Aggregate Formation of Gangliosides at Low Concentrations in Aqueous Media

Mraz, W.; Schwarzmann, Günter; Sattler, Jörg; Momoi, Takashi; Seemann, Brigitte; Wiegandt, Herbert

doi:10.1515/bchm2.1980.361.1.177

Cited by 44 publications

(12 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At the end of chase, the cell medium was carefully collected, and cells were repeatedly washed with EMEM, BME, or DMEM, and then with 10% FCS-containing medium (30 min) to remove ganglioside aggregates loosely bound to the cell surface. In some experiments a final 5-min treatment with 0.1% trypsin was performed to remove sphingolipids bound to surface proteins (11,24).…”

Section: Methodsmentioning

confidence: 99%

Sphingolipid Uptake by Cultured Cells

Chigorno

Giannotta

Ottico

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

Human fibroblasts, rat neurons, and murine neuroblastoma cells, cultured in the presence of fetal calf serum, were fed with [1-3 H]sphingosine to radiolabel sphingolipids. The fate of cell sphingolipids, the release of sphingolipids in the culture medium, the interaction of sphingolipids with the proteins and lipoproteins of fetal calf serum, and the fate of sphingolipids taken up by the cells were investigated. For this latter purpose, the culture medium containing radioactive sphingolipids was delivered to nonlabeled cells. The presence of tritium at position 1 of sphingosine allowed us to follow the extent of sphingolipid catabolism by measuring the production of radioactive phosphatidylethanolamine and proteins by recycling the radioactive ethanolamine formed during sphingosine catabolism and the production of tritiated water. We confirmed that in cells the recycling of sphingosine occurred to a high extent and that only a minor portion of cell sphingolipids was catabolized to the small fragments of ethanolamine and water. Cell sphingolipids were released in the culture medium, where they formed large lipoproteic aggregates at a rate of about 12% per day. Released sphingolipids were taken up by the cells and catabolized to the sphingosine and then to ethanolamine, and recycling of sphingosine was not observed. This suggests that in the presence of fetal calf serum in the culture medium, exogenous sphingolipids directly reach the lysosomes, were they are entirely catabolized. Thus, the trafficking of sphingolipids from cells to the extracellular environment and from this to other cells does not allow the modification of the plasma membrane composition.Membrane sphingolipids are complex lipids displaying a strong amphiphilic character (1). This implies an equilibrium between plasma membrane molecules and free monomers present in the extracellular environment. The critical aggregative concentration of sphingolipids is in the nano-or picomolar range (1, 2), thus the amount of free monomers in solution is low. Nevertheless, the quantity of free monomers should increase when a favorable equilibrium leads to the formation of stable lipoprotein complexes (3-5). This process should also occur in living organisms, where significant amounts of sphingolipids are detectable in serum and cerebrospinal fluid from normal and pathological subjects (6 -8), as components of serum lipoproteins or associated with albumin (4).The presence of sphingolipids in biological fluids leads to the following considerations. Exogenous sphingolipids administered to cells are taken up by the cells, thus entering the sphingolipid metabolic pathway. Several studies have been carried out by administering sphingolipids to cells (9). Gangliosides administered to cells that are cultured in the absence of fetal calf serum (FCS) 1 or proteins have been shown to become components of the cell membranes, modifying in some cases the quantity and the quality of plasma membrane gangliosides, and to enter into the endogenous sphingolipid metabolic pathways ...

show abstract

Section: Methodsmentioning

confidence: 99%

Sphingolipid Uptake by Cultured Cells

Chigorno

Giannotta

Ottico

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Taurine conjugation is a well known mechanism in biological systems that facilitates the clearance of xenobiotics from the body by increasing their polarity and aqueous solubility (34). GM2 is very insoluble in water (35). The pK a of the carboxylic acid of Neu5Ac is 2.6 (36), whereas that of the sulfonic acid of taurine has been reported to be between 1 and 1.5 (37,38).…”

Section: Immunological Detection Of the Presence Of Tauro-gm2 Inmentioning

confidence: 99%

Presence of an Unusual GM2 Derivative, Taurine-conjugated GM2, in Tay-Sachs Brain

Maskos

Chou

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 Nacetylneuraminic 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. Tay-Sachs disease (TSD)1 is a classical inborn lysosomal glycosphingolipid (GSL) storage disease characterized by massive cerebral accumulation of ganglioside GM2 due to the deficiency of either ␤-hexosaminidase A or GM2-activator protein (1). Although the genetic mutations and biochemical basis of TSD have been well established (1, 2), the mechanism that leads to the neuropathological manifestations in TSD is still not fully understood (1). Based on the premise that GSLs specifically found in TS brain are associated with the pathogenesis of TSD, we carried out studies of GSLs in brain samples from patients with TSD. This report describes the detection and structural elucidation of a novel GM2 derivative in TS brain samples. By using NMR spectroscopy, mass spectrometry, and chemical synthesis, the structure of this GM2 derivative isolated from a 75-g variant B TS brain sample was established to be tauro-GM2 in which the carboxyl group of Neu5Ac is amidated by taurine. We have also raised a rabbit anti-tauro-GM2 serum and immunologically detected the presence of tauro-GM2 in additional small brain samples (1-2 g) from one variant B and two variant O TSD patients. Under the same conditions, we did not detect the presence of tauro-GM2 in three normal human brain samples. EXPERIMENTAL PROCEDURES Isolation of an Unknown Compound (UC) from a Variant B TS BrainSample-Crude GSL extract was prepared from the brain sample (75 g) of a variant B TS patient as described (3). The GSL extract was dissolved in 70 ml of chloroform/methanol/water (30/60/8, v/v/v) and applied onto a Q-Sepharose column (2.5 ϫ 75 cm) that had been equilibrated with the same solvent. After washing the column with the same solvent, the gangliosides were eluted with a linear gradient of sodium acetate (4). The flow rate was 1.0 ml/min, and 7-ml fractions were collec...

show abstract

“…In aqueous solutions, gangliosides occur in the form of micelles, monomers, and oligomers in an equilibrium at concentrations above the CMC of about 10 Ϫ 9 M or less (Formisano et al 1979;Mraz et al 1980). Over many years, extensive studies on the binding to and uptake of gangliosides by biological membranes were performed (for an overview see Saqr et al 1991).…”

Section: Insertion Of Biotin-gm1 Into Cell Membranesmentioning

confidence: 99%

Intracellular Distribution of a Biotin-labeled Ganglioside, GM1, by Immunoelectron Microscopy After Endocytosis in Fibroblasts

Möbius

Herzog

Sandhoff

et al. 1999

J Histochem Cytochem.

Self Cite

View full text Add to dashboard Cite

A radioactive and biotin-labeled analogue of GM1 (biotin-GM1) was synthesized which enabled us to analyze its intracellular distribution in the compartments of the endocytic route by electron microscopic immunocytochemistry using thin sections of human skin fibroblasts labeled with gold-conjugated antibiotin antibodies. Metabolic studies with the biotin-GM1 showed its partial degradation to the corresponding GM2 and GM3 derivatives. Further degradation was inhibited by the biotin residue. The distribution of biotin-GM1 after uptake by cells was studied by postembedding labeling techniques. On the plasma membrane the biotin-GM1 was detectable in the form of patches (0.1 micrometer in diameter), in caveola-like structures and, to a much lesser extent, in coated pits or vesicles. During endocytic uptake, the biotin-GM1 became detectable in organelles identified as late endosomes and lysosomes. The intracellular distribution of the biotin-GM1 was compared to the localization of the EGF receptor in EGF-stimulated fibroblasts. Both the biotin-GM1 and the EGF receptor were transported to intraendosomal and intralysosomal membranes, indicating that both membrane constituents follow the same pathway of endocytosis. Our observations show that biotin-GM1 can be successfully incorporated into the plasma membrane and be used as a tool for morphological detection of its pathway to lysosomes.

show abstract

Aggregate Formation of Gangliosides at Low Concentrations in Aqueous Media

Cited by 44 publications

References 16 publications

Sphingolipid Uptake by Cultured Cells

Sphingolipid Uptake by Cultured Cells

Presence of an Unusual GM2 Derivative, Taurine-conjugated GM2, in Tay-Sachs Brain

Intracellular Distribution of a Biotin-labeled Ganglioside, GM1, by Immunoelectron Microscopy After Endocytosis in Fibroblasts

Contact Info

Product

Resources

About