EVIDENCE FOR DIRECT BINDING OF INTRACELLULARLY DISTRIBUTED GANGLIOSIDE GM2 TO ISOLATED VIMENTIN INTERMEDIATE FILAMENTS IN NORMAL AND TAY-SACHS-DISEASE HUMAN FIBROBLASTS

Although some intracellularly distributed glycosphingolipids are repor ted to be associated with vimentin intermediate filaments or colchicin e sensitive cytoskeleton, no direct evidence for such an association h as yet been shown. In this report we demonstrated that the intracellul arly distributed ganglioside GM2 directly binds to isolated vimentin i ntermediate filaments in normal and Tay-Sachs disease human fibroblast s. Indirect immunofluorescence microscopy using a GM2-specific monoclo nal antibody demonstrated filamentously distributed GM2 in the cytopla sm. A double staining of Tay-Sachs fibroblasts with anti-GM2 and anti- vimentin monoclonal antibodies strongly suggested that the GM2 positiv e filaments are vimentin intermediate filaments. We then isolated vime ntin, in the presence of a detergent and urea, from the normal human s kin fibroblasts and murine mastocytoma cells. In a solid phase enzyme- linked immunosorbent assay, the isolated vimentin dose-dependently rea cted with both anti-vimentin and anti-GM2 monoclonal antibodies but no t with anti-GM3 or anti-GM1 monoclonal antibody. The molar ratio of GM 2 to vimentin was approximately 20 : 1. The lipid fraction extracted f rom the purified vimentin preparation was immunostained with anti-GM2 on a thin-layer chromatography plate. Furthermore, only one band was d etected at the molecular weight of 57 kDa, after electroblotting and s imultaneous immunostaining with anti-GM2 and anti-vimentin monoclonal antibodies. These results clearly indicated that ganglioside GM2 direc tly binds to vimentin.