Stimulation of acid sphingomyelinase activity by lysosomal lipids and sphingolipid activator proteins

Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catal yzes the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide, Sphingomyelin itself is an important component of the extrac ellular leaflet of various cellular membranes, The aim of the present inves tigation was to study sphingomyelin hydrolysis as a membrane-bound process, We analyzed the degradation of sphingomyelin by recombinant, highly purifi ed acid sphingomyelinase in a detergent-free, liposomal assay system. In or der to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(mo noacylglycero)phosphate and phosphatidylinositol were incorporated into sub strate-carrying liposomes, Dolichol and its phosphate ester dolicholphospha te were also included in this study. Bis(monoacylglycero)phosphate and phos phatidylinositol were both effective stimulators of sphingomyelin hydrolysi s, Dolichol and dolicholphosphate also significantly increased sphingomyeli n hydrolysis, The influence of membrane curvature was investigated by incor porating the substrate into small (SUVs) and large unilamellar vesicles (LU Vs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs, Surface plasmon resonance experiments demonstrated t hat acid sphingomyelinase binds strongly to lipid bilayers, This interactio n is significantly enhanced by anionic lipids such as bis(monoacylglycero)p hosphate, Under detergent-free conditions only the sphingolipid activator p rotein SAP-C had a pronounced influence on sphingomyelin degradation in bot h neutral and negatively charged liposomes, catalyzed by highly purified ac id sphingomyelinase, while SAP-A, -B and -D had no noticeable effect on sph ingomyelin degradation.