Exploring Supramolecular Aspects of the Effect of Sphingomyelinase D On Sphingomyelin-Containing Membranes

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 a spider venom enzyme 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, 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 two orders of magnitude more active towards fluid- than gel-phase liposomes. The presence of cholesterol evens out this difference in activity at an intermediate level. The effect of SMD on fluid-phase giant unilamellar vesicles (GUVs) is observed by confocal fluorescence microscopy. GUVs composed of lauroyl-SM show a macroscopic domain formation and/or shrinking and buckling accompanied by the multiple formation of membrane tubes. GUVs composed of egg-SM display a beveling of the membrane and the formation of caps (outside curvature) approx. three days after the addition of SMD. Which membrane morphology evolves is likely a question of enzyme kinetics vs. the dynamics of lipid reorganization.GUVs of raft-like mixtures exhibit a single homogenous phase after the addition of SMD. 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.