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1.
Anal Biochem ; 458: 20-6, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24769373

ABSTRACT

GM2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of GM2 gangliosides (GM2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify GM2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of GM2 was achieved using LC/ESI-MS/MS with d35GM1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all GM2 gangliosidosis patients were demarcated from controls by elevated GM2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in GM2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted ß-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for GM2 gangliosidosis by allowing quantification of a reduction in the primary storage burden.


Subject(s)
Chromatography, High Pressure Liquid , G(M2) Ganglioside/analysis , Tandem Mass Spectrometry , Cell Line , Fibroblasts/chemistry , G(M2) Ganglioside/blood , G(M2) Ganglioside/isolation & purification , Humans , Leukocytes/chemistry , Solid Phase Extraction , Spectrometry, Mass, Electrospray Ionization , beta-N-Acetylhexosaminidases/metabolism
2.
Article in English | MEDLINE | ID: mdl-14552811

ABSTRACT

GM2 ganglioside, beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)-]beta-Gal-(1-4)-beta-Glc-(1-1)-Cer, is the main ganglioside in the brain of Tay-Sachs patients. In this work, GM2 ganglioside was extracted from a Variant B Tay-Sachs human brain, purified to homogeneity of the oligosaccharide moiety by silica gel chromatography. It was further fractionated for the first time into the molecular species differing in the ceramide structures by reverse-phase flash chromatography. The GM2 ganglioside species were characterized by gas-chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. The major GM2 species contained the ceramides with d18:1-18:0 (40.5% of the total GM2 species), d20:1-18:0 (31%) and d18:1-20:0 (12%). We also found minor GM2 species with the ceramides with d18:1-24:1 (4%), d18:1-22:0 (2%) and d18:2-24:1 (1%), which have not been reported previously.


Subject(s)
Chromatography, Gel/methods , G(M2) Ganglioside/isolation & purification , Chromatography, Gas , G(M2) Ganglioside/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Silica Gel , Silicon Dioxide/chemistry
3.
J Biol Chem ; 278(37): 35286-91, 2003 Sep 12.
Article in English | MEDLINE | ID: mdl-12829702

ABSTRACT

Tay-Sachs disease (TSD) is a classical glycosphingolipid (GSL) storage disease. Although the genetic and biochemical bases for a massive cerebral accumulation of ganglioside GM2 in TSD have been well established, the mechanism for the neural dysfunction in TSD remains elusive. Upon analysis of GSLs from a variant B TS brain, we have detected a novel GSL that has not been previously revealed. We have isolated this GSL in pure form. Using NMR spectroscopy, mass spectrometry, and chemical synthesis, the structure of this unusual GSL was established to be a taurine-conjugated GM2 (tauro-GM2) in which the carboxyl group of N-acetylneuraminic acid was amidated by taurine. Using a rabbit anti-tauro-GM2 serum, we also detected the presence of tauro-GM2 in three other small brain samples from one variant B and two variant O TSD patients. On the other hand, tauro-GM2 was not found in three normal human brain samples. The presence of tauro-GM2 in TS brains, but not in normal brains, indicates the possible association of this unusual GM2 derivative with the pathogenesis of TSD. Our findings point to taurine conjugation as a heretofore unelucidated mechanism for TS brain to cope with water-insoluble GM2.


Subject(s)
Brain Chemistry , G(M2) Ganglioside/analogs & derivatives , G(M2) Ganglioside/genetics , Taurine , Tay-Sachs Disease/metabolism , Chromatography, Thin Layer , G(M2) Ganglioside/chemistry , G(M2) Ganglioside/isolation & purification , Humans , N-Acetylneuraminic Acid/chemistry , N-Acetylneuraminic Acid/isolation & purification , Spectrometry, Mass, Electrospray Ionization , Tay-Sachs Disease/genetics
4.
J Am Soc Nephrol ; 10(3): 435-43, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10073593

ABSTRACT

This study describes the molecular characterization of an antigen defined by an autoantibody from a woman with habitual abortion as GM2-activator protein. The patient showed no disorder of renal function. Accidentally with routine serum screening for autoantibodies, an immunoreactivity was found in kidney collecting duct intercalated cells. Three distinct patterns of immunostaining of intercalated cells were observed: staining of the apical pole, basolateral pole, and diffuse cytoplasmic labeling. Ultrastructurally, the immunoreactivity was associated with "studs," which represent the cytoplasmic domain of the vacuolar proton pump in intercalated cells. This pump is subjected to a shuttling mechanism from cytoplasmic stores to the cell membrane, which exclusively occurs in intercalated cells. Peptide sequences of a 23-kD protein purified from rat kidney cortex showed complete identity with corresponding sequences of GM2-activator protein. In the brain, GM2-activator protein is required for hexosaminidase A to split a sugar from ganglioside GM2. Because neither ganglioside GM2 nor GM1 (its precursor) is present in significant amounts in the kidney, the previous finding that this tissue contains the highest level of activator protein in the body was confusing. In this study, a novel role for GM2-activator protein in intercalated cells is proposed, and possible roles in the shuttling mechanism are discussed.


Subject(s)
G(M2) Ganglioside/metabolism , Kidney Tubules, Collecting/metabolism , Kidney Tubules, Collecting/ultrastructure , Proteins/metabolism , Abortion, Habitual/immunology , Animals , Antigen-Antibody Reactions , Autoantibodies/analysis , Base Sequence , Biological Transport , Cell Membrane/ultrastructure , Cells, Cultured , Cytoplasm/ultrastructure , Female , G(M2) Activator Protein , G(M2) Ganglioside/genetics , G(M2) Ganglioside/isolation & purification , Humans , Immunohistochemistry , Kidney Tubules, Collecting/chemistry , Kidney Tubules, Collecting/cytology , Microscopy, Confocal , Microscopy, Fluorescence , Microscopy, Immunoelectron , Molecular Sequence Data , Polymerase Chain Reaction , Pregnancy , Protein Binding , Proteins/genetics , Proteins/isolation & purification , Proton Pumps/ultrastructure , Rats , Rats, Sprague-Dawley , Reference Values , Sensitivity and Specificity
5.
J Biol Chem ; 272(5): 2828-33, 1997 Jan 31.
Article in English | MEDLINE | ID: mdl-9006924

ABSTRACT

GM2 activator protein is a protein cofactor that has been shown to stimulate the enzymatic hydrolysis of both GalNAc and NeuAc from GM2 (Wu, Y. Y., Lockyer, J. M., Sugiyama, E., Pavlova, N.V., Li, Y.-T., and Li, S.-C. (1994) J. Biol. Chem. 269, 16276-16283). To understand the mechanism by which GM2 activator stimulates the hydrolysis of GM2, we examined the interaction of this activator protein with GM2 as well as with other glycosphingolipids by TLC overlay and Sephacryl S-200 gel filtration. The TLC overlay analysis unveiled the binding specificity of GM2 activator, which was not previously revealed. Under the conditions optimal for the activator protein to stimulate the hydrolysis of GM2 by beta-hexosaminidase A, GM2 activator was found to bind avidly to acidic glycosphingolipids, including gangliosides and sulfated glycosphingolipids, but not to neutral glycosphingolipids. The gangliosides devoid of sialic acids, such as asialo-GM1 and asialo-GM2, and the GM2 derivatives whose carboxyl function in the NeuAc had been modified by methyl esterification or reduction, were only very weakly bound to GM2 activator. These results indicate that the negatively charged sugar residue or sulfate group in gangliosides is one of the important sites recognized by GM2 activator. For comparison, we also studied in parallel the complex formation between glycosphingolipids and saposin B, a separate activator protein with broad specificity to stimulate the hydrolysis of various glycosphingolipids. We found that saposin B bound to neutral glycosphingolipids and gangliosides equally well, and there was an exceptionally strong binding to sulfatide. In contrast to previous reports, we found that GM2 activator formed complexes with GM2 and other gangliosides in different proportions depending on the ratio between the activator protein and the ganglioside in the incubation mixture prior to gel filtration. We were not able to detect the specific binding of GM2 activator to GM2 when GM2 was mixed with GM1 or GM3. Thus, the specificity or the mode of action of GM2 activator cannot be simply explained by its interaction with glycosphingolipids based on complex formation. The binding of GM2 activator to a wide variety of negatively charged glycosphingolipids may indicate that this activator protein has functions other than assisting the enzymatic hydrolysis of GM2.


Subject(s)
G(M2) Ganglioside/metabolism , Glycosphingolipids/metabolism , Oligosaccharides/chemistry , Proteins/chemistry , Proteins/metabolism , Brain Chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, Gel , Chromatography, Thin Layer , G(M2) Activator Protein , G(M2) Ganglioside/isolation & purification , Glycoproteins , Glycosphingolipids/chemistry , Glycosphingolipids/isolation & purification , Humans , Kinetics , Molecular Sequence Data , Proteins/isolation & purification , Saposins , Sphingolipid Activator Proteins , Structure-Activity Relationship , Substrate Specificity , Tay-Sachs Disease/metabolism
6.
Anal Biochem ; 254(2): 221-5, 1997 Dec 15.
Article in English | MEDLINE | ID: mdl-9417780

ABSTRACT

A rapid, simple, and efficient chloroform/methanol-free method of isolating gangliosides is described. The nonionic polyoxyethylene detergent, hexaethyleneglycol mono-n-tetradecyl ether (C14EO6), forms clear micellar solutions in water, but two-phase separation can be achieved by centrifugation in the presence of ammonium sulfate at room temperature. A mixture of pure gangliosides, metabolically labeled gangliosides obtained from cultured hippocampal neurons, and gangliosides from rat cerebellar tissue were quantitatively recovered in the detergent-rich upper (coacervate) phase, with a partition coefficient K > 60. Gangliosides were subsequently separated from the detergent using an Iatrobead column prior to analysis by thin-layer chromatography. The procedure described here is as efficient as other methods of ganglioside extraction, such as that using chloroform/methanol/water/pyridine, but is less time-consuming inasmuch as extraction, purification, and TLC analysis can be completed within 1 day.


Subject(s)
Detergents/metabolism , Ethylene Glycols/metabolism , Gangliosides/isolation & purification , Animals , Cerebellum/chemistry , Chemistry Techniques, Analytical/methods , Chromatography, Thin Layer , G(M1) Ganglioside/isolation & purification , G(M2) Ganglioside/isolation & purification , G(M3) Ganglioside/isolation & purification , Hippocampus/chemistry , Neurons/chemistry , Rats , Sphingomyelins/metabolism , Tritium/metabolism
7.
Diabetes ; 45(9): 1193-6, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8772721

ABSTRACT

Recently, the GM2-1 pancreatic islet ganglioside, proposed as a potential autoantigen in type I diabetes autoimmunity, has been biochemically characterized and found to be a novel ganglioside structure. In the present study, we aimed to determine whether an autoimmune response toward this novel islet molecule is 1) present in type I diabetes and is specifically directed against this molecule and not to gangliosides in general and 2) predictive of disease in high-risk subjects. To this end, the following patients have been studied: 1) 24 newly diagnosed type I diabetic subjects, 20 islet cell autoantibody (ICA)-negative first-degree relatives of type I diabetic subjects, and 25 age-matched normal control individuals; and 2) 31 prospectively evaluated ICA+ first-degree relatives of type I diabetic subjects who were followed for up to 10 years, during which 14 of them developed type I diabetes. A direct assay for autoantibodies to GM2-1 and to other pancreatic gangliosides (GM3, GD3, GD1a) was developed using an indirect immunoperoxidase technique performed directly on thin layer chromatography plates. Anti-GM2-1 autoantibodies (all belonging to the IgG class) were expressed in a high percentage of newly diagnosed type I diabetic subjects (71%), while no significant difference was found in the expression of antibodies directed against other pancreatic gangliosides (GM3, GD3, GD1a) among the different groups studied. Anti-GM2-1 autoantibodies were also present in ICA+ relatives (64%) (P < 0.001 vs. control subjects and ICA-relatives): in this group, life table analysis of progression to diabetes showed that anti-GM2-1 autoantibodies were significantly (P < 0.001) associated with disease, occurring in all relatives developing type I diabetes within 5 years and thus identifying a cohort of ICA+ subjects with markedly increased diabetes risk.


Subject(s)
Autoantibodies/blood , Autoantigens/immunology , Autoimmunity , Diabetes Mellitus, Type 1/immunology , G(M2) Ganglioside/immunology , Islets of Langerhans/immunology , Prediabetic State/immunology , Autoantigens/isolation & purification , Diabetes Mellitus, Type 1/genetics , Family , G(M2) Ganglioside/isolation & purification , Gangliosides/immunology , Humans , Islets of Langerhans/chemistry , Reference Values
8.
Glycoconj J ; 13(3): 391-9, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8781970

ABSTRACT

The human glioma D-54MG cell line grown in vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3'-isoLM1, an alteration that was dose dependent, with the highest dose (1 x 10(8)) resulting in a phenotype that was most like that of the inoculum. After one passage in vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grown in vitro than in those grown in vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors.


Subject(s)
G(M2) Ganglioside/biosynthesis , Gangliosides/biosynthesis , Glioma/metabolism , N-Acetylgalactosaminyltransferases/biosynthesis , Animals , Carbohydrate Sequence , Cell Division , Cell Line , Culture Media , G(M2) Ganglioside/chemistry , G(M2) Ganglioside/isolation & purification , Gangliosides/chemistry , Gangliosides/isolation & purification , Glioma/pathology , Humans , Mice , Mice, Nude , Molecular Sequence Data , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Spectrometry, Mass, Fast Atom Bombardment , Transcription, Genetic , Transplantation, Heterologous , Tumor Cells, Cultured , Polypeptide N-acetylgalactosaminyltransferase
9.
Proc Natl Acad Sci U S A ; 92(7): 2805-9, 1995 Mar 28.
Article in English | MEDLINE | ID: mdl-7708728

ABSTRACT

Gangliosides, such as GM2, GD2, GD3, and 9-O-acetyl GD3, are receiving attention as targets for antibody-based and vaccine-based therapies of melanoma. GM2 appears to be a particularly immunogenic ganglioside in humans, as indicated by the presence of naturally occurring IgM anti-GM2 antibodies in approximately 5% of humans and the fact that immunization with irradiated GM2-expressing melanoma cells or purified GM2 adherent to bacillus Calmette-Guérin elicits GM2 antibodies of low to moderate titers in a high proportion of vaccinated patients. To develop vaccines that consistently induce high titers of IgM as well as IgG anti-GM2 antibodies, vaccines containing GM2 conjugated to keyhole limpet hemocyanin as the carrier protein and QS-21 as the adjuvant have been constructed. The serological response of vaccinated patients was monitored by ELISA using purified GM2 ganglioside for IgM and IgG anti-GM2 antibodies and for GM2 cell surface-reactive antibodies by immune adherence assays and cytotoxic tests (IgM antibodies) and mixed hemadsorption assays (IgG antibodies). The majority of vaccinated patients developed IgM and IgG antibodies detectable by ELISA. In most cases, the results of IgM ELISA correlated with assays for cell surface-reactive IgM antibodies. This was not true for IgG anti-GM2 antibodies, where strong discrepancies were seen between high titers in ELISA and little or no reactivity in mixed hemadsorption tests for cell surface-reactive antibodies. These IgG antibodies (and the less frequent IgM antibodies that show similar discrepancies) may be directed against GM2 determinants that are buried, hidden, or not present on GM2-expressing target cells. With regard to a major objective of ganglioside vaccines--i.e., generation of cytotoxic antibodies--the GM2-keyhole limpet hemocyanin/QS-21 vaccine is clearly superior to the previously tested GM2/bacillus Calmette-Guérin vaccine. However, variability in patient response and lack of persistence of high-titered IgM cytotoxic antibodies in many patients are problems that remain to be solved.


Subject(s)
G(M2) Ganglioside/immunology , Melanoma/therapy , Vaccines, Synthetic/therapeutic use , Adjuvants, Immunologic , Animals , Brain/metabolism , Cats , Cattle , Cyclophosphamide/therapeutic use , Enzyme-Linked Immunosorbent Assay , G(M2) Ganglioside/isolation & purification , Hemocyanins/immunology , Immunoglobulin G/blood , Immunoglobulin M/blood , Melanoma/blood , Melanoma/immunology , Monitoring, Immunologic , Neoplasm Staging , Tay-Sachs Disease/metabolism , Time Factors
10.
Biokhimiia ; 55(1): 43-51, 1990 Jan.
Article in Russian | MEDLINE | ID: mdl-2140519

ABSTRACT

Hexosaminidase A (HA) was isolated from human kidney and purified to an electrophoretically homogeneous state. The purification procedure included ion-exchange chromatography on DEAE-cellulose, gel filtration on Toyopearl HW-55 and chromatofocusing on PBE 94 (enzyme yield 26.6%, 1133.6-fold purification). The physico-chemical and kinetic properties of HA are as follows: Mr of the purified enzyme is approximately 100,000; Km for 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside is 0.6 mM; pH optimum is at pH 4.4-4.6; pI is 5.0. The amino acid composition of the purified enzyme was determined. A specific anti-HA antiserum was raised, which did not immunoprecipitate with fibroblast extracts characterized by a mutational blockade of HA synthesis. GM2 was isolated and purified from murine liver as well as from the brain of a female patient who died of Tay-Sachs disease. The label was introduced by way of treatment of GM2 with tritiated acetic anhydride. The specific radioactivity of [3H]GM2 was 521 and 2065 Ci/M, respectively. The label was introduced into the N-acetylneuraminic acid and GalNAc residues of these GM2 preparations. An activator protein capable of solubilizing the natural substrate of HA was isolated from human kidney and partially purified (with a 19.9% yield and 480-fold purification). The Mr of the purified activator protein was approximately 21,000. Purified HA hydrolyzed [3H]GM2 only in the presence of the activator protein. An addition of the activator to the incubation medium containing normal fibroblast culture extracts and [3H]GM2 caused an increase in the rate of substrate hydrolysis, tenfold, on the average.


Subject(s)
G(M2) Ganglioside/isolation & purification , Gangliosides/isolation & purification , Glycoproteins/isolation & purification , Kidney/enzymology , beta-N-Acetylhexosaminidases/isolation & purification , Amino Acids/analysis , Animals , Brain Chemistry , Catalysis , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Enzyme Stability , G(M2) Activator Protein , Hexosaminidase A , Humans , Mice , Molecular Weight , Proteins/isolation & purification , Saposins , Sphingolipid Activator Proteins , Tay-Sachs Disease/enzymology , Tay-Sachs Disease/metabolism , beta-N-Acetylhexosaminidases/metabolism
11.
J Biol Chem ; 264(21): 12122-5, 1989 Jul 25.
Article in English | MEDLINE | ID: mdl-2473071

ABSTRACT

A monoclonal antibody produced by immunization with cells of the human glioma cell line D-54 MG reacted with ganglioside GM2. The binding epitope of the antibody was found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal. Immunological detection of glycolipid antigens on thin-layer plates with this monoclonal antibody, DMAb-1, revealed the presence of a new ganglioside. This ganglioside, co-migrating with NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer(6'-LM1) and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GalNAc beta 1-4Gla beta 1-4Glc beta 1-1Cer (GalNAc-isoGM1) at chromatographic separation was isolated from human meconium. Its structure was determined by permethylation and fast atom bombardment-mass spectometry analyses. The new ganglioside was found to be a combination of the lacto and ganglio series gangliosides, and the structure found to be GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-3GlcNAc alpha 1-3Gal beta 1-4Glc beta 1-1Cer(GalNAc-3'-isoLM1).


Subject(s)
Gangliosides/isolation & purification , Meconium/analysis , Antibodies, Monoclonal , Carbohydrate Conformation , Carbohydrate Sequence , Epitopes/analysis , G(M2) Ganglioside/isolation & purification , Gangliosides/immunology , Glycolipids/immunology , Humans , Infant, Newborn , Mass Spectrometry , Molecular Sequence Data
12.
Int J Cancer ; 43(5): 857-62, 1989 May 15.
Article in English | MEDLINE | ID: mdl-2714890

ABSTRACT

Ganglioside GM2 is expressed on cell surface membranes of a variety of human malignant cells and has been demonstrated to be immunogenic in humans. We have assessed the role of the antigen GM2 on melanoma cells as a recognition structure for lymphokine-activated killer (LAK) cells. LAK cells were generated by stimulation of non-adherent peripheral blood lymphocytes (PBL) from human donors with recombinant interleukin-2 (IL-2). The selection of target cells was based on GM2 content and included 11 human melanoma cell lines and 2 human leukemia lines. Using a single-cell binding assay, LAK cell binding to target lines expressing high levels of GM2 was significantly greater than to those expressing minimum GM2. This cell-binding was specifically inhibited by addition of purified GM2 but not by other gangliosides. LAK-melanoma cell-binding was also specifically inhibited by anti-GM2 monoclonal antibody (MAb). For further analysis LAK cell lysis of melanoma target cells expressing various amounts of GM2 was assessed. A significant correlation occurred with GM2 expression and LAK cell lysis (p less than 0.025; r = 0.623). Three other gangliosides commonly expressed on human melanoma, GM3, GD3 and GD2, had no correlation with LAK cell lysis. These studies suggest that GM2 on melanoma cells is a marker for LAK cell sensitivity, as well as indicate that GM2 is a potential target recognition structure for human LAK cells.


Subject(s)
Cytotoxicity, Immunologic , G(M2) Ganglioside/biosynthesis , Gangliosides/biosynthesis , Killer Cells, Natural/immunology , Lymphocyte Activation , Lymphokines/immunology , Melanoma/immunology , Membrane Lipids/biosynthesis , Tumor Cells, Cultured/immunology , Antibodies, Monoclonal/immunology , Cell Line , Cell Membrane/metabolism , G(M2) Ganglioside/isolation & purification , Humans , Melanoma/metabolism , Membrane Lipids/isolation & purification , Tumor Cells, Cultured/metabolism
13.
J Lipid Res ; 25(10): 1132-9, 1984 Oct.
Article in English | MEDLINE | ID: mdl-6512419

ABSTRACT

A method for the separation of molecular species of brain monosialogangliosides by high-performance liquid chromatography is described. GM4, GM3, GM2, and GM1 were purified from human brain and their individual molecular species were separated on a C18 reversed-phase column. Peaks were identified by mass spectrometry of the intact ganglioside, by gas-liquid chromatography of the fatty acids, and by high-performance liquid chromatography of the long chain bases. A characteristic elution sequence of molecular species permitted their identification based upon their retention times on the reversed-phase column.


Subject(s)
Brain Chemistry , Gangliosides/isolation & purification , Ceramides/analysis , Chromatography, High Pressure Liquid/methods , Fatty Acids/analysis , G(M1) Ganglioside/isolation & purification , G(M2) Ganglioside/isolation & purification , G(M3) Ganglioside/isolation & purification , Humans , Mass Spectrometry/methods
14.
J Biol Chem ; 259(14): 8980-5, 1984 Jul 25.
Article in English | MEDLINE | ID: mdl-6746635

ABSTRACT

The major ganglioside from the roe of striped mullet (Mugil cephalus) has been isolated and purified. Compositional analysis of this ganglioside revealed that it contained an equimolar ratio of the following residues: N-acetylneuraminic acid, N-acetylgalactosamine, galactose, glucose, and the long-chain base. Further structural studies by sequential enzymatic hydrolysis, permethylation analysis, and proton NMR spectroscopy indicated that the structure of the oligosaccharide moiety was identical to that of GM2 ganglioside from human brain: GalNAc beta 1----4Gal beta 1----4(3----2 alpha NeuAc)-Glc----ceramide. This ganglioside, however, differed from brain GM2 in its ceramide portion. The most striking differences are the presence of large amounts of C18 and C20 phytosphingosine (over 80% of the total long-chain bases) and the preponderance of monounsaturated alpha-hydroxy fatty acids (over 80%). Such a phytosphingosine-containing GM2 ganglioside has never been reported.


Subject(s)
G(M2) Ganglioside/isolation & purification , Gangliosides/isolation & purification , Ovum/analysis , Sphingosine/analogs & derivatives , Animals , Carbohydrate Conformation , Carbohydrate Sequence , Carbohydrates/analysis , Chromatography, Thin Layer , Fatty Acids/analysis , Female , Fishes , Hydroxy Acids/analysis , Sphingosine/analysis
15.
J Chromatogr ; 195(2): 257-64, 1980 Jul 18.
Article in English | MEDLINE | ID: mdl-6773970

ABSTRACT

Total ganglioside extracts prepared from brain tissue were concentrated either by dialysis against Carbowax or by employing Millipore filter cones. Thin-layer chromatography was then carried out using silica gel plates. After location of the various fractions quantitation was effected by direct densitometry. The methods that have been adopted are rapid and suitable for the study of brain gangliosides in post mortem and biopsy material in a clinical chemistry laboratory.


Subject(s)
Brain/metabolism , Chromatography, Thin Layer/methods , Gangliosides/isolation & purification , Animals , Cats , G(M1) Ganglioside/isolation & purification , G(M2) Ganglioside/isolation & purification , Gangliosides/analysis , Gangliosides/metabolism , Gangliosidoses/metabolism , Humans , Mucopolysaccharidosis III/metabolism , Tay-Sachs Disease/metabolism
16.
J Lipid Res ; 20(5): 678-81, 1979 Jul.
Article in English | MEDLINE | ID: mdl-490046

ABSTRACT

GM2 and GA2 gangliosides from the brain of a patient who died of Sandhoff's disease were purified by solvent partition, silicic acid and silica gel column chromatography, and silica gel preparative thin-layer chromatography. They were tritiated in the terminal N-acetylgalactosamine residue using galactose oxidase and sodium [3H]borohydride with the inclusion of catalase and peroxidase into the oxidation reaction. The specific activities were 4.62 X 10(8) dpm/mumol of GM2 ganglioside and 5.54 X 40(7) dpm/mumol of GA2 ganglioside. The addition of catalase and peroxidase to the tritiation procedure is recommended.


Subject(s)
G(M2) Ganglioside , Gangliosides , Tritium , Brain Chemistry , G(M2) Ganglioside/isolation & purification , Gangliosides/isolation & purification , Humans , Isotope Labeling , Sandhoff Disease
17.
J Natl Cancer Inst ; 60(6): 1313-27, 1978 Jun.
Article in English | MEDLINE | ID: mdl-206706

ABSTRACT

Hyperplastic nodules and hepatocellular carcinomas were induced in livers of rats by a low-protein diet containing 0.05% of the carcinogen N-2-fluorenylacetamide. Ganglioside amounts and composition were determined for histologically different hepatocellular carcinomas and compared with those for control livers, hyperplastic nodules, and liver tissue surrounding hepatomas and nodules as well as those for livers of fetal, newborn, 1-week-old, weanling, and adult Sprague-Dawley rats. Ganglioside sialic acid levels were elevated above those of normal adult liver in all liver tissues following the carcinogen treatment regimen. Livers of fetal and newborn rats contained nearly twice the amount of ganglioside sialic acid on a protein or DNA basis as did livers of adult rats. Analyses of individual nodules and hepatomas revealed two populations of tumors in which the levels of ganglioside sialic acid were 2.3 and 3.8 times normal. Ganglioside sialic acid content was at hepatoma levels in small nodules. Individual gangliosides were evenly distributed between products of the monosialoganglioside and disialoganglioside pathways in normal liver with a ratio of [N-acetylneuraminic acid (sialic acid)] (NAN)-galactose (Gal)-N-acetylgalactosamine (GalNAc)-(NAN)-Gal-glucose (Glc)-ceramide (Cer) (GD1a) to Gal-GalNAc-(NAN)2-Gal-Glc-Cer (GD1b) of about one. In contrast, the monosialogangliosides predominated in liver tissues following administration of the carcinogen. Increased levels of specific monosialogangliosides were present in nodules, in liver of carcinogen-treated animals prior to the appearance of tumors, and in the liver tissues surrounding nodules and hepatomas. In single hepatomas, ganglioside patterns correlated with tumorigenicity. A well-differentiated hepatoma had a normal complement of most gangliosides but was deficient in trisialogangliosides. In a poorly diferentiated but well-circumscribed hepatoma, the relative levels of all higher gangliosides were reduced. The monosialoganglioside Gal-GalNAc-(NAN)-Gal-Glc-Cer (GM1) accounted for 80% of the total ganglioside in a poorly circumscribed and poorly differentiated hepatoma. The ganglioside pattern of fetal livers most closely resembled that of a poorly differentiated hepatoma. During the first week post natum, levels of all higher monosialogangliosides and disialogangliosides declined, but the decline was most pronounced for gangliosides GM1 and GD1a. The ratio of GM1 + GD1a to GD1b + NAN-Gal-GalNAc-(NAN)2-Gal-Glc-Cer or (NAN)3-Gal-Glc-Cer (GT), used as an index of the relative predominance of the monoslaloganglioside and disialoganglioside pathways, fell from 2.7 for fetal liver to 0.4 for adult liver. Pools of precursor gangliosides increased during development, transiently for GalNAc-(NAN)-Gal-Glc-Cer and for more than 3 weeks for NAN-Gal-Glc-Cer. When hyperplastic nodules and hepatocellular carcinomas were compared, a reverse pattern was observed. The ratio of GM1 + GD1a to GD1b + GT rose steadily to values of 2.7 and 11...


Subject(s)
2-Acetylaminofluorene , Carcinoma, Hepatocellular/metabolism , Fluorenes , Gangliosides/metabolism , Liver Neoplasms/metabolism , Animals , Animals, Newborn/metabolism , Carcinoma, Hepatocellular/chemically induced , G(M1) Ganglioside/isolation & purification , G(M1) Ganglioside/metabolism , G(M2) Ganglioside/isolation & purification , G(M2) Ganglioside/metabolism , G(M3) Ganglioside/isolation & purification , G(M3) Ganglioside/metabolism , Hyperplasia/metabolism , Liver/embryology , Liver/metabolism , Liver Neoplasms/chemically induced , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/metabolism , Precancerous Conditions/metabolism , Rats , Sialic Acids/metabolism
18.
Proc Natl Acad Sci U S A ; 72(7): 2520-4, 1975 Jul.
Article in English | MEDLINE | ID: mdl-1058471

ABSTRACT

Ganglioside GM1 was isolated from the small intestinal mucosa of man, pig, and beef and amounted to 0.1, 2.0, and 43 nmol per g fresh weight, respectively. These differences in GM1 content were associated with a quantitatively differing ability of the mucosal cells to bind cholera toxin. Human cells bound about 15,000 toxin molecules when saturated with the toxin, porcine cells 120,000, and bovine cells 2,600,000 molecules. The association constant (KA) of the cholera toxin binding was, for cells of all three species, about 10(9) liters/mol. Exogenously added GM1 ganglioside was incorporated in intestinal mucosal cells as well as in intact rabbit small bowel. The increment in GM1 was associated with a correspondingly increased number of binding sites for cholera toxin, whereas KA was unchanged. GM1 incorporation increased the sensitivity of the rabbit small bowel to the diarrheogenic action of cholera toxin. Vibrio cholerae sialidase hydrolyzed isolated intestinal diand trisialogangliosides to GM1. However, the enzyme did not change the ganglioside pattern of intestinal mucosa, had very little influence on the number of toxin binding sites on intestinal cells, and did not alter the sensitivity of the small bowel to the diarrheogenic action of the toxin. These results demonstrate a relationship in the intestinal mucosa between the GM1 ganglioside concentration, the number of binding sites for cholera toxin, and the sensitivity to the biologic action of the toxin. Thus, the study strongly supports the concept that the GM1 ganglioside is the intestinal binding receptor for cholera toxin.


Subject(s)
G(M2) Ganglioside , Gangliosides , Intestine, Small/analysis , Toxins, Biological , Animals , Binding Sites , Cattle , Cell Membrane/analysis , Cerebral Cortex/analysis , Cholera , G(M2) Ganglioside/isolation & purification , Humans , Intestinal Mucosa/analysis , Jejunum/analysis , Kinetics , Neuraminidase , Organ Specificity , Protein Binding , Rabbits , Receptors, Drug , Species Specificity , Swine , Vibrio cholerae/enzymology
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