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

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.

Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization.

Biochemical and structural studies on human acid sphingomyelinase (haSMase) depend on the access to homogeneous biologically active enzyme. Due to the low abundance of native haSMase (n-haSMase) in human tissue, conventional purification strategies are not suitable for the isolation of preparative amounts of the enzyme. We describe a novel approach to the functional expression and purification of haSMase employing the baculovirus expression vector system. Infection of Spodoptera frugiperda 21 cells with recombinant baculovirus encoding haSMase leads to the expression of a glycosylated 75 kDa precursor protein, which is subsequently processed to an enzymatically active secreted 72 kDa haSMase. Variations in N-glycosylation and proteolytic maturation account for the difference in molecular mass between mature recombinant (72 kDa) and human placental haSMase (75 kDa). N-terminal amino acid sequencing of recombinant haSMase (r-haSMase) reveals a 23-residue N-terminal extension compared to the placental enzyme. The apparent K(m) and Vmax values for sphingomyelin degradation by r-haSMase in a micellar assay system are 32 microM and 0.56 mmol h-1 mg-1, respectively. In conclusion, the established baculovirus expression vector system provides an efficient tool for the expression and functional characterization of haSMase.

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis.

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.

Purification of acid sphingomyelinase from human placenta: characterization and N-terminal sequence.

Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A-Sepharose, octyl-Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaffinity chromatography employing polyclonal anti-ASM antibodies. The antibodies also allowed specific detection of ASM by Western blotting at various stages of purification. The ASM activity was enriched about 110,000-fold over that of the crude extract, yielding an enzyme preparation with a specific activity of about 1 mmol/h per mg protein in a detergent-containing assay system. Analysis of the final preparation by SDS-PAGE resulted in a single protein band with a molecular mass of approximately 75 kDa, which was reduced to approximately 60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N-terminal amino acid sequence of the mature placental enzyme.