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Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

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Abstract

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells.

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Abbreviations

 :

Ganglioside and glycosphingolipid nomenclature is in accordance with Svennerholm [1], and the IUPAC-IUBMB recommendations [2].

GlcCer:

ß-Glc-(1-1)-Cer

LacCer:

ß-Gal-(1-4)-ß-Glc-(1-1)-Cer GM3, II3Neu5AcLacCer, α-Neu5Ac-(2-3)-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer

GM1:

II3Neu5AcGg4Cer, ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer

GD3:

II3(Neu5Ac)2LacCer, α-Neu5Ac-(2-8)-α-Neu5 Ac-(2-3)-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer

GD1a:

IV3Neu5AcII3Neu5AcGgOse4Cer, α-Neu5Ac-(2-3)-ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer

GD1b:

II3(Neu5Ac)2GgOse4Cer, ß-Gal-(1-3)-ß-Gal NAc-(1-4)-[α-Neu5Ac-(2-8)-α-Neu5Ac-(2-3)]ß-Gal-(1-4)-ß-Glc-(1-1)-Cer; O-Ac-GT1b, sIV3 Neu5AcII3[α-Neu5, 9Ac2-(2-8)-α-Neu5Ac-(2-3)]GgOse4Cer

GT1b:

IV3Neu5AcII3(Neu5Ac)2GgOse4Cer, α-Neu5 Ac-(2-3)-ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-8)-α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer

GQ1b:

IV3(Neu5Ac)2II3(Neu5Ac)2GgOse4Cer, α-Neu 5Ac-(2-8)-α-Neu5Ac-(2-3)-ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-8)-α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc-(1-1)-Cer

Neu5Ac:

N-acetylneuraminic acid

Cer:

ceramide, N-acyl-sphingosine

Sph:

sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy -octadecene; [1-3H]sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy-[1-3H]octadecene

PE:

phosphatidylethanolamine

SM:

sphingomyelin

BME:

Basal modified Eagle’s medium

EMEM:

Eagle’s minimum essential medium

FCS:

fetal calf serum

HPTLC:

high-performance thin-layer chromatography

PMSF:

phenyl-methylsulfonyl fluoride

SDS:

sodium dodecylsulfate

PAGE:

polyacrylamide gel electrophoresis

PBS:

phosphate-buffered saline

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Authors and Affiliations

  1. Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, 20090, Segrate, Italy

    Vanna Chigorno, Mariateresa Sciannamblo, Joanna Mikulak, Alessandro Prinetti & Sandro Sonnino

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  1. Vanna Chigorno
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  2. Mariateresa Sciannamblo
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  3. Joanna Mikulak
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  4. Alessandro Prinetti
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  5. Sandro Sonnino
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Correspondence to Sandro Sonnino.

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This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.

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Chigorno, V., Sciannamblo, M., Mikulak, J. et al. Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture. Glycoconj J 23, 159–165 (2006). https://doi.org/10.1007/s10719-006-7921-7

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