Crude Ganglioside Research Articles

Enhancement of bioconversion gangliosides to monosialotetrahexosylganglioside by in situ sialic acid removal and recovery

Monosialotetrahexosylganglioside (GM1) production via bioconversion from gangliosides is promising for industrial application because it has the advantages of a high GM1 yield and an environmentally friendly process. Sialidase hydrolyzes gangliosides to GM1 producing sialic acid as a by‐product, which inhibits the sialidase activity, while the incomplete conversion of gangliosides was indicated by thin‐layer chromatography (TLC) in the presence of sialic acid. The sialic acid showed competitive inhibition on the sialidase activity with an inhibition constant of 0.75 mmol/L. By harnessing the in situ product removal (ISPR) technique, 50 g/L of crude gangliosides was completely converted to GM1 after a 12 h conversion. The GM1 concentration increased from 0.42 to 10.88 g/L in the ISPR system, which was 59.1 % higher than that of the control (6.84 g/L GM1). In addition, sialic acid was recovered simultaneously with a yield of 74.7 %. In summary, the ISPR system improved the bioconversion from gangliosides to GM1 and recovered sialic acid within a one‐step bioprocess.

3-O-sulfo-β-d-galactose moiety of endogenous sulfoglycolipids is a potential ligand for immunoglobulin-like receptor LMIR5

Axonal Guillain–Barré syndrome (GBS) is an autoimmune neuropathy characterized by limb weakness and/or paralysis due to the presence of autoantibodies against brain glycolipids. The immune receptors that recognize these autoimmune targets have not been described. In this study, 12 C-type lectin and 10 immunoglobulin-like receptors were screened for their potential ligands from the brain glycolipids, which are the binding targets for GBS autoantibodies. These glycolipids were GM1, GM2, GD1a, GD1b, GQ1b, crude gangliosides, and 3-O-sulfo-β-d-galactosylceramide C24:1 (designated as C24:1). A direct interaction between ligand and receptor was examined using an ELISA-based binding assay. C-type lectin (CLEC5a, SIGNR3) and immunoglobulin-like receptors (TREM2, TREM3, LMIR2, LMIR5, LMIR7, LMIR8) interacted with C24:1. In addition, TREM3 did bind to GQ1b. LMIR5 interacted with GD1a, GQ1b, and crude gangliosides. Binding with highest affinity was observed for the LMIR5–C24:1 interaction, which was selected for further verification. C24:1 was found to induce MCP-1 production, but not proinflammatory cytokines, in basophils. C24:1-induced MCP-1 production was significantly reduced in DAP12−/− basophils. Importantly, LMIR5 ligation by C24:1 resulted in NFAT activation through DAP12 in LMIR5-expressing reporter cells. Structural analysis showed that LMIR5 recognized the 3-O-sulfo-β-d-galactose moiety of C24:1. The findings indicated that C24:1 is a potential ligand for DAP12-coupled LMIR5.

Development of a cell immobilization technique for the conversion of polysialogangliosides to monosialotetrahexosylganglioside

Context: Monosialotetrahexosylganglioside (GM1) prepared from the brain of pig or bovine is an effective clinical drug in the treatment of different nervous system diseases. Generally, polysialogangliosides are transformed into GM1 by enzymic or chemical hydrolysis due to the very poor level of natural GM1.Objective: To continuously obtain GM1 by cell immobilization in a packed-bed reactor.Materials and methods: Brevibacterium casei, which is Gram-positive bacteria belonging to the order Actinomyces and family Brevibacteriaceae, can produce high-activity sialidase, are encapsulated in silk fibroin hydrogel, and subsequently packed into a reactor. The crude ganglioside is pumped into the reactor and continuously turned to GM1.Results: The optimal silk fibroin concentration for hydrogel preparation is 6.0% (w/v). The optimal initial biomass for immobilization is ~12% (wet weight). The optimal conversion conditions are 35°C and 6 mL/min of flow rate. Under above conditions, the maximum GM1 productive strength and conversion ratio can reach to 4.2 g/L·h and 313.5%, respectively.Discussion: Silk fibroin is a promising material for cell immobilization because it has predominant characteristics of higher permeability and intensity. Cell immobilization for continuous GM1 transformation could eliminate the asialo GM1 and decrease the foreign matter from transfer medium and metabolism product.Conclusion: In the packed-bed reactor, continuous production of GM1 had been under effective running at least for 15 days indicating a potential for industrial production. It is significant that this is a first report on cell immobilization for GM1 production.

Efficient conversion from polysialogangliosides to monosialotetrahexosylganglioside using Oerskovia xanthineolytica YZ-2

A new sialidase-producing strain isolated from soil was identified as Oerskovia xanthineolytica YZ-2. Sialidase was produced when Oerskovia xanthineolytica YZ-2 was exposed to polysialogangliosides. The sialidase of Oerskovia xanthineolytica YZ-2 hydrolyzed sialic acid linkages in polysialogangliosides, and released monosialotetrahexosylganglioside (GM1). The sialidase had the capability of product specificity because it did not attack the sialic acid linkage in GM1. Therefore, Oerskovia xanthineolytica YZ-2 was used for GM1 production from polysialogangliosides. In flasks cultivation phase, it was proved that Oerskovia xanthineolytica YZ-2 could convert polysialogangliosides to GM1 efficiently. Scaling-up the bioprocess with 8% crude ganglioside, polysialogangliosides was converted to GM1 by Oerskovia xanthineolytica YZ-2 in 30 L bioreactor after 18 h. The relative content of GM1 increased from 16.3% in crude ganglioside to 83.7% after Oerskovia xanthineolytica YZ-2 conversion. Therefore, a simple, large-scale conversion process for GM1 production from polysialogangliosides was achieved using Oerskovia xanthineolytica YZ-2 as a biocatalyst.

Mass spectrometry of fluorocarbon‐labeled glycosphingolipids

A method for generation of novel fluorocarbon derivatives of glycosphingolipids (GSLs) with high affinity for fluorocarbon phases has been developed, and their potential applications to mass spectrometry (MS)-based methodologies for glycosphingolipidomics have been investigated. Sphingolipid ceramide N-deacylase (SCDase) is used to remove the fatty acid from the ceramide moiety, after which a fluorocarbon-rich substituent (F-Tag) is incorporated at the free amine of the sphingoid. In initial trials, a neutral GSL, globotriaosylceramide (Gb(3)Cer), three purified bovine brain gangliosides, and four fungal glycosylinositol phosphorylceramides (GIPCs) were de-N-acylated, derivatized by prototype F-Tags, and recovered by solid phase extraction on fluorocarbon-derivatized silica (F-SPE). The efficacy of SCDase treatment of GIPCs was here demonstrated for the first time. Compatibility with subsequent per-N,O-methylation was established for the F-tagged Gb(3) Cer and purified gangliosides, and extensive mass spectra (MS(1) and MS(2)) consistent with all of the expected products were acquired. The potential use of F-tagged derivatives for a comprehensive MS based profiling application was then demonstrated on a crude ganglioside mixture extracted from bovine brain. Finally, a simple trial in microarray format demonstrated fixation of F-tagged G(M1) ganglioside to a fluorous glass surface, with the glycan intact and available for interaction with a fluorescent derivative of cholera toxin B chain. The methods described thus provide a new avenue for rapid GSL recovery or cleanup, potentially compatible with a variety of platforms for mass spectrometric profiling and structure analysis, as well as parallel analysis of functional interactions.

Antibody to glycolipid in a patient with IgM paraproteinemia and polyradiculoneuropathy

Serum from a patient with IgM paraproteinemia and polyradiculoneuropathy, diagnosed as malignant lymphoma reacted specifically with a lipid component in a crude ganglioside fraction isolated from human peripheral nerve, but did not react with myelin proteins including myelin-associated glycoprotein (MAG). The reactive glycolipid migrated between GM1 and GD1a gangliosides on TLC, and there was no distinct band corresponding to this glycolipid on examination with resorcinol reagent. Antibody to glycolipid in this case may play a role in the pathogenesis of neuropathy.

Development of a large scale process for the conversion of polysialogangliosides to monosialotetrahexosylganglioside with a novel strain of Brevibacterium casei producing sialidase

A bioconversion process of producing GM1 (monosialotetrahexosylganglioside) on an industrial scale was developed with a novel sialidase-producing strain Brevibacterium casei. The sialidase hydrolyzed polysialogangliosides to produce GM1 but did not act on GM1. When Brevibacterium casei was cultured in a synthetic medium containing crude pig brain gangliosides (10% w/v) at 30 degrees C for 24 h in a 50 l fermenter, most of the polysialogangliosides were converted to GM1. The content of GM1 was increased from 9% in crude gangliosides to 45% with 70% (w/w) yield.

Highly efficient conversion of polysialoganglioside to GM1 with Brevibacterium casei as a microbial biocatalyst

A novel bacterium, which can be used to convert polysialoganglioside to GM1 efficiently, was isolated from soil with ganglioside as the sole source of carbon. It was identified tentatively as Brevibacterium casei. In a typical experiment, 0.8 g of highly purified GM1 (93%) was obtained from 1.5 g free GM1 crude ganglioside after chromatographic separation. The overall yield of this procedure was estimated to be more than 50% (w/w).

Large scale purification of gangliosides G M3(Neu5Ac) and G M3(Neu5Gc) by trimethylaminoethyl-Fractogel high-performance liquid chromatography

A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides G M3(Neu5Ac) and G M3(Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure G M3(Neu5Ac) and G M3(Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of G M3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two G M3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl- versus N-glycolylneuraminic acid).

Preparation of GM1 ganglioside with sialidase-producing marine bacteria as a microbial biocatalyst.

This paper describes the preparation of monosialoganglioside GM1 with sialidase-producing marine bacteria as a microbial biocatalyst. A new sialidase-producing bacterium, identified tentatively as Pseudomonas sp. strain YF-2, was isolated from seawater by enrichment culture with ganglioside as the sole source of carbon. When YF-2 was cultured in a synthetic medium containing crude bovine brain gangliosides at 25 degrees C for 3 days, 80 to 90% of the gangliosides were converted to GM1. GM1 was then purified from the supernatant of YF-2 culture by C18 reverse-phased chromatography, followed by DEAE-Sephadex A25 anion-exchange chromatography. In a typical experiment, 178 mg of highly purified GM1 was obtained from 500 mg of the crude ganglioside fraction. The GM1 induced neurite outgrowth of neuroblastoma Neuro2a cells at a concentration of 33 to 100 microM in the presence of fetal calf serum. Sialidase was purified 33-fold with 13.3% recovery from the culture supernatant of YF-2. The purified enzyme hydrolyzed polysialogangliosides to produce GM1 but did not act on GM1. It was therefore concluded that polysialogangliosides in the culture of strain YF-2 were converted to GM1 by this sialidase.

A novel ganglioside, 9-O-acetyl GD1b, is recognized by serum antibodies in Guillain-Barré syndrome

A hitherto undescribed ganglioside was detected in a crude ganglioside fraction of bovine brain using an IgM M-protein binding to Ga1β 1,3Ga1NAc residue. We purified and identified it as 9-O-acetyl GD1b based on results of alkali treatment that yielded GD1b and results of fast atom bombardment-mass and gas chromatography-mass spectrometries. 9-O-acetyl GD1b was also found to be present in human peripheral nerve tissue. The reactivities of the serum antibodies from patients with Guillain-Barré syndrome to 9-O-acetyl GD1b, GD1b, and GM1 were determined by ELISA and TLC immunostaining. Nineteen of 85 serum samples from Guillain-Barré syndrome patients had antibodies that bound to 9-O-acetyl GD1b: 14 of the positive samples also reacted with GM1 and GD1b, three reacted with GM1 but not with GD1b, one with GD1b but not with GM1, and one with neither GM1 nor GD1b. These results show that a subset of patients with Guillain-Barré syndrome had antibodies that react with 9-O-acetyl GD1b; therefore, this ganglioside can serve as a target antigen against the antibodies present in Guillain-Barré syndrome.

N‐acetylgalactosaminyl GD1a is a target molecule for serum antibody in Guillain‐Barré syndrome

Serum antibodies against such major glycolipids as GM1, GD1b, and LM1 have been reported in patients in the acute phase of Guillain-Barré syndrome (GBS). Because minor unidentified glycolipids also may be targets of antibodies in GBS sera, we assayed serum antibody against a crude ganglioside fraction using thin-layer chromatogram immunostaining. Antibody activity was detected against a band that migrated just below GD1a in 6 of the 50 patients with GBS tested. Antibody titer, as determined by enzyme-linked immunosorbent assay, decreased during the course of the disease. All 6 patients had suffered gastrointestinal infection before the neurological onset of GBS and showed low amplitudes for the compound muscle action potentials and normal or only slightly decreased nerve conduction velocities. Thin-layer chromatogram immunostaining did not show this antibody activity in any of the 16 normal and 119 disease controls. The unidentified glycolipid was isolated by DEAE-Sephadex A-25 column chromatography, sialidase treatment, and Iatrobeads column chromatography. Fast atom bombardment-mass spectra showed it to be N-acetyl-galactosaminyl GD1a.

Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition.

A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100 degrees C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.

Immunocytochemical Study of the Distribution of a Ganglioside in Sea Urchin Eggs1

An antibody against M5 ganglioside (NeuGc alpha 2-6Glc beta 1-1Cer), the dominant ganglioside in the eggs of the sea urchin, Anthocidaris crassispina, was purified by affinity chromatography from rabbit antiserum against crude ganglioside of the eggs. The specificity of the antibody was verified by enzyme-linked immunosorbent assay and TLC immunostaining. M5 ganglioside was also the major one in the eggs of another sea urchin, Hemicentrotus pulcherrimus, as judged from TLC analyses including immunostaining. Cryostat-sections of H. pulcherrimus eggs were examined to determine the distribution of M5 ganglioside by indirect immunofluorescence microscopy with the antibody. Before fertilization, the egg cortex was highly stained, while the other part of cytoplasm was uniformly but much more weakly stained. After fertilization, the staining rapidly decreased in the cortex and was restricted to a very thin peripheral layer and to cytoplasmic patches. The immunoreactivity was also observed in the esophagus and the somatic cells of the testis, but the spermatozoa were never stained with the antibody.

Determination of Gangliosides in Human Cerebrospinal Fluid by High-Performance Thin-Layer Chromatography and Direct Densitometry

A method for the separation and quantification of a complex ganglioside mixture from a clinically available amount (5 ml) of human cerebrospinal fluid (CSF) is described. After reduction of the CSF volume by ultrafiltration, gangliosides are extracted with methanol/chloroform, then separated and quantified by high performance thin layer chromatography (HPTLC) and direct densitometry. For purification of crude ganglioside extract, the method of choice was microdialysis against water. Recovery for the present method including all methodological steps was 78%. No delective loss of gangliosides was demonstrated. The CSF ganglioside pattern from 'normal' CSF samples resembles that of brain gangliosides, particularly cerebellum gangliosides. Based on chromatographic comparison with standards, the percentages of lipid-bound NeuAc-positive fractions were: GM1 = II3NeuAc-GgOse4Cer (3%), GD3 = II3NeuAc2-Lac-Cer (3%), GD1a = IV3NeuAc,II3NeuAc-GgOse4Cer (15%), X1 (3%), GD1b = II3(NeuAc)2-GgOse4Cer (16%), X2 (3%), GT1b = IV3NeuAc,II3NeuAc2-GgOse4-Cer (41%), and GQ1b = IV3NeuAc2-,II3NeuAc2-GgOse4-Cer (16%). The total ganglioside content varied between 616-944 micrograms/l. Within-run and between-run assay precision (relative standard deviation) for 'normal' pooled CSF ranged from 0.04 to 0.12 for the predominant CSF ganglioside fractions (GD1a, GD1b, GT1b, GQ1b), and from 0.13 to 0.23 for the less pronounced fractions (GM1, GD3).

High-performance liquid chromatographic separation of gangliosides, combined with immunological detection and fast atom bombardment mass spectrometric characterization

A complete strategy for the isolation of individual mono- and disialogangliosides has been elaborated. We have used straight-phase silica gel chromatography or partitioning to obtain a crude ganglioside fraction. This fraction was then peracetylated and run through a second silica gel column. After anion-exchange chromatography the gangliosides were separated by straight-phase high-performance liquid chromatography with chloroform-methanol-water mixtures as eluting solvents. The method is suitable for preparative isolation of gangliosides and subsequent structural characterization by thin-layer chromatography-enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry and/or gas chromatography-mass spectrometry, which is demonstrated by several examples, including the separation of GalNAc-II3NeuAc-GgOse4Cer from GalNAc-isoII3NeuAc-GgOse4Cer.

Identification of a novel ganglioside on erythrocytes with blood group Cad specificity.

The blood group Cad antigen is a carbohydrate structure well characterized on the sialoglycoproteins of the red cell membrane from some rare individuals (Blanchard, D., Cartron, J. P., Fournet, B., Montreuil, J., Van Halbeck, H., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 7691-7695). However, protease treatment of whole cells did not destroy their antigenic activity which indicated that glycolipid might also be involved in the antigenic reaction. A crude ganglioside fraction was prepared from Cad cells and found to inhibit the hemagglutination reaction, whereas neutral glycolipids were inactive. Further analysis of the ganglioside extract from Cad erythrocytes by thin layer chromatography revealed an unusual profile characterized by a lower content of sialosylparagloboside and the presence of a novel ganglioside of slower mobility. Immunochemical studies demonstrate that this ganglioside binds Helix pomatia lectin and inhibits human anti-Sda antibody. In addition, a ganglioside with identical chromatographic mobility can be obtained by the enzymatic transfer of GalNAc from UDP-GalNAc to sialosylparagloboside using a microsomal preparation from human kidney. These results together with cell surface labeling experiments suggest that the major ganglioside of Cad erythrocytes might be derived from sialosylparagloboside by substitution with an additional N-acetylgalactosamine residue.

Further characterization of the tracheal receptor for Pseudomonas aeruginosa.

The purpose of this study was to obtain more information on the nature of the macromolecule to which Pseudomonas aeruginosa adheres. Acid-injured tracheal epithelium was treated with trypsin or lipase to determine whether the receptor molecule was a protein or a lipid. Lipase treatment significantly reduced adherence to these cells, whereas trypsin had no effect. Since the receptor appeared to be a lipid containing sialic acid, gangliosides were used to test whether they would inhibit adherence. Crude ganglioside preparations inhibited adherence in a dose-dependent manner when added to the bacteria before exposure to tracheal cells. Lastly, fibronectin, which presumably binds to gangliosides, significantly reduced the adherence of these organisms. According to these findings Pseudomonas aeruginosa appears to adhere to a sialic acid-containing glycolipid on cell surfaces, probably a ganglioside.

Ganglioside‐Induced Neuritogenesis: Verification That Gangliosides Are the Active Agents, and Comparison of Molecular Species

Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility that neurite-stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three-step purification procedure that eliminated at least 95% of the contaminating peptides. These purified preparations retained their capacity to induce extensive neurite growth in neuro-2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of their inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.

Antibodies against viruses and structural brain components in oligoclonal IgG obtained from multiple sclerosis brain.

Immunoglobulin G (IgG) was isolated from three multiple sclerosis (MS) and two control brains by Protein A Sepharose affinity chromatography and was characterized by thin-layer polyacrylamide gel isoelectric focusing (PAG IEF) and antiserum immunofixation. The three MS brains contained oligoclonal IgG. Immunofixation with measles, herpes simplex, varicella, rubella, mumps and cytomegalovirus as antigens and autoradiography revealed that some of the oligoclonal IgG bands separated by PAG IEF contained antibodies against herpes simplex in one, measles in two and varicella virus in all three MS brains. No antibodies were detected with this technique against structural human (crude saline, lipid-proteolipid, ganglioside, and myelin basic protein extracts of MS and normal human brain) and bovine (purified myelin myelin basic protein and oligodendrocytes of bovine brain) brain components. The finding of viral antibodies and the absence of antibodies against structural brain proteins in oligoclonal MS brain IgG is similar to that previously recorded in MS cerebrospinal fluid (CSF).