Evidence for Direct Binding of Intracellularly Distributed Ganglioside GM2 to Isolated Vimentin Intermediate Filaments in Normal and Tay-Sachs Disease Human Fibroblasts

Masaharu Kotani; Hiroshi Hosoya; Hideo Kubo; Kohji Itoh; Hitoshi Sakuraba; Masashi Kusubata; Masaki Inagaki; Kazumori Yazaki; Yoshiyuki Suzuki; Tadashi Tai

doi:10.1247/csf.19.81

Masaharu Kotani, Hiroshi Hosoya, Hideo Kubo, Kohji Itoh, Hitoshi Sakuraba, Masashi Kusubata, Masaki Inagaki, Kazumori Yazaki, Yoshiyuki Suzuki, Tadashi Tai

Author information

Masaharu Kotani
Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science
Present address : Department of Biology, Hiroshima University
Hiroshi Hosoya
Department of Ultrastructural Research, The Tokyo Metropolitan Institute of Medical Science
Hideo Kubo
Department of Membrane Biochemistry, The Tokyo Metropolitan Institute of Medical Science
Kohji Itoh
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science
Hitoshi Sakuraba
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science
Masashi Kusubata
Department of Neurophysiology, The Tokyo Metropolitan Institute of Gerontology
Masaki Inagaki
Department of Neurophysiology, The Tokyo Metropolitan Institute of Gerontology
Kazumori Yazaki
Department of Ultrastructural Research, The Tokyo Metropolitan Institute of Medical Science
Yoshiyuki Suzuki
Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science
Tadashi Tai
Department of Tumor Immunology, The Tokyo Metropolitan Institute of Medical Science

Keywords: ganglioside GM2, vimeiitin intermediate filaments, Tay-Sachs fibroblasts, human fibroblasts

JOURNAL FREE ACCESS

1994 Volume 19 Issue 2 Pages 81-87

DOI https://doi.org/10.1247/csf.19.81

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Abstract

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.

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