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.

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 reticuIudGolgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticuludGolgi. 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.

Al~stract Human placental acid sphingomyelinase (ASM) was purified by sequential chromatography on Con A-Sepharose, octyI-Sepharose and Matrex gel red A. Final purification to apparent homogeneity was achieved by immunoaff'mity 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 ll0000-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 ~ 75 kDa, which was reduced to ,-~60 kDa after complete deglycosylation. Microsequencing of the purified ASM revealed the N-terminal amino acid sequence of the mature placental enzyme.

No abstract

Human acid sphingomyelinase (haSMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to ceramide and phosphorylcholine. An inherited haSMase deficiency leads to Niemann–Pick disease, a severe sphingolipid storage disorder. The enzyme was purified and cloned over 10 years ago. Since then, only a few structural properties of haSMase have been elucidated. For understanding of its complex functions including its role in certain signaling and apoptosis events, complete structural information about the enzyme is necessary. Here, the identification of the disulfide bond pattern of haSMase is reported for the first time. Functional recombinant enzyme expressed in SF21 cells using the baculovirus expression system was purified and digested by trypsin. MALDI‐MS analysis of the resulting peptides revealed the four disulfide bonds Cys120‐Cys131, Cys385‐Cys431, Cys584‐Cys588 and Cys594‐Cys607. Two additional disulfide bonds (Cys221‐Cys226 and Cys227‐Cys250) which were not directly accessible by tryptic cleavage, were identified by a combination of a method of partial reduction and MALDI‐PSD analysis. In the sphingolipid activator protein (SAP)‐homologous N‐terminal domain of haSMase, one disulfide bond was assigned as Cys120‐Cys131. The existence of two additional disulfide bridges in this region was proved, as was expected for the known disulfide bond pattern of SAP‐type domains. These results support the hypothesis that haSMase possesses an intramolecular SAP‐type activator domain as predicted by sequence comparison [Ponting, C.P. (1994) Protein Sci., 3, 359–361]. An additional analysis of haSMase isolated from human placenta shows that the recombinant and the native human protein possess an identical disulfide structure.