopolysaccharides from phenol-water extraction of cells of Campylobacter jejuni serotype O:19 were separated into a water-soluble gel of low M(r) and a water-soluble component of high M(r). Acetic acid hydrolysis of the ketosidic linkages to lipid A furnished respectively a core oligosaccharide, the structure of which is reported herein, and an O antigenic polysaccharide. Structural investigations were performed on the O-deacetylated lipopolysaccharide of low M(r), the liberated core oligosaccharide and the various products from removal of neuraminic acid and phosphate residues, and from the Smith degradation. It is concluded that the lipopolysaccharide from the serostrain has a core region with two types of closely related oligosaccharide chains showing striking homologies with gangliosides, the first with a single N-acetylneuraminic acid residue in an outer chain resembling GM1 and the second with two N-acetyl-neuraminic acid residues with a terminal region resembling GD1a. Similar experiments were carried out on lipopolysaccharides of low M(r) from bacterial isolates OH 4384 and OH 4382 serotyped as O:19 that had been obtained from two patients who subsequently developed the Guillain-Barré syndrome. The core oligosaccharide region of lipopolysaccharide from the former isolate differed only slightly from that of the serostrain, whereas that from the latter isolate was distinctly shorter.

Chemical structures of the core regions of Campylobacter jejuni serotypes O:1, O:4, O:23, and O:36 lipopolysaccharides.

Complete structures, including the location of N-acetylneuraminic acid (Neu5Ac) residues, were assigned for the core regions of Campylobacter jejuni serotypes O:1, O:4, and O:23 and O:36 lipopolysaccharides (LPS). In continuation of earlier studies, structure determinations of liberated oligosaccharides and, where necessary, of intact LPS, were by 1H-NMR spectroscopy, Smith degradation, chromium trioxide and enzymic degradations, in conjunction with methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analyses by gas chromatography/mass spectrometry. It was concluded on the basis of the following structures, in which each was linked 1-->5 to a terminal 3-deoxy-D-manno-octulosonic acid residue, that the core regions with qualitatively similar sugar compositions showed serotypic differences in one or more of their sequences, linkage types, and anomeric configurations: [formula: see text] [corrected]. The outer regions of each structure carry Neu5Ac residues linked 2-->3 to available beta-D-Galp residues and show striking similarities with various glycosphingolipids of the ganglioside family. However, Neu5Ac epitopes are not apparently involved in determining serospecificity.

A sub-population of keratan sulphates derived from bovine articular cartilage is capped with α(2–6)-linked N-acetylneuraminic acid residues. Affinity chromatography using immobilized Sambucus nigra lectin and characterization using 1H n.m.r. spectroscopy

Alkaline borohydride-reduced keratan sulphate (KS) chains derived from bovine femoral head cartilage were fractionated by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) into binding and non-binding populations. Analysis of the SNA-binding and non-binding KS chains using 600 MHz 1H n.m.r. spectroscopy showed that the former population contained alpha(2-6)-N-acetylneuraminic acid residues and the latter contained primarily alpha(2-3)-N-acetylneuraminic acid residues as chain terminators. Both populations contained a similar proportion of alpha(2-3)-N-acetylneuraminic acid residues within their protein-linkage regions, and similar sulphation and fucosylation levels. Analysis of these two fractions by gel-permeation chromatography (g.p.c.) on a TSK-30 XL column showed them to have the same size distributions. It was concluded from the n.m.r. spectra and g.p.c. data that the populations differed primarily in the mode of linkage of the chain-terminating sialic acids.