Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.
Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.
Prinetti
A
A
Study Center for the Functional Biochemistry and Biotechnology of Sphingolipids, Department of Medical Chemistry and Biochemistry, University of Milano, Segrate, Milan, Italy.
Prioni
S
S
Chigorno
V
V
Karagogeos
D
D
Tettamanti
G
G
Sonnino
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D000081247:CSK Tyrosine-Protein Kinase; D015816:Cell Adhesion Molecules, Neuronal; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D058969:Contactin 2; D005732:Gangliosides / Q000276:immunology / Q000302:isolation & purification*; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D021962:Membrane Microdomains / Q000201:enzymology*; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D011233:Precipitin Tests / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification; D051076:Proto-Oncogene Proteins c-fyn; D051381:Rats; D017207:Rats, Sprague-Dawley; D013110:Sphingosine / Q000302:isolation & purification; D014316:Tritium; D019061:src-Family Kinases / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D015816:Cell Adhesion Molecules, Neuronal; C548676:Cntn2 protein, rat; D058969:Contactin 2; D005732:Gangliosides; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D014316:Tritium; C026226:ganglioside, GD3; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D013110:Sphingosine