An approach to understand the glycosphingolipid-protein interactions

INTRODUCTION: Lipid-rafts are membrane microdomains particularly enriched in glycosphingolipids (GSLs) and proteins involved in trasduction processes across the membrane. GSL-protein interactions can be investigated by cell photolabelling experiments using radioactive photoactivable GSLs, which yield, when illuminated, a very reactive intermediate that covalently binds to the molecules in the environment, i.e. proteins (1,2). The preparation of a GSL probe with two photoactivable groups, one at position 2 and the other at the end of the acyl chain of ceramide, could be a tool for the simultaneous identifications of the proteins involved in the biological recognition and belonging to both the extracellular and the cytoplasmatic leaflets of the membrane. RESULTS: The preparation of an α,ω-diaminoacid as a bifunctional fatty acid useful for the conjugation of GLSs to photoactivable groups, i.e. nitrophenylazide, through the amino functionalities has been performed. This lipid chain has been conjugated to a radioactive lyso-ganglioside GM1, tritium labeled at position 3 of sphingosine, obtained by an enzymatic reaction with a SCDase. The purpose is to verify the capability of this new kind of probes to enter the cells and participate to metabolic processes. So, the photolabelled 3H-GM1 will be administer to cells and its metabolic fate will be studied.