Lyn-coupled LacCer-enriched lipid rafts in neutrophilis : a possible organization

Glycosphingolipids (GSL) are membrane components consisting in a hydrophobic ceramide (Cer) and in a hydrophilic sugar moieties. They cluster to form GSL-enriched domains on cellular plasma membrane (lipid rafts, caveolar domains, and glycosynapses) providing a microenvironment within the plasma membrane for reciprocal interactions between lipids and proteins. Biochemical analyses have demonstrated that GSL-enriched microdomains contain several kinds of transducer molecules, especially membrane-anchored signal transduction molecules, such as Src family kinases. It has been speculated that GSLs are closely associated with cell processes like differentiation, proliferation and phagocytosis, but there are quite few evidences that GSLs by themselves directly mediate signal transductions, which lead to cell functions. Lactosylceramide (LacCer), a neutral GSL, is abundantly expressed on human neutrophils, and specifically recognizes several pathogenic microorganisms. We previously demonstrated that LacCer forms detergent resistant domains (DRM) coupled with the Src family kinase Lyn, on the PM. Ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration. The presence of a LacCer molecular species with Cer containing a very long fatty acid chain is necessary for the association of Lyn with LacCer-enriched DRM and LacCer-mediated functions. Lyn molecules are attached to the inner leaflet while LacCer is component of the outer leaflet of membrane bilayer. So, how does LacCer interact with signal transducer molecules? To explore the molecular mechanism responsible for the association between fatty acid chains of LacCer and Lyn in LacCer-enriched DRM, we prepared analogues of C24-LacCer and C16-LacCer containing tritium and photoactivable group. Similar analogues of glycosphingolipids have been successfully used in the past to study sphingolipid-protein interactions. We found that (i) LacCer is immunoprecipitated with Lyn in DRM fraction, when cells are loaded with C24-LacCer analogue, but not with C16-LacCer analogue; (ii) Lyn has a direct interaction with LacCer in DRM fraction, because Lyn became radiolabeled after the loading with C24-LacCer analogue followed by illumination. For the first time, at the best of our knowledge, we show a direct connection, across the plasma membrane, between GSLs and palmitoylated proteins. These results suggest that LacCer with a long fatty acid chain in Cer moiety could be the key-player of the transduction of information across the plasma membrane, modulating membrane interdigitation, through the long acyl chain, and forming specific plasma membrane microdomains.