Characterization of a nonspecific activator protein for the enzymatic hydrolysis of glycolipids.

We have studied the substrate specificities of a non-specific activator protein on the enzymatic hydrolyses of the following compounds: GM1 and GM2, as well as several of their derivatives including oligosaccharides, GgOse3Cer-II3-sulfate and LacCer-II3-sulfate, Gb-Ose3Cer and GbOse4Cer, three neolacto-series glycosphingolipids, and two non-ceramide glycolipids. Our results show that this activator protein has a broad spectrum of activity and exhibits the properties of a nonspecific natural detergent. The evidence of non-specificity was the ability of this activator protein to stimulate the hydrolyses of glycolipids, regardless of glycosphingolipids or non-ceramide glycolipids, carried out by glycosidases from animals, plants, and microorganisms.

The antitumor monoclonal antibody MOv2 recognizes the Lewis A hapten.

Monoclonal antibody MOv2, produced against ovarian carcinoma, was previously found to bind a carbohydrate epitope (CAMOv2) present on mucins, glycoproteins and a neutral glycolipid. In this paper, the structure of the carbohydrate epitope is determined by immunological reactivity with purified glycolipids and oligosaccharides. Using solid-phase radioimmunoassay and immunostaining of thin layer chromatograms, MOv2 binds strongly to Le(a)-active pentasaccharide ceramide.

Semisynthetic preparation of N-glycolylneuraminic acid containing GM1 ganglioside: chemical characterization, physico-chemical properties and some biochemical features.

N-Glycolylneuraminic acid containing GM1, GM1(NeuGc), was prepared by semisynthetic procedure. The procedure makes use of GM1 ganglioside deacetylated at the level of sialic acid residue (deAc-GM1) and of 1,3-dioxalan-2,4-dione. DeAc-GM1 is prepared from GM1 by alkaline hydrolysis in the presence of tetramethylammonium hydroxide and the glycolylating compound by reaction of glycolic acid with phosgene in dioxane, followed by cyclization under vacuum.

New trends in ganglioside chemistry.

New methods have been developed for the preparation of highly purified gangliosides, homogeneous in the saccharide, long chain base, and fatty acid moieties and gangliosides carrying different kinds of labelled probes. Gangliosides, homogeneous in the oligosaccharide portion, were prepared by preparative normal phase HPLC on a Lichrosorb-NH-2 column, using a gradient of acetonitrile-phosphate buffer, pH 5.6, as solvent system.

The sialic acid residue of exogenous GM1 ganglioside is recycled for biosynthesis of sialoglycoconjugates in rat liver.

In order to assess metabolic recycling of sialic acid, GM1 ganglioside [nomenclature of Svennerholm (1964) J. Lipid. Res.

New chemical trends in ganglioside research.

A report is given of recent progress in the methodology for isolation of gangliosides from natural sources, for the preparation of molecular species of gangliosides homogeneous in both the oligosaccharide and ceramide portions of the molecule, for chemical manipulation and derivatization of gangliosides, and for the preparation of gangliosides radiolabelled in different parts of the molecule. Particular emphasis has been given to: high performance liquid chromatographic procedures capable to separate gangliosides on the basis of their oligosaccharide or ceramide moieties and yielding completely homogeneous compounds, that is gangliosides with a single oligosaccharide, a single long chain base and a single fatty acid; two-dimensional thin-layer chromatographic procedures, provided with a fully computerized quantification system, particularly suitable to identifying gangliosides containing alkali-labile linkages, including ganglioside lactones; chemical procedures of high yield for reducing gangliosides at the double bond of long chain base, for selective removal of the fatty acyl moiety and replacement with a novel fatty acid, and for the synthesis of ganglioside lactones; chemical procedures for inserting fluorescent, paramagnetic or photoreactive probes at the fatty acyl part of the ganglioside molecule; procedures for chemical isotopic radiolabelling of gangliosides at the level of sialic acid acetyl group and at the fatty acid moiety. Examples are provided evidencing the significance and potential use of a variety of ganglioside derivatives in the study of ganglioside metabolism and functional implications.

Light scattering measurements on gangliosides: dependence of micellar properties on molecular structure and temperature.

Static and dynamic laser light scattering measurements on micellar aqueous solutions of gangliosides GM2, GM1, GD1a are reported. The aggregation number, the hydrodynamic radius and the micellar shape depend on the type of ganglioside and the unsaturation degree of the hydrocarbon chains. At a temperature of 25 degrees C the molecular weights of GM2, GM1 and GD1a are 740,000, 470,000 and 418,000 DA respectively.

Incorporation and metabolism of exogenous GM1 ganglioside in rat liver.

The pathways of metabolic processing of exogenously administered GM1 ganglioside in rat liver was investigated at the subcellular level. The GM1 used was 3H-labelled at the level of long-chain base ([Sph(sphingosine)-3H]GM1) or of terminal galactose ([Gal-3H]GM1). The following radioactive compounds, derived from exogenous GM1, were isolated and chemically characterized: gangliosides GM2, GM3, GD1a and GD1b (nomenclature of Svennerholm [(1964) J.

Natural occurrence of ganglioside lactones. Isolation and characterization of GD1b inner ester from adult human brain.

A new ganglioside containing an inner ester linkage was extracted from adult brain specimens, obtained at the time of surgery on 51-70-year-old subjects, purified, and analyzed. It contains glucose, galactose, N-acetylgalactosamine, an N-acetylneuraminic acid in the molar ratio 1:2:1:2, but, on ion-exchange chromatography, behaves as a monosialoganglioside. Structural analyses showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose ceramide, carrying a disialosyl residue on the 3-position of internal galactose.

A new chemical procedure for the preparation of gangliosides carrying fluorescent or paramagnetic probes on the lipid moiety.

A new chemical procedure is described for preparing labelled GM1 molecular species, carrying as acyl moiety pyrene-decanoic acid, 5-doxyl-stearic acid and 16-doxyl-stearic acid. It makes use of a mixed anhydride formed by ethylchloroformate and the labelled acyl chain, as the reagent for N-acylation of a deacetylated, deacylated GM1 ganglioside, which is prepared by alkaline hydrolysis of natural GM1. The reaction performed with a unitary GM1 derivative/mixed anhydride molar ratio, occurs with a yield of above 40%.

A radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in cultured human fibroblasts.

A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GDla isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated [3H]GM1 was determined by computer-assisted radiochromatoscanning.

Carbohydrate epitope defined by an antitumor monoclonal antibody detected on glycoproteins and a glycolipid by immunoblotting.

The murine monoclonal antibody (MAb) MOv2 was found to be directed against the carbohydrate moieties of different kinds of molecules expressed on a human ovarian cystoadenocarcinoma. To define further the glycoconjugates carrying the MOv2-defined epitope, different procedures were used to analyze materials from surgical specimens and carcinoma cell lines. SDS-PAGE and immunoblotting showed glycoprotein molecules migrating in the gel as high and intermediate molecular weight components.

Normal-phase high-performance liquid chromatographic separation of non-derivatized ganglioside mixtures.

A new analytical and semi-preparative high-performance liquid chromatographic method for the separation of a brain ganglioside mixture into individual components is described. Gangliosides were applied to a LiChrosorb-NH2 column and eluted with the solvent system acetonitrile-phosphate buffer at different volume ratios and ionic strengths. The elution profile was monitored by flow-through detection of UV absorbance at 215 nm.

Analysis of gangliosides using fast atom bombardment mass spectrometry.

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues.

Characterization of two molecular species GD3 ganglioside from bovine buttermilk.

Two gangliosides, representing 85% of total lipid-bound sialic acid, have been isolated from bovine buttermilk and characterized. Both contained long-chain base, glucose, galactose and sialic acid in the molar ratio 1:1:1:2, and gave, upon sialidase treatment, a neutral glycolipid, characterized as lactosylceramide. Partial acid hydrolysis, permethylation analysis and chromium trioxide oxidation indicated their basic oligosaccharide portion to be NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc.

Preparation of GM1 ganglioside molecular species having homogeneous fatty acid and long chain base moieties.

A new procedure is described for preparing the molecular species of GM1 ganglioside that carry a single fatty acid (myristic (C14:0), stearic (C18:0), arachidic (C20:0) or lignoceric (C24:0) acid) and a single long chain base (C18 or C20 sphingosine, C18 or C20 sphinganine, each of them in natural 3D(+)erythro or unnatural 3L(-)threo form). The procedure consisted of the following steps: a) alkaline hydrolysis of GM1 ganglioside in the presence of tetramethylammonium hydroxide, which produces de-N-acylation of the ceramide and de-N-acetylation of the sialic acid residue; b) specific re-N-acylation at the long chain base amino group with a new fatty acid (myristic, stearic, arachidic, or lignoceric) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride; and c) final re-N-acetylation at the level of the sialic acid residue. GM1 ganglioside molecular species, completely homogeneous in the ceramide portion, were prepared by reversed phase high performance liquid chromatography.

Biochemical analysis of human ovarian cancer-associated antigens defined by murine monoclonal antibodies.

Two monoclonal antibodies (MOv1 and MOv2) raised against a membrane preparation of a human surgical specimen from a mucinous ovarian cystoadenocarcinoma were used to biochemically define their target antigens. The heating of peritumoral mucus-soluble extracts and the sialidase treatment of crude membrane preparations did not affect the binding capacity of MOv1 and MOv2, which, on the contrary, was significantly reduced by periodate oxidation of the same materials. Pronase digestion completely solubilized MOv1-defined antigens, whereas MOv2-defined antigens were only partially solubilized.

Characterization of a glycosphingolipid antigen defined by the monoclonal antibody MBr1 expressed in normal and neoplastic epithelial cells of human mammary gland.

The antigen defined by a monoclonal antibody, MBr1, was found to be expressed in normal human mammary gland epithelia and human mammary carcinoma cells (Ménard, S., Tagliabue, E., Canevari, S.

Ganglioside pattern of normal human brain, from samples obtained at surgery. A study especially referred to alkali labile species.

The ganglioside pattern from normal human temporal cortex and cerebellum has been studied in fresh specimens obtained at surgery. The analyses have been performed by two-dimensional thin layer chromatography with an intermediate ammonia treatment which is a methodology particularly suitable for resolving alkali labile gangliosides. Alkali labile gangliosides were detected in all the analyzed specimens and their content contributed to 23% and 11% of total lipid bound sialic acid, in temporal cortex and cerebellum, respectively.

High performance liquid chromatography preparation of the molecular species of GM1 and GD1a gangliosides with homogeneous long chain base composition.

A semi-preparative, analytical high performance liquid chromatographic (HPLC) procedure is described for the isolation of molecular species of GM1 and GD1a gangliosides containing a single long chain base, C18 or C20 sphingosine, C18 or C20 sphinganine, each in its natural erythro or unnatural threo form. The threo forms were obtained from 2,3-dichloro-5,6-dicyanobenzoquinone/NaBH4 -treated gangliosides. The ganglioside molecular species separated by HPLC were analyzed for carbohydrate, fatty acid, and long chain base composition.

Interaction of cholera toxin with gangliosides: differential effects of the oligosaccharide of ganglioside GM1 and of micellar gangliosides.

Ultraviolet difference absorption spectra of cholera toxin and its B protomer produced by the oligosaccharide moiety of the monosialoganglioside GM1 were measured as a function of the oligosaccharide concentration. In the presence of oligosaccharide, the spectrum is characterized by three peaks at 282, 288, and 292 nm. A linear increase in difference absorption was observed at these wavelengths vs.