The Adhesion Protein TAG-1 Has a Ganglioside Environment in the Sphingolipid-Enriched Membrane Domains of Neuronal Cells in Culture

J Neurochem. 2003 Apr;85(1):224-33. doi: 10.1046/j.1471-4159.2003.01655.x.

Abstract

We studied the interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains by ganglioside photolabeling combined with cell surface biotin labeling. After cell photolabeling with radioactive photoactivable derivatives of GM3, GM1 and GD1b gangliosides, followed by cell surface biotin labeling, sphingolipid-enriched domains were prepared and immunoprecipitated with streptavidin-coupled beads, under experimental conditions preserving the integrity of the lipid domain. About 50% of the total radioactivity linked to proteins was associated with acylated tubulin, about 10% with a 135-kDa protein present as a series of species with pI ranging from 6.5 to 8.0, about 5% with a protein of about 70 kDa and with pI near to 6.5. By immunoprecipitation with streptavidin-coupled beads under conditions disrupting the integrity of the lipid domain, the 135 kDa protein was recovered in the immunoprecipitate, that did not contain tubulin. Thus, the 135 kDa protein has an exoplasmic domain, and it was then identified as the GPI-anchored neural cell adhesion molecule TAG-1. Remarkably, TAG-1 was cross-linked in a similar extent by the photoactivated ganglioside GM3, GM1 and GD1b. The three gangliosides bear different oligosaccharide chains, suggesting that the ganglioside/TAG-1 interaction is not specifically associated with the ganglioside oligosaccharide structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Adhesion Molecules, Neuronal / metabolism*
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Cerebellum / cytology
  • Contactin 2
  • Electrophoresis, Polyacrylamide Gel
  • G(M1) Ganglioside / chemistry
  • G(M1) Ganglioside / metabolism
  • G(M3) Ganglioside / chemistry
  • G(M3) Ganglioside / metabolism
  • Gangliosides / chemistry
  • Gangliosides / metabolism*
  • Neurons / chemistry
  • Neurons / cytology
  • Neurons / metabolism*
  • Photochemistry
  • Precipitin Tests
  • Rats
  • Rats, Sprague-Dawley
  • Sphingolipids / chemistry
  • Sphingolipids / metabolism*
  • Subcellular Fractions / chemistry

Substances

  • Cell Adhesion Molecules, Neuronal
  • Contactin 2
  • G(M3) Ganglioside
  • Gangliosides
  • Sphingolipids
  • ganglioside, GD1b
  • G(M1) Ganglioside