Kayoko Fukuda

Saponins can cause the agglutination of phospholipid vesicles.

The interaction of saponins with phospholipid vesicles was investigated by means of liposomal agglutination or a precipitation assay. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue at the non-reducing terminus, exhibited remarkable agglutinability toward egg yolk phosphatidylcholine vesicles, while other saponins lacking this characteristic sugar residue showed less or no agglutinability. The molar ratio of ginsenoside-Rc to egg phosphatidylcholine in the aggregates was estimated to be 0.4-0.5 by a precipitation assay using 14C-labeled egg phosphatidylcholine vesicles. The agglutination was inhibited by p-nitrophenyl alpha-L-arabinofuranoside but not by p-nitrophenyl beta-D-glucopyranoside or arabinogalactan. The results indicated that the alpha-L-arabinofuranose residue in ginsenoside-Rc should be important for the expression of the agglutinability. The agglutinability of ginsenoside-Rc toward lipid vesicles depended on both the polar head groups and fatty acyl chains of phospholipids. Egg yolk phosphatidylcholine vesicles were strongly agglutinated by ginsenoside-Rc, although sphingomyelin, phosphatidylethanolamine, phosphatidic acid and phosphatidylserine were less agglutinated. The agglutinability of ginsenoside-Rc was effective for phosphatidylcholines with short or unsaturated fatty acyl chains. The results suggested that the interaction of ginsenoside-Rc with phospholipid membranes should be affected not only by the chemical structure of the phospholipid but also by the membrane fluidity.

Structural studies of the acidic oligosaccharide units from bovine glycophorin

The O-glycosidically-linked carbohydrate units of glycophorin from bovine erythrocyte membrane were released by alkaline borohydride treatment. These oligosaccharides were separated into the neutral fractions and the acidic fractions by ion-exchange chromatography followed by gel filtration. The two acidic fractions (fractions 10 and 13) which have the smallest molecular weight in acidic oligosaccharides, were further purified by gel filtration on Bio-Gel P-4 column. Two acidic oligosaccharides (fractions 10-I and 10-II), heptasaccharides, were separated by gel filtration on a Bio-Gel P-4 column from fraction 10. These structures were determined by methylation analyses, nitrous acid deamination after hydrazinolysis and Smith degradation after desialylation. In addition, the structures were also analyzed by direct-probe mass spectrometry of the permethylated derivatives before and after desialylation. These studies indicated that one of them (fraction 10-I) was NeuNGc alpha(2 leads to 3)Gal beta(1 leads to 4)GlcNac beta(1 leads to 3)Gal beta(1 leads to 4)GlcNAc beta (1 leads to 3)Gal beta(1 leads to 3)GalNAcol and another heptasaccharide (fraction 10-II) was Gal beta (1 leads to 4)GlcNAc beta (1 leads to 3)Gal beta (1 leads to 3)[NeuNGc alpha(2 leads to 3)Gal beta(1 leads to 4)GLcNAc beta(1 leads to 6)]GalNAcol. Although another acidic fraction (fraction 13, was obtained as a single peak on a Bio-Gel P-4 column, it appeared to be the mixture of a heptasaccharide, NeuNGc alpha(2 leads to 3)Gal beta(1 leads to 4)GlcNAc beta(1 leads to 3 or 6)[Gal beta (1 leads to 4)GLcNAc beta (1 leads to 6 or 3)]Gal beta (1 leads to 3)GalNAcol and an oligosaccharide similar to fraction 10-II, by analysis of two products obtained by Smith degradation after desialylation.

Isolation and structural studies of the neutral oligosaccharide units from bovine glycophorin

The O-glycosidically linked carbohydrate units of glycophorin from bovine erythrocyte membranes were released as reduced oligosaccharides by alkaline borohydride treatment. These oligosaccharides were separated by ion-exchange chromatography followed by gel filtration. Three oligosaccharides, a penta- a hepta- and a decasaccharide were obtained as the major components from the neutral fraction, and seven fractions were separated from the acidic fractions. All of the fractions were found to contain galactose and N-acetylglucosamine in variable amounts, as well as N-acetylgalactosaminitol. Studies of the neutral oligosaccharides by methylation analyses, nitrous acid deamination and Smith degradation, indicated the structure of the pentasaccharide to be Gal(1 leads to 3)Gal(1 leads to 4)GlcNAc(1 leads to 3)GalNAcol and that of the heptasaccharide to be Gal(1 leads to 3)Gal(1 leads to 4)GlcNAc(1 leads to 3)Gal(1 leads to 4)GlcNAc(1 leads to 3)Gal(1 leads to 3)GalNAcol. The highest molecular weight fraction, decasaccharide in the neutral fraction had a branching point at C-6 of a galactose residue.

3-O-Methyl galactose found in polysaccharides of Lampteromyces japonicus.

A polysaccharide fraction from Lampteromyces japonicus contained a sugar (4%), which was identified as 3-O-methyl galactose by demethylation with boron trichloride, periodate oxidation of the methyl glycoside derivative and gas chromatography-mass spectrometry of the alditol acetate derivative.

Alkali-labile oligosaccharide units of a sialoglycoprotein from rabbit erythrocyte membranes

Two glycoproteins (apparent molecular weights 120,000 and 70,000) were extracted from rabbit erythrocyte membranes, and only one (Mr 120,000), which is a sialoglycoprotein, contained O-glycosidically linked sugar chains. Alkali-labile oligosaccharide units of the sialoglycoprotein were released as reduced oligosaccharides by NaOH-NaB3H4 treatment, and then purified by gel filtration on a Bio-Gel P-4 column followed by ion-exchange chromatography. From the results of methylation analysis, mass spectrometry and chromium trioxide oxidation, the main oligosaccharide unit was determined to be a linear trisaccharide (85% by weight), NeuNGc alpha(2----3)Gal beta(1----3)GalNAcol. In addition, small amounts of a tetrasaccharide (11% by weight) and a disaccharide (4% by weight) were found, which were determined to have the following structures, NeuNGc alpha(2----3)Gal beta(1----3)[NeuNGc alpha(2----6)] GalNAcol and Gal-GalNAcol, respectively.

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the majorO-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey,Macaca fuscata) erythrocyte membranes were characterized.N-Glycolylneuraminic acid (neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. ThreeO-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose andN-acetylgalactosaminitol in the molar ratios of 1∶1∶1 (trisaccharide), 2∶1∶1 (tetrasaccharide) and 1∶1∶1 (pentasaccharide). The content of oligosaccharide units was estimated to be 1∶12∶5 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gcα2-3Galβ1-3[Neu5Gcα2-6]GalNAcol.

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl alpha-D-mannopyranoside and appeared to be alpha-mannan, while that which passed through the column was virtually all beta-glucan. Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the alpha-mannan contained predominantly alpha-(1 leads to 2)-linked side chains branching from an alpha-(1 leads to 6)-linked backbone at the (1 leads to 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The beta-glucan seemed to contain mainly (1 leads to 6)-linked side chains branching from a (1 leads to 3)-linked backbone at the (1 leads to 3,6)-linked glucopyranosyl residues.

Evidence for the occurence of oligosaccharides from bovine glycophorin having a branching point at C-6 of N-acetyl-galactosamine

Abstract Some large acidic oligosaccharides from bovine glycophorin were purified and their structures were determined by methylation analysis, nitrous acid deamination and selective acid hydrolysis after hydrazinolysis, Smith degradation and acetolysis. One oligosaccharide was a mixture of two decassacharides: Another acidic fraction was an undecasaccharide: The mixture of oligosaccharides released by NaOH-NaBH 4 treatment was subjected to selective acid hydrolysis after hydrazinolysis. The occurence of N -acetylglucosamine residue attached to C-6 of the reducing terminal N -acetylgalactosaminitol was demonstrated by isolating the dissccharide, GlcNAc(1→ 6)GalNAcol.

Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199-206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb2, Rb1, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb2 and p-nitrophenyl alpha-L-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb2 interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.