Stimulation of Acid Sphingomyelinase Activity by Lysosomal Lipids and Sphingolipid Activator Proteins

Abstract

Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catalyzes the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellular membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a membrane-bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergent-free, liposomal assay system. In order to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(monoacylglycero)phosphate and phosphatidylinositol were incorporated into substrate-carrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidylinositol were both effective stimulators of sphingomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigated by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon resonance experiments demonstrated that acid sphingomyelinase binds strongly to lipid bilayers. This interaction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under detergent-free conditions only the sphingolipid activator protein SAP-C had a pronounced influence on sphingomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAP-A, -B and -D had no noticeable effect on sphingomyelin degradation.

Heat Shock Protein 70.1 (Hsp70.1) Affects Neuronal Cell Fate by Regulating Lysosomal Acid Sphingomyelinase

“…To address whether the increase or decrease in the expression of ASM proteins correlates with the changes of its enzyme activity, the ASM activity in vivo was assessed by the colocalization of ASM and BMP because the presence of anionic lipids, such as BMP, can stimulate ASM activity (8,23,24). ASM colocalization with BMP was detected in the postischemic day 3 motor cortex (Fig.…”

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

“…Pseudomonas strains were grown at 30°C and all other bacteria were grown at 37°C in 10 mL B medium for 48 h. Cells were removed by centrifugation and the supernatants were assessed for the presence of SMase and CDase, using qualitative assays [19,24] based on the hydrolysis of the fluorescent C6-NBD sphingomyelin and C6-NBD ceramide analogues (Molecular Probes). In short, the fluorescent substrates (1 lg/mL) were dissolved in buffer H containing extra 2.5 mM MgCl 2 to determine CDase and SMase activities.…”

“…The enzyme acts on ceramide at the lipid-water interface and is activated by anionic phospholipids and sphingolipid activator proteins [19]. In lysosomes, acid CDase plays a critical role in the catabolism of ceramides, and the hereditary deficiency of this CDase leads to the accumulation of ceramide in the lysosomes as it occurs in the lethal Farber disease.…”

Why is Hippocampal CA1 Especially Vulnerable to Ischemia?

“…It contributes to lysosomal stabilization by binding to the anionic phospholipid, Bis(Monoacylglycero) Phosphate (BMP), a co-factor essential for sphingomyelin metabolism [4]. Hsp70.1-BMP binding enhances activity of acid sphingomyelinase, which mediates the sphingolipid degradation at the internal membrane in the acidic (pH 4.5) compartment to generate ceramide [26][27][28]. Ceramide protects the lysosomal limiting membrane from rupturing [4,29,30], presumably because the increased concentration of lysosomal ceramide can facilitate fusion of lysosomes with other intracellular vesicles and membranes, and strengthen limiting membranes [31].…”