The adhesion protein TAG‐1 has a ganglioside environment in the sphingolipid‐enriched membrane domains of neuronal cells in culture

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. Keywords: gangliosides, lipid domains, neuronal cells, photolabeling, TAG-1. Abbreviations used: Cer, ceramide, N-acyl-sphingosine; GM3, II[11-3 H-(Neu5Ac)]GM1-N 3 (position 11 of sialic acid is the acetyl carbon), b-Gal-2-nitro-4-azidophenyl)amino-dodecanoyl]amino-3-hydroxyoctadec-4-ene}; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; SEMF, sphingolipid-enriched membrane fraction; SNIP, supernatant obtained after separation of immunoprecipitate.

Membrane lipid domains and membrane lipid domain preparations: are they the same thing?

“…Sphingosine is very rapidly and extensively taken up by cells, and it is immediately and e‹cient-ly acylated to ceramide, which is then converted into more complex sphingolipids. Under these conditions, in all the cell types so far investigated, all sphingolipids (including ceramide, sphingomyelin, neutral glycolipids and gangliosides), and phosphatidylethanolamine (obtained by recycling of radioactive ethanolamine formed in the catabolism of [1-3 H]sphingosine) were metabolically radiolabeled (56,74,77,(94)(95)(96)(97)(98)(99)(100)(101). The great extent of sphingosine recycling for the biosynthesis of complex sphingolipids allows the incorporation of a large amount of radioactivity into sphingolipids.…”

Dynamics of membrane lipid domains in neuronal cells differentiated in culture

“…These domains are involved in the trafficking and sorting of specific proteins, as well as in signal transduction processes (2,3). The lipid domain composition and the machinery of signal transduction were extensively studied in neurons (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In neurons and neuronal cell lines, many lines of evidence suggest that lipid membrane domains or multimolecular complexes localized within such domains are directly involved in the process of neuronal differentiation.…”

“…Particularly significant seem to be the interactions of sphingolipids with proAbbreviations: BME, basal modified Eagle's medium; HPTLC, high-performance thin-layer chromatography; [1- 2 To whom correspondence should be addressed. e-mail: alessandro.prinetti@unimi.it tein kinases of the Src family, probably the most typical protein family localized in lipid domains, as well as with other proteins implicated in the Src signaling cassette (4,7,(8)(9)(10)(11)(12)(13)(14). Interestingly, the lipid composition changes were accompanied by changes in the association of signaling molecules with the lipid membrane domain during neuronal cell differentiation (9).…”

Direct interaction, instrumental for signaling processes, between LacCer and Lyn in the lipid rafts of neutrophil-like cells

“…Photolabeling with photoactivable GM3 provided evidence for the specifi c role of GM3 in reducing insulin receptor-mediated processes in adipocytes ( 13 ) and showed that, following its interaction with CD9, GM3 downregulated tumor cell motility and malignancy ( 14 ). GM1 was found to interact with several membrane and cytosolic proteins (15)(16)(17)(18), two of which were characterized as caveolin-1 ( 16 ) and tubulin ( 17 ), and that GM3, GM1, and GD1b interacted with TAG-1 in the plasma membrane lipid rafts of neuronal cells ( 19,20 ).…”

“…The presence of Lyn and G ␣ i was assessed by immunoblotting with specifi c antibodies, followed by reaction with secondary HRP-conjugated antibody ( 11,12,15,18,19,27,30,33 ).…”