Utilization of Ganglioside-Degrading Paenibacillus sp. Strain TS12 for Production of Glucosylceramide

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a → asialo GM1 → asialo GM2 → lactosylceramide → glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, β-galactosidases, and β-hexosaminidases. TS12 also produced β-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.     

Isolation and characterization of extremely minor gangliosides, GM1b and GD1 alpha, in adult bovine brains as developmentally regulated antigens

In addition to ganglioside GM1b, an unusual and extremely minor ganglioside, GD1 alpha, was efficiently isolated from bovine brain by combination of Q-Sepharose and Iatrobeads column chromatographies. In the course of purification steps, the presence of the sialidase-labile ganglioside was proved by a highly sensitive TLC/enzyme-immunostaining method. The structure was characterized by gas-liquid chromatography, permethylation study, sialidase degradation, immunostaining with specific antibodies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry. The content of the ganglioside was very small (0.016%) in the total gangliosides. This finding suggests that a synthetic pathway of asialo GM1----GM1b----GD1 alpha may exist in mammalian brains. A monoclonal antibody NA-6 that was obtained by immunizing mice with purified GM1b reacted specifically with GM1b but showed no cross-reactivity with other structurally related gangliosides such as GM1a, GD1a, and so on. Using the method of TLC/immunostaining with NA-6, GM1b was found to be strongly expressed during embryonic days 14-17 in chick brains. Thus, it is assumed that extremely minor gangliosides like GM1b and GD1 alpha found in adult brains are characterized as embryonic molecules.     

Adhesion of Bordetella pertussis to sulfatides and to the GalNAc beta 4Gal sequence found in glycosphingolipids

The adherence of the human respiratory pathogen, Bordetella pertussis, to purified glycosphingolipids was investigated using thin layer chromatography overlay assays. Both virulent and avirulent strains of B. pertussis bound to asialo GM1. The bacterium did not bind to the gangliosides GM1, GD1a, GD1b, and GT1b, nor to lactosylceramide, trihexosylceramide, globoside, or Forssman antigen. However, after treatment of the chromatography plates with sialidase, B. pertussis bound to the gangliosides GM1, GM2, GD1a, GD1b, and GT1b but not to GM3. Comparison of the oligosaccharide structures of these gangliosides suggests that the minimum sugar structure needed for avid bacterial binding is GalNAc beta 4Gal. This structure has been previously implicated as a receptor for other human respiratory pathogens (Krivan, H. C., Roberts, D. D., Ginsburg, V. (1988) Proc. Natl. Acad. Sci. U.S.A 85, 6157-6161). Virulent strains of B. pertussis also bound specifically to sulfatide. This response was dose-dependent and inhibited by the anionic polysaccharide dextran sulfate. The sulfated-sugars dextran sulfate, fucoidan, and heparin inhibited the attachment of virulent strains of B. pertussis to human WiDr cells and to hamster trachea cells indicating that sulfatides on the surface of mammalian cells may function as a receptor for B. pertussis. The occurrence of both sulfatides and asialo GM1 in human lung and trachea suggests that these glycolipids may serve as specific receptors for B. pertussis.     

Identification of asialo GM1 as a binding structure for Escherichia coli colonization factor antigens

We have examined the binding of colonization factor antigens, (CFAs) of enterotoxigenic Escherichia coli to gangliosides and asialo gangliosides by using immuno-thin layer chromatography. CFA/II and its subcomponents (CS1, CS2 and CS3) as well as the subcomponent CS4 of the CFA/IV complex bound to asialo ganglioside GM1.     

Modulation of Ganglioside Biosynthesis in Primary Cultured Neurons

Murine cerebellar cells were pulse labeled with [14C]galactose, and the incorporation of radioactivity into gangliosides and neutral glycosphingolipids was examined under different experimental conditions. In the presence of drugs affecting intracellular membrane flow, as well as at 15 degrees C, labeled GlcCer was found to accumulate in the cells, whereas the labeling of higher glycosphingolipids and gangliosides was reduced. Monensin and modulators of the cytoskeleton effectively blocked biosynthesis of the complex gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, whereas incorporation of radioactivity into neutral glycosphingolipids, such as glucosylceramide and lactosylceramide, as well as GM3, GM2, and GD3 was either increased or unaltered. As monensin has been reported to interfere with the flow of molecules from the cis to the trans stacks of the Golgi apparatus, this result highlights at least one subcompartmentalization of ganglioside biosynthesis within the Golgi system. Inhibitors of energy metabolism affected, predominantly, the biosynthesis of the b-series gangliosides, whereas a reduced temperature (15 degrees C) more effectively blocked incorporation of radiolabel into the a-series gangliosides, a result suggesting the importance of GM3, as the principal branching point, for the regulation of ganglioside biosynthesis.     

Recycling of glucosylceramide and sphingosine for the biosynthesis of gangliosides and sphingomyelin in rat liver.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.     

产唾液酸酶微生物的筛选及产酶条件的优化

以无单唾液酸四己糖神经节苷脂（GM1）的神经节苷脂为唯一C源,从土壤中筛选出1株能以多唾液酸神经节苷脂为底物,转化生成GM1的产唾液酸酶微生物,经鉴定为溶黄嘌呤厄菌（Oerskovia xanthineolytica）。利用单因素法和响应面分析法对溶黄嘌呤厄菌产生唾液酸酶的条件进行优化,酶活提高了4.89倍。利用优化后培养条件,以神经节苷脂混合体为底物,经过微生物生物转化后,单唾液酸神经节苷脂GM1的含量从10%提高到83.7%。     

Involvement of gangliosides in glucosamine-induced proliferation decrease of retinal pericytes

The hexosamine pathway (HP) is a biochemical hypothesis recently proposed explaining cellular alterations occurring during diabetic microvascular complications. Diabetic retinopathy is a common microvascular complication of diabetes, and it is known that cell proliferation is severely affected during the development of the disease. Particularly, early stages are characterized by death of the retinal microvascular cells, pericytes. Gangliosides have often been described to regulate cell growth; however, very few studies focused on the potential role of gangliosides in diabetic microvascular alterations. The aim of this article was to investigate the effect of the HP activation on pericyte proliferation and determine the potential implication of gangliosides in this process. Results indicate first that HP activation, mimicked by glucosamine treatment, decreased pericyte proliferation. Second, glucosamine treatment induced a modification of gangliosides pattern, particularly GM1 and GD3 were significantly increased. Next, results showed that exogenous addition of a-series gangliosides (GM3, GM2, GM1, GD1a) and b-series ganglioside (GD3) caused a decrease of pericyte proliferation, whereas nonsialylated precursors glucosylceramide and lactosylceramide were without effect. Furthermore, when ganglioside biosynthesis was blocked using PPMP, a glucosylceramide synthase inhibitor, the effects of glucosamine on pericyte proliferation were partially reversed. Our results suggest that in retinal pericytes, gangliosides and particularly GM1 and GD3 that are increased in response to glucosamine, are involved in the antiproliferative effect of glucosamine. These observations also underlie the potential involvement of gangliosides in a pathological context, such as diabetic microvascular complications.     

Development of new ganglioside probes and unraveling of raft domain structure by single-molecule imaging

Gangliosides are involved in a variety of biological roles and are a component of lipid rafts found in cell plasma membranes (PMs). Gangliosides are especially abundant in neuronal PMs and are essential to their physiological functions. However, the dynamic behaviors of gangliosides have not been investigated in living cells due to a lack of fluorescent probes that behave like their parental molecules. We have recently developed, using an entirely chemical method, four new ganglioside probes (GM1, GM2, GM3, and GD1b) that act similarly to their parental molecules in terms of raft partitioning and binding affinity. Using single fluorescent-molecule imaging, we have found that ganglioside probes dynamically enter and leave rafts featuring CD59, a GPI-anchored protein. This occurs both before and after stimulation. The residency time of our ganglioside probes in rafts with CD59 oligomers was 48 ms, after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 ms and 12 ms, respectively. In this review, we introduce an entirely chemical-based ganglioside analog synthesis method and describe its application in single-molecule imaging and for the study of the dynamic behavior of gangliosides in cell PMs. Finally, we discuss how raft domains are formed, both before and after receptor engagement. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains

Glycosphingolipids (GSLs) and their sialic acid-containing derivatives, gangliosides, are important cellular components and are abundant in the nervous system. They are known to undergo dramatic changes during brain development. However, knowledge on the mechanisms underlying their qualitative and qualitative changes is still fragmentary. In this investigation, we have provided a detailed study on the developmental changes of the expression patterns of GSLs, GM3, GM1, GD3, GD1a, GD2, GD1b, GT1b, GQ1b, A2B5 antigens (c-series gangliosides such as GT3 and GQ1c), Chol-1alpha (GT1aalpha and GQ1balpha), glucosylceramide, galactosylceramide (O1 antigen), sulfatide (O4 antigen), stage-specific embryonic antigen-1 (Lewis x) glycolipids, and human natural killer-1 glycolipid (sulfoglucuronosyl paragloboside) in developing mouse brains [embryonic day 12 (E12) to adult]. In E12-E14 brains, GD3 was a predominant ganglioside. After E16, the concentrations of GD3 and GM3 markedly decreased, and the concentrations of a-series gangliosides, such as GD1a, increased. GT3, glucosylceramide, and stage-specific embryonic antigen-1 were expressed in embryonic brains. Human natural killer-1 glycolipid was expressed transiently in embryonic brains. On the other hand, Chol-1alpha, galactosylceramide, and sulfatide were exclusively found after birth. To provide a better understanding of the metabolic basis for these changes, we analyzed glycogene expression patterns in the developing brains and found that GSL expression is regulated primarily by glycosyltransferases, and not by glycosidases. In parallel studies using primary neural precursor cells in culture as a tool for studying developmental events, dramatic changes in ganglioside and glycosyltransferase gene expression were also detected in neurons induced to differentiate from neural precursor cells, including the expression of GD3, followed by up-regulation of complex a- and b-series gangliosides. These changes in cell culture systems resemble that occurring in brain. We conclude that the dramatic changes in GSL pattern and content can serve as useful markers in neural development and that these changes are regulated primarily at the level of glycosyltransferase gene expression.     

Hormonal effects on the development changes of mouse small intestinal glycolipids

The composition of intestinal glycosphingolipids during normal and hormone-perturbed development was investigated. The concentrations of glycosphingolipids of mouse small intestine were affected by the injection of thyroxine or cortisone during suckling and weaning periods. GDla was reduced by the hormonal treatment among major gangliosides, GM3, GM1 and GD1a, of mouse small intestine during the suckling period. In contrast, asialo GM1 was precociously produced by the treatment, which scarcely found in control suckling mouse small intestine. The results showed that these hormones were related to developmental alteration of small-intestinal glycolipids.     

Structural heterogeneity of terminal glycans in Campylobacter jejuni lipooligosaccharides

Lipooligosaccharides of the gastrointestinal pathogen Campylobacter jejuni are regarded as a major virulence factor and are implicated in the production of cross-reactive antibodies against host gangliosides, which leads to the development of autoimmune neuropathies such as Guillain-Barré and Fisher Syndromes. C. jejuni strains are known to produce diverse LOS structures encoded by more than 19 types of LOS biosynthesis clusters. This study demonstrates that the final C. jejuni LOS structure cannot always be predicted from the genetic composition of the LOS biosynthesis cluster, as determined by novel lectin array analysis of the terminal LOS glycans. The differences were shown to be partially facilitated by the differential on/off status of three genes wlaN, cst and cj1144-45. The on/off status of these genes was also analysed in C. jejuni strains grown in vitro and in vivo, isolated directly from the host animal without passaging, using immunoseparation. Importantly, C. jejuni strains 331, 421 and 520 encoding cluster type C were shown to produce different LOS, mimicking asialo GM(1), asialo GM(2) and a heterogeneous mix of gangliosides and other glycoconjugates respectively. In addition, individual C. jejuni colonies were shown to consistently produce heterogeneous LOS structures, irrespective of the cluster type and the status of phase variable genes. Furthermore we describe C. jejuni strains (351 and 375) with LOS clusters that do not match any of the previously described LOS clusters, yet are able to produce LOS with asialo GM(2)-like mimicries. The LOS biosynthesis clusters of these strains are likely to contain genes that code for LOS biosynthesis machinery previously not identified, yet capable of synthesising LOS mimicking gangliosides.     

Metabolism of Exogenous Gangliosides in Cerebellar Granule Cells, Differentiated in Culture

The metabolism of exogenous gangliosides in the CNS has been investigated using cerebellar granule cells in culture as a model. For this purpose, GM2 and GM1, both isotopically radiolabeled at the level of the terminal sugar residue or of the long chain base moiety, were administered to differentiated cells for a 15-h pulse, and their metabolic fate was followed in a time course protocol. At each time investigated (1, 2, and 4 days after the pulse), several compounds, besides the ones administered, were detected: (a) GM2 (only after GM1 was given), GM3, lactosylceramide, glucosylceramide, and ceramide, all products of ganglioside stepwise catabolism; (b) GM1 (only after GM2 was given), GD1a, GD1b, O-Ac-GT1b, and GT1b, that is, gangliosides more complex than the one administered; and (c) sphingomyelin. The compounds derived from ganglioside catabolism and sphingomyelin were detected only after administration of long chain base-labeled precursors, whereas the others were found regardless of the labeling position of the precursor. In addition, radioactivity was incorporated in the delipidized residue when sugar-labeled gangliosides were given to cells. Besides qualitative differences, quantitative ones were found after administration of the different precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of sialic acid carboxyl group of ganglioside

A simple and quantitative method for the modification of sialic acid carboxyl group in ganglioside is described. Methyl iodide was added to the ganglioside in dimethylsulfoxide. The reaction was completed quickly at room temperature, giving the methyl ester with practically no by-products. Reduction of the methyl ester was achieved by sodium borohydride. These modified gangliosides were chemically characterized. Reduced GM1 has a strong antigenicity compared with the original GM1, and it raised high titer antisera which did not crossreact with the original GM1 nor with its methyl ester. Peanut agglutinin, which binds strongly to asialo GM1 but weakly to GM1, bound to the methyl ester of GM1 and reduced GM1. Cholera toxin, which is specific for GM1, also reacted with the methyl ester of GM1 and reduced GM1 to a certain extent.     

Glycosphingolipids of human aorta

The structures of the main gangliosides of human aorta (intima and media) were elucidated. The main component (67%) was identified as N-acetylneuraminosyl-lactosylceramide (ganglioside GM3). The aorta tissue contained also gangliosides GM1, GD3, GD1a, and GT1. All sialic acid residues in gangliosides were present as N-acetyl-neuraminosyl derivatives. Among neutral glycosphingolipids of human aorta, the main components were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The preliminary data suggest that the composition of the investigated glycosphingolipids in tissue might vary upon atherosclerosis lesions of aorta.     

Gangliosides stimulate astroglial cell proliferation in the absence of serum

Previous studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 X 10(-5)M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12-24 hr. The ganglioside-induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10(-5)M--while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.     

Characterization of the glycosphingolipids of pig cortical bone and cartilage

Neutral glycosphingolipids and gangliosides were extracted from pig cortical bone and cartilage. To ensure the completeness of extraction, the cortical bone was demineralized and reextracted. Globotriaosylceramide and globoside were noted to be present at high content in the cortical bone. It contained glucosylceramide, lactosylceramide, globotriaosylceramide and globoside as neutral glycosphingolipids at a ratio of 1:0.7:3.1:2.7. In articular cartilage, the ratio was 1:0.7:0.4:0.8. GM3 and GD3 were the major gangliosides in both these tissues. GM3, GM1, GD3, GD1 and GT1 were present at ratios of 1:0.9:0.9:0.1:0.1 in the cortical bone and 1:0:1.2:0.06:0.02 in the cartilage. Neutral glycosphingolipids could be extracted from the cortical bone without the need for demineralization, while most of the gangliosides were extracted after this treatment, implying the occurrence of interactions between gangliosides and minerals in the bone.     

Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.     

Characterization of neutral sphingolipids and gangliosides from chicken liver

The neutral sphingolipids and gangliosides were isolated from 62- and 63-day-old chicken livers and characterized. The total concentration of neutral sphingolipids was 59 nmol/g of liver, and that of gangliosides was 330 nmol/g of liver. The major neutral sphingolipids were free ceramide, galactosylceramide, glucosylceramide, lactosylceramide, galabiosylceramide, and Forssman glycolipid. Galactosylceramide was the most abundant and free ceramide was the second most abundant. The major gangliosides were sialosylgalactosylceramide (GM4) and sialosyllactosylceramide (GM3), each of which contained only N-acetylneuraminic acid as a sialic acid. Sphingosine (d18:1) was a major long-chain base in all the sphingolipids. Considerable amounts of 2-hydroxy fatty acids were present in free ceramide, galactosylceramide, and GM4.     

Abrupt induction of GDP-fucose: Asialo GM1 fucosyltransferase in the small intestine after conventionalization of germ-free mice

We studied the time-course of the induction of GDP-fucose: asialo GM1 fucosyltransferase and its product, i.e. fucosyl asialo GM1, of the small intestine after introduction of microorganisms to germ-free mice (conventionalization). We found that the fucosyltransferase activity was abruptly induced and asialo GM1 was converted into fucosyl asialo GM1 within a few days after conventionalization. However, two weeks after conventionalization this enzyme activity dropped to approximately 10^?2 level of the maximum value and asialo GM1 appeared again as one of the major glycolipids. These results showed that the microbial colonization in the gut evoked a drastic change of the glycolipid pattern at the intestinal epithelial cell-surface via the induction of a fucosyltransferase.