Interactions between gangliosides and proteins in the exoplasmic leaflet of neuronal plasma membranes: a study performed with a tritium-labeled GM1 derivative containing a photoactivable group linked to the oligosaccharide chain.
Interactions between gangliosides and proteins at the exoplasmic surface of the sphingolipid-enriched membrane domains can be studied by ganglioside photolabeling combined with cell surface biotin labeling. In the present paper, we report on the results obtained using a novel radioactive photoactivable derivative of GM1 ganglioside, carrying the photoactivable nitrophenylazide group at the external galactose. After cell photolabeling with the radioactive photoactivable derivative of GM1 and cell surface biotin labeling, sphingolipid-enriched domains were prepared from rat cerebellar neurons differentiated in culture and further purified by immunoprecipitation with streptavidin-coupled beads. Among proteins belonging to the sphingolipid-enriched domains that were biotin labeled, thus bearing an exoplasmic domain, a few were also cross-linked by the radioactive photoactivable ganglioside. In particular, two protein bands showing apparent molecular mass of 135 and 35 kDa were intensely photolabeled. The 135 kDa protein was immunologically identified as the GPI-anchored neural cell adhesion molecule TAG-1. These data suggest that hydrophilic interaction between the exoplasmic domains of the protein and the ganglioside sialooligosaccharide chain could exist.
Prioni
Simona
S
Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, 20090, Segrate, Italy.
Mauri
Laura
L
Loberto
Nicoletta
N
Casellato
Riccardo
R
Chigorno
Vanna
V
Karagogeos
Domna
D
Prinetti
Alessandro
A
Sonnino
Sandro
S
D000818:Animals; D002240:Carbohydrate Sequence; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D058969:Contactin 2; D005677:G(M1) Ganglioside / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009474:Neurons / Q000737:chemistry*; D019920:Photoaffinity Labels; D010777:Photochemistry; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling; D014316:Tritium
D015816:Cell Adhesion Molecules, Neuronal; C548676:Cntn2 protein, rat; D058969:Contactin 2; D008565:Membrane Proteins; D019920:Photoaffinity Labels; D014316:Tritium; D005677:G(M1) Ganglioside