Large scale enzymatic synthesis of oligosaccharides and a novel purification process.

Herein we report the practical chemo enzymatic synthesis of trisaccharide and derivatives of iGb3 and Gb3, and a novel purification process using immobilized yeast to remove the monosaccharide from the reaction mixture. High purity oligosaccharide compounds were achieved in large scale. This study represents a facile enzymatic synthesis of and novel purification process of oligosaccharide.

Corrugoside, a new immunostimulatory alpha-galactoglycosphingolipid from the marine sponge Axinella corrugata.

Corrugoside (1a), a new immunostimulatory triglycosilated alpha-galactoglycosphingolipid, was isolated from the marine sponge Axinella corrugata, and its structure determined by spectral analysis and chemical degradation. Compound 1a activated murine NKT cells in vitro, with a potency of about 2 logs lower than that of alphaGalCer. Four stereoisomeric glycosphingolipids (2a-2d) were also obtained, beta-glucosylceramides bearing unusual endoperoxide and allylic hydroperoxide functionalities on the sphinganine chain. They were shown to be photooxidation artifacts of the known glycosphingolipids 3, also present in the sponge. A possible role of compound 3 as a singlet oxygen scavenger to protect the organism from oxidative damage is proposed.

Cloned Shiga toxin 2 B subunit induces apoptosis in Ramos Burkitt's lymphoma B cells.

The Shiga toxins (Stx1 and Stx2), produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli, consist of one A subunit and five B subunits. The Stx1 and Stx2 B subunits form a pentameric structure that binds to globotriaosylceramide (Gb3-Cer) receptors on eukaryotic cells and promotes endocytosis. The A subunit then inhibits protein biosynthesis, which triggers apoptosis in the affected cell. In addition to its Gb3-Cer binding activity, the data in the following report demonstrate that the Stx2 B pentamer induces apoptosis in Ramos Burkitt's lymphoma B cells independently of A subunit activity. Apoptosis was not observed in A subunit-free preparations of the Stx1 B pentamer which competitively inhibited Stx2 B pentamer-mediated apoptosis. The pancaspase inhibitor, Z-VAD-fmk, prevented apoptosis in Ramos cells exposed to the Stx2 B subunit, Stx1 or Stx2. Brefeldin A, an inhibitor of the Golgi transport system, also prevented Stx2 B subunit-mediated apoptosis. These observations suggest that the Stx2 B subunit must be internalized, via Gb3-Cer receptors, to induce Ramos cell apoptosis. Moreover, unlike the two holotoxins, Stx2 B subunit-mediated apoptosis does not involve inhibition of protein biosynthesis. This study provides further insight into the pathogenic potential of this family of potent bacterial exotoxins.

Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk.

In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.

Interaction of Shiga toxins with human brain microvascular endothelial cells: cytokines as sensitizing agents.

Neurologic abnormalities are among the most serious extraintestinal complications of infection with Shiga toxin (Stx)-producing bacteria. Histopathologic examination of tissues from patients with extraintestinal sequelae suggested that Stxs damage endothelial cells. It is shown here that human brain microvascular endothelial cells (HBMECs) are relatively resistant to purified Stxs (50% cytotoxic doses [CD50s] >/=10 microgram/mL). Pretreatment of HBMECs with tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, n-butyric acid, or a cAMP analogue resulted in a 103- to 104-fold decrease in CD50 values and a 2- to 4-fold increase in fluoresceinated Stx binding to HBMECs. Treatment of HBMECs with lipopolysaccharides did not significantly alter cytotoxicity or toxin binding. TNF-alpha and IL-1beta treatment was associated with the increased HBMEC expression of the toxin-binding glycolipid globotriaosylceramide. HBMECs did not produce IL-1beta and produced only trace amounts of TNF-alpha when stimulated with purified Stx1 in vitro.

HIV-1-induced perturbations of glycosphingolipid metabolism are cell-specific and can be detected at early stages of HIV-1 infection.

The metabolism of glycosphingolipids (GSL) has been investigated in peripheral blood mononuclear cells (PBMC) from 8 patients at an early stage of HIV-1 infection. Following metabolic labeling of these cells with [14C]galactose, the GSL were purified and the radioactivity incorporated into each individual GSL quantitated by phosphoimaging. Compared with PBMC from seronegative donors, the GSL metabolism in PBMC from HIV-1-infected individuals was characterized by an increased synthesis of two GSL: the B-lymphocyte differentiation antigen globotriaosylceramide (Gb3, also referred to as CD77), and the monosialoganglioside GM3, a marker of T-lymphocytes and macrophages. The accumulation of Gb3 and GM3 in PBMC from HIV-1-infected patients was associated with the appearance of anti-Gb3 and anti-GM3 antibodies. Because these GSL are involved in the control of cell proliferation and signal transduction, such anti-GSL autoantibodies may contribute to the immune suppression during the course of HIV-1 infection. Studies on purified cell populations showed that GM3 accumulation occurred preferentially in HIV-1-infected monocytes/macrophages, whereas the synthesis glucosylceramide, the common precursor of complex GSL, was enhanced in both macrophages and CD4+ lymphocytes. Taken together, our data suggest that the dysregulation of GSL metabolism is an early event of HIV-1 pathogenesis that can induce important effects on immune cells homeostasis.

Retrograde transport from the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is regulated by butyric acid and cAMP.

Endocytosed Shiga toxin is transported from the Golgi complex to the endoplasmic reticulum in butyric acid-treated A431 cells. We here examine the extent of this retrograde transport and its regulation. The short B fragment of Shiga toxin is sufficient for transport to the ER. The B fragment of cholera toxin, which also binds to glycolipids, is transported to all the Golgi cisterns, but cannot be localized in the ER even after butyric acid treatment. Under all conditions the toxic protein ricin was found predominantly in the trans-Golgi network. There is no transport of endocytosed fluid to the Golgi apparatus or to the ER even after butyric acid treatment and in the presence of Shiga toxin, indicating that transport to the ER, through the trans-Golgi network and the cisterns of the Golgi apparatus, involves several sorting stations. Since Shiga toxin receptors (Gb3) in butyric acid-treated A431 cells seem to have a ceramide moiety with longer fatty acids than in untreated cells, the possibility exists that fatty acid composition of the receptor is important for sorting to the ER. Both retrograde transport and intoxication with Shiga toxin can also be induced by cAMP, supporting the idea that retrograde transport from the Golgi to the ER is required for intoxication. The data suggest that transport to the ER in cells in situ may depend on fatty acid composition and is regulated by physiological signals.

Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome.

The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin-producing Escherichia coli infection. The endothelial cell is believed to play an important role in the pathogenesis of HUS. Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha). Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1. Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding. Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days. The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide. Glycolipid extracts of TNF-alpha-treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells. We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer. These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS.

A novel ceramide trihexoside from the eggs of the sea urchin, Hemicentrotus pulcherrimus.

Glucosylceramide (Glc beta 1-1Cer) and a novel ceramide trihexoside (Gal beta 1-6Gal beta 1-6Glc beta 1-1Cer) were purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, chromic acid oxidation, enzymatic hydrolysis, enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The ceramide trihexoside has a novel carbohydrate structure, and its core structure, Gal beta 1-6Glc, is also novel. The ceramide moieties of these glycolipids are almost identical. Two fatty acids, 22:1 and 22h:1, constitute more than 80% of the total acids. Long-chain bases are all phytosphingosine, approximately 90% of which is n-t18:0. The finding of melibiosylceramide (Gal alpha 1-6Glc beta 1-1Cer) from the eggs of another sea urchin species [Kubo, H. et al. (1988) J. Biochem. 104, 755-760] and the present finding of the novel ceramide trihexoside suggest that there are a variety of unique sugar structures in sea urchin glycosphingolipids.

Isolation and partial characterization of fucose- and N-acetylglucosamine-containing neutral glycosphingolipids from human senile cataracts.

Five neutral glycosphingolipids were isolated from human cataracts using silicic acid column chromatography and preparative thin-layer chromatography. Three of these glycolipids were partially identified by gas-liquid chromatography as glucosylceramide, lactosylceramide, and trihexosylceramide. Two glucosamine-containing glycosphingolipids (one of which contained fucose) were also detected. One of these two lipids contained galactose, glucose, N-acetylglucosamine in the molar ratio of 2:1:1, while the other contained fucose, galactose, glucose, and N-acetylglucosamine with the molar ratio of 1:2:1:1. Dihydrosphingosine (sphinganine) was the major long-chain base detected in all these fractions. The fatty acids of these neutral glycosphingolipids were variable in chain length, although the majority of them were greater than 20 carbons. This represents the first time whereby a family of neutral glycosphingolipids has been detected in human cataracts. This is also the first demonstration of the existence of a neutral fucolipid in the lenses of any species.

Glycolipids of rat small intestine. Characterization of a novel blood group H-active triglycosylceramide.

A novel blood group H-active triglycosylceramide has been isolated from rat small intestine. It was present exclusively in the epithelial cells. The structure was established by mass spectrometry, NMR spectroscopy and degradative methods to the Fucp alpha 1 leads to 2Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. The lipophilic part was made up of mainly trihydroxy base (phytosphingosine) and 16 : 0--24 : 0 fatty acids.

The structure of canine intestinal trihexosylceramide.

One of the neutral glycosphingolipids isolated from dog intestine has a mobility on thin-layer chromatography and a carbohydrate composition similar to trihexosylceramides. Structural analysis has shown that it consists largely of isoglobotriaosylceramide, galactosyl(alpha-1-3)galactosyl(beta-1-4)glucosyl(beta 1-1')ceramide.