g(m1)-ganglioside and bis(sulfosuccinimidyl)suberate

Glycosylphosphatidylinositol (GPI)-anchored proteins are clustered mainly in sphingolipid-cholesterol microdomains of the plasma membrane. The distribution of GPI-anchored fusion yellow fluorescent protein (GPI-YFP) in the plasma membrane of Chinese hamster ovary (CHO)-K1 cells with different glycolipid compositions was investigated. Cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity or cell lines expressing different gangliosides caused by stable transfection of appropriate ganglioside glycosyltransferases or exposed to exogenous GM1 were transfected with GPI-YFP cDNA. The distribution of GPI-YFP fusion protein expressed at the plasma membrane was studied using the membrane-impermeable cross-linking agent bis(sulfosuccinimidyl)suberate. Results indicate that GPI-YFP forms clusters at the surface of cells expressing GM3, or cells depleted of glycolipids, or transfected cells expressing mainly GD3 and GT3, or GM1 and GD1a, or mostly GM2, or highly expressing GM1. However, no significant changes in membrane microdomains of GPI-YFP were detected in the different glycolipid environments provided by the membranes of the cell lines under study. On the other hand, wild type CHO-K1 cells exposed to 100 microm GM1 before cross-linking with bis(sulfosuccinimidyl)suberate showed a dramatic reduction in the amount of GPI-YFP clusters. These findings clearly indicate that manipulating the glycolipid content of the cellular membrane, just by changing the ganglioside biosynthetic activity of the cell, did not significantly affect the association of GPI-YFP on the cell surface of CHO-K1 cells. The effect of exogenous GM1 gangliosides on GPI-YFP plasma membrane distribution might be a consequence of the ganglioside level reached in plasma membrane and/or the effect of particular ganglioside species (micelles) that lead to membrane architecture and/or dynamic modifications.

Topics: Animals; Bacterial Proteins; Cell Membrane; CHO Cells; Cricetinae; Cross-Linking Reagents; Culture Media, Serum-Free; G(M1) Ganglioside; Glucosyltransferases; Glycolipids; Glycosylphosphatidylinositols; Humans; Luminescent Proteins; Membrane Glycoproteins; Recombinant Fusion Proteins; Succinimides; Viral Envelope Proteins

Exogenous application of gangliosides to cells affects many cellular functions. We asked whether these effects could be attributed to the influence of gangliosides on the properties of sphingolipid-cholesterol microdomains on the plasma membrane, also termed rafts. The latter are envisaged as lateral assemblies of sphingolipids (including gangliosides), cholesterol, and a specific set of proteins. Rafts have been implicated in processes such as membrane trafficking, signal transduction, and cell adhesion. Recently, using a chemical cross-linking approach with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) as a model system, we could show that GPI-anchored proteins are clustered in rafts in living cells. Moreover, this clustering was dependent on the level of cholesterol in the cell. Here we show that incubation of MDCK cells with gangliosides abolished subsequent chemical cross-linking of GH-DAF. Furthermore, insertion of gangliosides into the plasma membrane of MDCK GH-DAF cells renders GH-DAF soluble when subjected to extraction with Triton X-114 at 4 degrees C. Our data suggest that exogenous application of gangliosides displaces GPI-anchored proteins from sphingolipid-cholesterol microdomains in living cells.

Topics: Animals; Carrier Proteins; CD55 Antigens; Cell Line; CHO Cells; Cricetinae; Cross-Linking Reagents; Dogs; Fluorescent Antibody Technique; Folate Receptors, GPI-Anchored; G(M1) Ganglioside; Gangliosides; Glucosides; Glycosylphosphatidylinositols; Growth Hormone; Membrane Lipids; Membrane Proteins; Orthomyxoviridae; Receptors, Cell Surface; Succinimides

Topics: Blotting, Western; Cross-Linking Reagents; Dimerization; G(M1) Ganglioside; Nerve Growth Factors; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Receptor, trkA; Receptors, Nerve Growth Factor; Succinimides

GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6-24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.

Topics: Animals; Cross-Linking Reagents; Dimerization; Drug Synergism; Enzyme-Linked Immunosorbent Assay; G(M1) Ganglioside; Gene Expression; Mice; Nerve Growth Factors; Neurons; PC12 Cells; Phosphorylation; Proto-Oncogene Proteins; Rats; Receptor Protein-Tyrosine Kinases; Receptor, Nerve Growth Factor; Receptor, trkA; Receptors, Nerve Growth Factor; Succinimides; Time Factors; Tyrosine