Interfacial regulation of acid ceramidase activity - Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins

The lysosomal degradation of ceramide is catalyzed by acid ceramidase and r equires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid cerami dase at the water-membrane interface in the presence of sphingolipid activa tor proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of S AP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosp hate, phosphatidylinositol, and dolichol phosphate were incorporated into s ubstrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated t hat acid ceramidase remained bound to the liposomal surface during catalysi s. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators o f ceramide hydrolysis in the presence of anionic phospholipids. This findin g was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffer ing from prosaposin deficiency. Strong protein-lipid interactions were obse rved for both SAP-D and acid ceramidase in surface plasmon resonance experi ments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occur red at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are r equired for efficient hydrolysis of ceramide by acid ceramidase.