MIMETIC MODEL OF MATRIX VESICLES TO STUDY THE SPHINGOMYELINASE ACTIVITY

Due to physical-chemical properties, ceramide (CER) is correlated as a possible key factor in a biogenesis class of extracellular vesicles: the matrix vesicles (MV) which were directly correlated to the bone mineralization process. It is suggested that sphingomyelinase (SMase) contributes to membrane curvature and vesiculation with the formation of MV, by the conversion of sphingomyelin (SM) to CER in plasma membranes. Despite all studies, the pivotal role of CER in MV biogenesis and the precise molecular mechanism underlying such role remains poorly understood. 

The advantage of using mimetic models is the possibility to control parameters that influence various natural phenomena. Therefore, the study of SMase interaction with bilayers is important to comprehend the mechanisms of this process. 

In this study were used liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and sphingomyelin (SM) in 8:2 molar ratio. The liposomes were incubated with one enzyme Unit in a 7.4 pH buffer (Tris-HCl 50 mM and 2.0 mM MgCl₂) at 37°C during 6 hours. These liposomes were characterized by dynamic lighting scattering (DLS), Zeta Potential and difference scanning calorimetry (DSC), before and after reaction. Also, liposomes with different molar proportions of DPPC, SM and CER were analyzed by DSC to comprehend the interaction between these three lipids and compare them with the liposome after the SMase action.

The transition temperature (T_c) is specific to each lipid, knowing that the pure DPPC, SM and CER-liposome are 39, 68°C and 63°C, respectively, was possible to identify the ceramide formation after the enzyme activity due to peaks appearing in this temperature at DSC analysis. Unfortunately, the Zeta Potential (-11,5 mV) and size (125,5 nm) alteration wasn’t significant that could indicate ceramide production. 

Furthermore, the mixtures of DPPC, SM and CER showed that with sphingomyelin the higher proportions of CER the more stabilized the liposomes were due the increase of transition enthalpy (ΔH_trans). However, when it lacked SM, the increase of ceramide didn't change ΔH_trans, only T_c which implies that probably the domains rich in SM-CER are responsible for stability of the liposome.