Rüdiger W. Veh

New chromatographic system for the rapid analysis and preparation of colostrum sialyloligosaccharides

A new thin-layer chromatographic system on silica gel for the separation of sialyloligosaccharides is described. Calibration of the system with standard milk and colostrum sialyloligosaccharides is presented. The use of the system in monitoring different oligosaccharides is demonstrated for the purification of bovine colostrum sialyllactose isomers and a commercial sialyllactose product, and is discussed with respect to other biological fluids. The large-scale preparation of pure sialyllactose isomers from bovine colostrum is achieved using an improved ion-exchange separation on Dowex 1-X2 (less than 400 mesh) employing isomolar elution at 20 mM for monosialyloligosaccharides and 200 mM for disialyllactose. The purification of four major monosialyltrisaccharides, the 2-3 and 2-6 isomers of N-acetylneuraminyllactose, N-glycolylneuraminyl2-3lactose and N-acetylneuraminyl2-6-N-acetyllactosamine, and the disialyltetrasaccharide di-N-acetylneuraminyllactose is reported. The detection and partial purification of three new minor monosialyloligosaccharides is described.

Neuraminic acid-specific modification and tritium labelling of gangliosides.

1. A crude ganglioside mixture and pure GM1 and GD1a from bovine brain grey matter were prepared on a large scale. 2. The C7- and G8-analogues of NeuNAc were prepared from Collocalia mucoid and their structures established by gas-liquid chromatography and mass spectrometry. 3. Using model compounds in addition to various gangliosides, the conditions for the periodate oxidation and subsequent borohydride reduction of gangliosides were investigated with regard to the yield of C7- and C8-analogues of NeuNAc and the integrity of other monosaccharides in the oligosaccharide chain. These conditions were optimised to yield maximum C8-NeuNAc production and low C7-NeuNAc formation. Thus products were obtained which closely resemble the native gangliosides. 4. Using boro [3H] hydride, ganglioside derivatives with high specific radioactivity were prepared for the first time, containing either NeuNAc and labelled C8-NeuNAc or mainly labelled C7-NeuNAc depending on the prevailing conditions.

“Neuraminidase-Resistant” Sialic Acid Residues of Gangliosides

Gangliosides have been found to contain up to five sialic acid moieties bound to galactose residues in α, 2->3 linkages either at the end of the oligosaccharide chain or within the chain, and may form di- and trisialyl groups with α,2->8 linkages (1,2). N-Acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuG1) have been identified as the commonly occurring sialic acids in gangliosides, while the O-acetylated sialic acids occur less frequently. The 9-O-acetyl-N-acetylneuraminic acid has been found in gangliosides from a variety of vertebrates including man (3), and 4-O-acetyl-N-glycolylneuraminic acid (4-OAc-NeuGl) has been isolated from horse erythrocyte hematoside (4).

Methylation analysis determination of acylneuraminic acid residue type 2-> 8 glycosidic linkage. Application to GT1b ganglioside and colominic acid

Acylneuraminic acids are known to occur in carbohydrates and glycoconjugates in different positions: at the non-reducing end (in general), at the reducing end (disaccharides [ 1,2] bacterial polysaccharides [ 1,3-51) and as inter-chain residues (polysaccharides [ 1,3-S] , glycoproteins [6] , gangliosides [7-91). Permethylation analysis of compounds containing acylneuraminic acids has been reported in literature [7,9-l 11. However, these studies inform only about the structure of the asialo-part of the molecules, as only partly methylated neutral and amino sugars have been investigated. Incorporation of neuraminic acid derivatives into methylation analysis is of great value for the elucidation of the complete structure of carbohydrates and glycoconjugates containing neuraminic acid. In this paper the analysis is described of (partly) methylated acylneuraminic acids, obtained on methanolysis and subsequent re-N-acetylation from

Subcellular site of the biosynthesis ofO-acetylated sialic acids in bovine submandibular gland

Bovine submandibular glands were homogenized and fractionated under conditions which yielded subcellular fragments from mainly one cell type, the mucous acinar cell, as judged by morphological analysis of the glands before and after homogenization. The majorN-acetylneuraminate-9(7)-O-acetyltransferase activity was detected in the cytosolic fraction, a result supported by the high specific radioactivity of free sialic acids isolated after [14C]acetate-labelling experiments. Separation of membranes on a Ficoll density gradient gave six fractions which were analyzed biochemically and morphologically. The particulate activities of acetyltransferase and sialyltransferase were found in fractions containing smooth and mitochondrial membranes. MembraneO-acetyl sialic acids were present at the highest levels in these fractions and also had the highest specific radioactivity after [14C]acetate-labelling experiments. Significant amounts of theO-acetyltransferase activity also occur in the cytosol and are consistent with a model ofO-acetyl sialic acid biosynthesis involving both cytosolic and smooth membrane sites ofO-acetylation.

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio ofN-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin.Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts ofN-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content ofN-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8×105 Da and aggregates in excess of 106 Da, while the minor mucin ranged from 3.0 × 105 to 106 Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.

Interaction of Human Brain Neuraminidase with Tritium—Labelled Gangliosides

Since the first demonstration of neuraminidase activity in mammals (1,2), the brain was recognised as a major source of this enzyme. In this organ soluble activity could be detected (3,4), however in most species the bulk of the enzyme proved to be membrane-bound. The first detailed description of a mammalian brain neuraminidase was given by Leibovitz and Gatt (5). Working with calf brain these investigators were able to solubilise about 30% of the neuraminidase activity by sequential treatment with sodium cholate and Triton X-100, and they obtained by this procedure an enzyme preparation purified sixfold over the starting material. Attempts to further purify the enzyme were unsuccessful. A modification of this method has been used in a detailed study of the neuraminidase present in human brain by Ohman et al. (3), but also in this case no further purification could be achieved.

Sulpho-N-hydroxysuccinimide activated long chain biotin: A new microtitre plate assay for the determination of its stability at different pH values and its reaction rate with protein bound amino groups

Biotinamidohexanoic acid N-hydroxysulphosuccinimide ester (N-hydroxysulphosuccinimide activated long chain biotin; sulpho-NHS-LC-biotin) has become an invaluable tool for the biotinylation of protein despite the absence of data concerning its stability and reaction velocity. A convenient, rapid and sensitive assay for this compound has been developed based on the sulpho-NHS-LC-biotin mediated biotinylation of bovine serum albumin following adsorption to the wells of a microtitre plate. Bound biotin was visualized by the sequential use of streptavidin and biotinylated horseradish peroxidase. This assay was used for the determination of the stability of sulpho-NHS-LC-biotin in aqueous solution of different pH values. Hydrolysis half lives were below 15 min at pH values above 8.0, but a pH values below 6.5 they exceeded 2 h. It is suggested, therefore, that biotinylations should be performed with sulpho-NHS-LC-biotin taken from a stock solution, prepared at pH values between 3.0 and 5.8. Reaction velocities with primary amino groups were also investigated by means of this ELISA procedure. As expected, biotinylation proceeds faster at pH 8.0 as compared to 7.2, but the increased reaction rate does not compensate for the decreased hydrolysis half life at the higher pH value. Thus, biotinylation with sulpho-NHS-LC-biotin at near neutral pH values appears to be optimal.

Demonstration of Glycoprotein- and Glycolipid-Specific Neuraminidases in Horse Liver

Publisher Summary This chapter discusses the demonstration of glycoprotein and glycolipid-specific neuraminidases in horse liver and other vertebrate species. It also presents the basic criteria to distinguish between different neuraminidases in the same tissue. In an experiment described in the chapter, glycoproteins and gangliosides were labeled in the C-6 side-chain of the neuraminic acid residue by periodate oxidation and borotritide reduction. T.l.c of radioactive labeled compound had shown the specificity of the neuraminidases with collocalia glycopeptides. The enzyme activity was stable under incubation conditions up to 4 h and increased linearly up to 12 mg of protein/ml. This chapter also presents a comparison of neuraminidases activities toward the two substrates and the influence of N-acety1-2, 3-dehydro-2-deoxy-neuraminic acid on neuraminidase activity with the help of graphs.

Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase-labeled anti-digoxigenin antibodies — A new, sensitive technique with the potential for single and double tracing of neuronal connections

For double tracing experiments, wheat germ agglutinin (WGA) molecules labeled with two different haptens are desirable. In the present report the suitability of digoxigenylated WGA (DIG-WGA) for retrograde tracing was investigated. For this purpose the new tracer was pressure injected into rat brains and the transported DIG-WGA visualized via its digoxigenyl group with an alkaline phosphatase linked anti DIG antibody in permanently stained sections of high quality. With fixatives containing 2.5% glutaraldehyde only few positive cells were found. However, at milder fixation conditions (4% paraformaldehyde, 0.05% glutaraldehyde 0.2% picric acid, 30 min) retrogradely labeled cells were detected with a sensitivity comparable to tetramethylbenzidine protocols for conventional WGA-HRP (horseradish peroxidase) tracing. Preliminary experiments suggest excellent suitability for double labeling.