Incorporation of ganglioside analogs into fibroblast cell membranes. A spin-label study

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Abstract
The uptake of ganglioside analogs by a permanent mouse fibroblast cell line [clone-1D] was studied by radiotracer techniques and ESR spectroscopy with 3H- and nitroxide-labeled compounds. Analogs of GM1, GM2 and GM3 monosialogangliosides and of GD1a and GD3 disialogangliosides were synthesized. The spin-label group was situated on the 5-, 9- or 13-carbon atom of the C18 fatty acid chain, and the 3H label was in the carbohydrate moiety. Part of the ganglioside associated with the cells could be removed by trypsin treatment and consisted of ganglioside micelles attached to the cell surface. The trypsin-resistant component displayed characteristic anisotropic ESR spectra which closely resembled those of the same spin-labeled analogs at low dilution in liposomes prepared from the extracted cell lipids. The flexibility gradient, polarity profile and temperature dependence displayed by the spectra were similar to those found for fluid phospholipid bilayer model membranes, and the high effective order parameters suggested a location in the cell plasma membrane. Similar results were obtained for all the different ganglioside analogs, indicating a common anchoring region in the hydrophobic interior of the membrane. Under the incubation conditions used the amount of trypsin-resistant ganglioside analog taken up by the cells was about 15 nmol/mg of cellular protein, irrespective of the nature of the oligosaccharide moiety. By use of the natural ganglioside [3H]GM3, the trypsin-resistant uptake was about 19 nmol/mg of cellular protein. Although these amounts are quite similar, the uptake kinetics differed between the true ganglioside GM3 and the ganglioside analogs.

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