The ganglioside composition of Ehrlich ascites carcinoma (EAC) cells and the role of the individual gangliosides in binding and penetration into the cell of influenza virus were determined. EAC gangliosides identical with or close t o G M~. GMZ, G M~, G T~~ and G T I~ were characterized by thin-layer chromarography, compositional analyses, methylation analysis and mass-spectrometry. The ganglioside uptake capacity of native and neuraminidase-treated EAC cells was studied with tritium-labeled gangliosides of definite structure and the binding of influenza virus to cells was determinated by using ["Hluridine-labeled virus and by heinagglutination studies. Treatment of the cells with Vihrio c l t o l~~r n~~ neuraminidase largely decreased binding of the virus. Exogenous gangliosides with a terminal galactose unit or a penultimate galactose masked by neuraminic acid were able to restore the virus-binding capacity of ncuraminidase-treated cells, however, the main ganglioside of EAC cells, Ghfz, which carbohydrate chain is terminated by N-acetylgalactosamine, was completely ineffective. The common carbohydrate sequence of the gangliosides showing binding activityis proposed to be the main recognition structure of the influenza virus receptor on the surface of EAC cells. Penetration of labeled influenza virus into the nuclei of EAC cells was evaluated by measuring the radioactivity of the nuclei of neuraminidase-treated ganglioside-loaded cells after exposition to the labeled virus. Of all gangliosides tested only trisialogangliosidcs of the G~l b type were able to induce increased entry of the virus into the cells and accumulation of its radioactive component into the nuclei. It is suggested that G T I~ gangliosides react specifically with the virus protein responsible for membrane fusion (apparently the hemagglutinin HA2 subunit) and thus are involved in virus penetration and delivery of the virus genome to the nuclei.When interacting with cells ortho-and paramyxoviruses first adsorb to specific host receptors and then penetrate thc cell. While nothing is known about the nature of the cell surface components involved in the penetration process, some information about the receptors responsible for adsorbtion of the viruses is available. The receptors contain neuraminic acid and their receptor activity is destroyed by neuraminidase [2]. It is therefore thought that glycoproteins [3 -51 or sialoglycolipids (gangliosides) [6 -81 serve as receptors.Comparing the virus-binding activity of glycoproteins isolated from HeLa cells and brain gangliosides, Wu et al.", I showed that adsorbtion of Sendai virus required much less glycoprotein than glycolipid and concluded that the former are responsible for virus attachment. However recently it hdS been demonstrated that di-, tri-and tetrasialogangliosides can function as receptors for Sendai virus in cultured bovine kidney cells [lo, 1 11. Thus, the existing evidence of participitation of gangliosides in virus-cell association is contradictive. Moreover, the data obtained so far d...
The glycolipid composition of Ehrlich ascites carcinoma cells was found to depend strongly on the cell density of the suspension. The general trend observed upon dilution of the cell suspension was a reduction of the less complex gangliosides GM3 and G,, with concomitant increase of the more complex gangliosides, especially GM1. The increase of the content of ganglioside GMi upon dilution was accompanied by a comparable decrease of the content of its immediate precursor, asialo-GM1, whereas the content of other neutral glycosphingolipids did not change very much. When the cell suspension was diluted with medium conditioned by dense cells the ganglioside profile of the diluted suspension remained similar to that of the dense cell suspension. It is postulated that the medium conditioned with dense cells contains a transferable factor inhibiting sialylation of asialo-GM,.Glycolipids are involved in cellular recognition, cell growth, and receptor events [1]. For cells growing in culture on solid supports the synthesis and catabolism of glycolipids has been shown to depend on the cell density. For example, the ganglioside composition of BHK and NIL cells has been found to change on the stage of confluency [2] whereas no cell-densitydependent changes were observed with several strains transformed by oncogenic viruses [3]. On this ground it has been suggested that cell-density-dependent changes of glycolipid composition are linked with contact inhibition of growth and development [4-51. No comparable data are available for single living cells. A convenient model for studying cell density dependence in single living cells is the Ehrlich ascites carcinoma (EAC). Earlier the structure of the neutral glycosphingolipids and gangliosides of these cells has been established [6, 71.111 the present work we investigate the changes in the glycosphingolipid composition of EAC cells occurring during incubation of the cells at different cell densities. We demonstrate the glycolipid composition of the cells to depend strongly on the cell density and present data suggesting that at higher densities the biosynthesis of gangliosides is impaired, possibly due to the presence of a humoral sialyltransferase-inhibiting factor. To our knowledge this is the first demonstration of cell density dependence of the glycolipid composition in single living mammalian cells. MATERIALS AND METHODSAll chemicals were of analytical reagent grade, and solvents were redistillated prior to use. CellsEAC cells taken from mature male CBAXC,~BI/F, mice maintained for 7 days were used. The ascites fluid was drown off from the peritoneal cavity and centrifuged at 500 revjmin for Smin. The cell pellet was washed thrce times with phyAbbreviations. EAC, bhrlich Ascites Carcinoina; Ganglioside nomenclature according to the recommendations of IUPAC-IUR Commission on Biochemical Nomenclature (nomenclature system of Svcnnerholm) [15].siological salt solution and suspended in Eagle's mediumiphysiological salt solution (1 : 1, viv) to obtain a volume of cell suspension e...
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