Membrane restructuring induced by the enzymatic generation of ceramides with very long chain PUFA

DANIEL A. PEÑALVA; SILVIA S. ANTOLLINI; ERNESTO AMBROGGIO; MARTA I. AVELDAÑO; MARIA LAURA FANANI

Córdoba Capital

Congreso; LII Reunion Annual SAIB; 2016

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

It is widely known that cellular membranes of different cell types have particular properties according to their function. Regarding lipids, their qualitative and quantitative composition varies between cells. Sphingolipids in most mammalian tissues typically contain saturated and monounsaturated acyl chains from C14 to C24. Notable exceptions are spermatozoa of various mammals, including man, which contain SM and Cer species with infrequent series of very long chain (C24 to C36) polyunsaturated fatty acids (VLCPUFA). In rat sperm, SM species containing PUFA with 28-32 carbon atoms are exclusively located on the heads. After inducing the acrosomal reaction almost complete hydrolysis such SMs occurs, leading to gametes enriched in the corresponding Cer species. In the present study we evaluated the effect on the bilayer integrity and structuring of the enzymatic formation of Cer in binary POPC/SM vesicles. The VLCPUFA-containing molecular species of SM (V-SM) were isolated from rat testes by a combination of chromatographic techniques. Egg-SM, was used for comparison. Unilamellar liposomes (LUVs) were prepared. Dynamic Light Scattering reveals a large size population occurring after enzymatic hydrolysis, which evidences liposome aggregation. This was confirmed by an increase of FRET signal using NBD-PE as donor and Rh-PE as acceptor. The in-plane lateral organization was affected by Cer generation, as checked by different fluorescence probes (laurdan, DPH anisotropy). We further generated GUVs and analysed the structural membrane reorganization after SMase treatment by visualization through fluorescence confocal microscopy. While, the enzymatic formation of egg-Cer resulted in condensed domains, V-Cer induces bilayer destabilization and vesicle collapse. Our results show an enhanced restructuring capacity for the resultant V-Cer enriched membranes. The longer and bulkier acyl chains of V-Cer may play a role in favouring the fusion/fission events that occur in the head of spermatozoa during the acrosomal reaction, a process that requires topological lipid intermediates with negative curvature.