Exploring Molecular and Supramolecular Aspects of Sphingomyelin-Containing Membranes Upon Action of Sphingomyelinase D

Abstract

Lipid-modifying enzymes play a vital role in the regulation of lipids as mediators of cell function. At the same time, the activity of these enzymes is highly affected by the lipid membrane structure. These processes at lipid membranes can be observed in situ through the application of different biophysical techniques. Thus, we are investigating an enzyme from spider venoms, which is termed sphingomyelinase D (SMD). SMD hydrolyses sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While SM is an integral constituent of many cell membranes, e.g., of red blood cells, Cer-1-P occurs in very low concentrations and is suggested to be a novel lipid second messenger. At present, the physiologically relevant mechanism following Cer-1-P formation by SMD is incompletely understood, but possibly related to the modulation of membrane properties.Our results show a strong dependency of SMD activity on the phase state of the substrate. SMD is one order of magnitude more active towards fluid- than gel-phase liposomes. The effect of SMD on fluid-phase giant unilamellar vesicles (GUVs) is observed by confocal fluorescence microscopy. GUVs composed of lauroyl-SM show a drastic shrinking and buckling accompanied by the multiple formation of membrane tubes, which are up to 80 μm long. Generalized-polarization measurements using the probe LAURDAN exhibit a macroscopic domain formation upon the hydrolysis of lauroyl-SM. This finding points to the induction of membrane curvature by lipid sorting in the simple, binary system of SM/Cer-1-P. SMD activity on GUVs composed of oleoyl-SM also shows tube formation followed by the immediate and complete disintegration of the vesicular membrane structure. The consequences of SMD activity and Cer-1-P formation on cellular systems are currently being examined. This will indorse the correlation between enzymatic activity and membrane structure influencing the regulation of physiological processes.