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 reported to be associated with vimentin intermediate filaments or colchicine sensitive cytoskeleton, no direct evidence for such an association has yet been shown. In this report we demonstrated that the intracellularly distributed ganglioside GM2 directly binds to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts. Indirect immunofluorescence microscopy using a GM2-specific monoclonal antibody demonstrated filamentously distributed GM2 in the cytoplasm. A double staining of Tay-Sachs fibroblasts with anti-GM2 and anti-vimentin monoclonal antibodies strongly suggested that the GM2 positive filaments are vimentin intermediate filaments. We then isolated vimentin, in the presence of a detergent and urea, from the normal human skin fibroblasts and murine mastocytoma cells. In a solid phase enzyme-linked immunosorbent assay, the isolated vimentin dose-dependently reacted with both anti-vimentin and anti-GM2 monoclonal antibodies but not with anti-GM3 or anti-GM1 monoclonal antibody. The molar ratio of GM2 to vimentin was approximately 20:1. The lipid fraction extracted from the purified vimentin preparation was immunostained with anti-GM2 on a thin-layer chromatography plate. Furthermore, only one band was detected at the molecular weight of 57 kDa, after electroblotting and simultaneous immunostaining with anti-GM2 and anti-vimentin monoclonal antibodies. These results clearly indicated that ganglioside GM2 directly binds to vimentin.
Kotani
M
M
Department of Tumor Immunology, Tokyo Metropolitan Institute of Medical Science, Japan.
Hosoya
H
H
Kubo
H
H
Itoh
K
K
Sakuraba
H
H
Kusubata
M
M
Inagaki
M
M
Yazaki
S
S
Suzuki
Y
Y
Tai
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D005455:Fluorescent Antibody Technique; D005678:G(M2) Ganglioside / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007382:Intermediate Filaments / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D012515:Mast-Cell Sarcoma / Q000737:chemistry / Q000473:pathology; D051379:Mice; D013661:Tay-Sachs Disease / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014746:Vimentin; D005678:G(M2) Ganglioside