A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species

Activation of neutral sphingomyelinase(s) and subsequent generation of ceramide has been implicated in a wide variety of cellular responses. Although this enzyme(s) has not been purified and cloned from higher organisms, one mammalian cDNA has been previously isolated based on its similarity to the bacterial enzyme. To further elucidate the function of this neutral sphingomyelinase, we studied its relationship with enzymes present in mammalian cells and tissues, its subcellular localization, and properties that could be important for the regulation of its activity. Using specific antibodies, it is suggested that the enzyme could represent one of several forms of neutral sphingomyelinases present in the extract from brain particulate fraction. In PC12 cells, the enzyme is localized in the endoplasmic reticulum and is not present in the plasma membrane. The same result has been obtained in several cell lines transfected or microinjected with plasmids encoding this enzyme. The molecular and enzymatic properties of the cloned neutral magnesium-dependent sphingomyelinase, produced using baculovirus or bacterial expression systems, have been analyzed, demonstrating the expected ion dependence and substrate specificity. The enzyme activity also has a strong requirement for reducing agents and is reversibly inhibited by reactive oxygen species and oxidized glutathione. The studies demonstrate that the cellular localization and some properties of this enzyme are distinct from properties previously associated with neutral magnesium-dependent sphingomyelinases in crude or partially purified preparations.     

Ceramide and sphingomyelinases in the regulation of stress responses

Ceramide and other sphingolipids are now recognized as novel intracellular signal mediators. One of the important and regulated steps in the metabolism of sphingolipids is the hydrolysis of sphingomyelin into ceramide by sphingomyelinases. Whereas some studies suggest a role for acid sphingomyelinase in cell regulation, several lines of investigation suggest that neutral sphingomyelinase (N-SMase) plays a critical role in stress responses including apoptosis. Recently the advanced purification of neutral membrane-bound magnesium-dependent sphingomyelinase from rat brain was reported on. The specific activity of the purified N-SMase was increased by approximately 3000-fold over the rat brain homogenate, and it is specifically activated by phosphatidylserine. In cells, N-SMase may be coupled to either the redox state and/or glutathione metabolism. The significance of N-SMase and ceramide in stress responses is discussed.     

Purification and Characterization of Neutral and Acid Sphingomyelinases from Rat Brain

Neutral and acid sphingomyelinases were copurified from a rat brain P2 fraction by extraction with 1% Triton X-100, followed by (NH4)2SO4 fractionation, acetone powdering, extraction with 1% Triton X-100, (NH4)2SO4 fractionation, Sepharose CL-6B chromatography, and chromatofocusing. The neutral sphingomyelinase was eluted with buffer containing 0.4 M NaCl after the acid sphingomyelinase had been eluted with Polybuffer at pH 5.3. The neutral sphingomyelinase exhibited specific activity of 48,300 nmol/h/mg of protein, with 254-fold purification; the corresponding value for acid sphingomyelinase was 25,300 nmol/h/mg protein, with 668-fold purification from the P2 fraction. The purified neutral sphingomyelinase had no acid sphingomyelinase activity, and vice versa. The properties of the two enzymes were examined. A single band corresponding to a molecular weight of 67,000 was obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both enzymes. The pI was estimated to be 5.5 for both on isoelectric focusing. The native molecular weights of the neutral and acid sphingomyelinases were found to be 434,000 and 284,000, respectively, on gel filtration with Sepharose CL-6B. The single band obtained for each enzyme on SDS-PAGE was identified as an antigen with antibody raised against the purified neutral sphingomyelinase. Their amino acid compositions were very similar. The neutral and acid sphingomyelinases probably consist of common polypeptides and are immunologically cross-reactive.     

Neutral Magnesium-Dependent Sphingomyelinase from Liver Plasma Membrane: Purification and Inhibition by Ubiquinol

Plasma membranes isolated from pig liver contained almost no acid sphingomyelinase but significant neutral magnesium-dependent sphingomyelinase that was activated by phosphatidylserine. We report here the purification to apparent homogeneity of neutral sphingomyelinase of about 87 kDa from liver plasma membranes. The purified enzyme strictly required magnesium and had a neutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma membrane was not inhibited by GSH and, strikingly, it was not activated by phosphatidylserine. Liver sphingomyelinase was inhibited by several lipophilic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective than alpha-tocopherol, alpha-tocopherylquinone, alpha-tocopherylquinone, and ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with different levels of protection against lipid peroxidation. The purified sphingomyelinase was not inhibited significantly by ubiquinone-10 and ubiquinol- 10, but ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our results demonstrate a direct inhibitory effect of ubiquinol on the plasma membrane n-SMase and support the participation of this molecule in the regulation of ceramide-mediated signaling.     

Cyclic GMP-dependent inhibition of acid sphingomyelinase by nitric oxide: an early step in protection against apoptosis

Activation of acid and neutral sphingomyelinases, and the ensuing generation of ceramide, contributes to the biological effects of tumour necrosis factor-alpha (TNF-alpha), one of which is apoptosis. While the mechanisms of activation of sphingomyelinases by the cytokine are being unravelled, less is known about regulation of their activity. Nitric oxide has previously been shown to exert a cyclic GMP-dependent inhibition of early apoptotic events triggered by TNF-alpha in the U937 monocytic cell line. We therefore investigated whether inhibition of sphingomyelinases by nitric oxide plays a role in regulating such early events. We found that activation of both acid and neutral sphingomyelinases, triggered in the first minutes after U937 cell stimulation with TNF-alpha, is regulated in an inhibitory fashion by nitric oxide, working through generation of cyclic GMP and activation of protein kinase G. Using a range of inhibitors selective for either sphingomyelinase we found that the acid sphingomyelinase contributes to activation of the initiator caspase-8 and early DNA fragmentation and that inhibition of the acid enzyme by nitric oxide accounts for cyclic GMP-dependent early protection from apoptosis. We also found that the protective effect by both cGMP and acid sphingomyelinase inhibitors progressively disappeared at later stages of the apoptotic process. Inhibition of sphingomyelinases represents a novel action of nitric oxide, which might be of physiological relevance in regulating initial phases of apoptosis as well as other biological actions of ceramide.     

Neutral and Acid Sphingomyelinases of Rat Brain: Somatotopographical Distribution and Activity Following Experimental Manipulation of the Dopaminergic System In Vivo

The activities of neutral, magnesium-stimulated, and acid sphingomyelinases were measured in five regions of rat brain. Neutral enzyme activity was 2-3-fold higher in striatum than in parietal cortex and 13-fold higher than in cerebral white matter. Acid sphingomyelinase activity was more evenly distributed throughout these regions. Striatal neutral sphingomyelinase activity was not affected by treatment of rats with reserpine or haloperidol and was reduced (16%) by 6-hydroxydopamine. Striatal acid sphingomyelinase was unaffected by reserpine and 6-hydroxydopamine, and was increased (17%) by haloperidol. We conclude that neutral, magnesium-stimulated sphingomyelinase activity differs in various regions of rat brain and is particularly enriched in the corpus striatum. However, it appears to be a constitutive component of tissue rather than a readily modulated regulatory element of the catecholaminergic system.     

Sphingomyelinases: enzymology and membrane activity

This paper reviews our present knowledge of sphingomyelinases as enzymes, and as enzymes acting on a membrane constituent lipid, sphingomyelin. Six types of sphingomyelinases are considered, namely acidic, secretory, Mg(2+)-dependent neutral, Mg(2+)-independent neutral, alkaline, and bacterial enzymes with both phospholipase C and sphingomyelinase activity. Sphingomyelinase assay methods and specific inhibitors are reviewed. Kinetic and mechanistic studies are summarized, a kinetic model and a general-base catalytic mechanism are proposed. Sphingomyelinase-membrane interactions are considered from the point of view of the influence of lipids on the enzyme activity. Moreover, effects of sphingomyelinase activity on membrane architecture (increased membrane permeability, membrane aggregation and fusion) are described. Finally, a number of open questions on the above topics are enunciated.     

The isolation and characterization of sphingomyelinase from human placental tissue.

Human placental sphingomyelinase activity was eluted as a single symmetrical peak from Sephadex G-200 with a molecular weight of 290000; however, the enzyme behaved heterogeneously on ion exchange chromatography. A specific species of sphingomyelinase was purified approx. 10 000-fold to a constant specific activity of 274 000 nanomol of sphingomyelin hydrolyzed per mg protein per h. When the purified enzyme was examined on sodium dodecyl sulfate disc gel electrophoresis, two distinct protein bands in approximately equal proportions with molecular weights of 36 800 and 28 300 were found. The specificity of the enzyme is directed towards both the hydrophilic phosphocholine and the hydrophobic ceramide moieties of sphingomyelin. Possible interrelationships between the heterogenous forms of placental sphingomyelinases are discussed.     

A comparative evaluation of the level of sphingomyelinase activity in liver cell nuclei and in the brain

It was discovered that there is sphingomyelinase activity in the rat liver nuclei. The maximum of enzyme activity is at pH 7.1. The data obtained demonstrated that the main part of sphingomyelinase is located in the nuclear membrane. Comparison of sphingomyelinase activity in cell nuclei, liver and brain homogenates shows high level of enzyme activity in the nuclei. The authors discuss possible participation of sphingomyelinases in changes of phospholipids composition in nuclear structure under different functional activity of cell nuclei.     

Sphingomyelinase activities in neuronal cell cultures.

Human and rat brains have been previously demonstrated to contain three sphinomyelinases, one lysosomal with a pH optimum of 5.0, one with a pH optimum of 7.4 and a dependence on magnesium and another with a pH optimum of 7.0 and no divalent cation requirement. Using brain cell cultures and clonal cell lines of both neuronal and glial origin the activities of the pH 5.0 and pH 7.4 (magnesium-dependent) sphingomyelinase were examined. Sphingomyelinase activity measured at pH 5.0 was found in all the cell lines tested including G26, C6, N18 (differentiated and undifferentiated), mouse “L” cells, human skin fibroblasts, fetal mouse brain surface cultures and fetal mouse brain reaggregate cultures. However, pH 7.4 (magnesium-dependent) sphingomyelinase activity was found only in the N18 cell lines and the reaggregate cultures suggesting the probable localization of this activity in neurons. Although the pH 7.4 sphingomyelinase activity was found in the fetal mouse brain used for the surface cultures this activity was rapidly lost. This enzyme may play an important role in neuronal development and maturation.     

Role of thyroid hormones in regulation of sphingomyelin metabolism in the liver.

Inhibition of thyroid gland function in rats with mercasolil sharply decreased thyroxine and triiodothyronine levels in blood serum and increased acid sphingomyelinase activity and sphingomyelin content in liver. Thyroxine injected into hypothyroid rats normalized the sphingomyelin content, reduced the free ceramide content, and further increased the acid sphingomyelinase activity in liver. Thyroxine stimulated de novo sphingomyelin synthesis. Changing the thyroid status of the rats did not influence the free sphingosine content. Thyroxine blocks the accumulation of free sphingosine in the liver during activation of sphingomyelinases.     

Activation of magnesium-dependent, neutral sphingomyelinase by phosphatidylserine

The plasma membrane isolated from rat ascites hepatoma, AH 7974 cells was treated with 1% Triton X-100, which resulted in a more than 80% reduction in the phospholipid content of the plasma membrane. The delipidized plasma membrane showed only 18% of the activity of the magnesium-dependent, neutral sphingomyelinase in the untreated plasma membrane. On the addition of acidic phospholipids, especially phosphatidylserine, however, the enzyme activity in the delipidized membrane was markedly restored up to 77% of that in the untreated membrane. It was suggested that, considering the phospholipid composition of the AH 7974 plasma membrane (Koizumi, K. et al. (1977) Cell Struct. Func. 2, 145-153), phosphatidylserine may be a natural activator of neutral sphingomyelinase.     

Structure and biological activities of beta toxin from Staphylococcus aureus

Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-Å resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.     

Molecular Cloning and Expression of Mn2+-Dependent Sphingomyelinase/Hemolysin of an Aquatic Bacterium, Pseudomonas sp. Strain TK4

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn(2+). The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.     

Purified Rat Brain Microvessels Exhibit Both Acid and Neutral Sphingomyelinase Activities

Purified rat brain microvessels have been shown to hydrolyze radiolabeled sphingomyelin by means of two different enzyme systems. Enzymatic activity was detected at pH 7.4 and was strongly stimulated by magnesium or manganese and inhibited by calcium. Activity at pH 5.1 could also be found and was not dependent on any of these cations. At neutral pH and in the presence of magnesium, the rate of sphingomyelin hydrolysis did not exhibit a linear relationship with protein concentration. In contrast, increasing the protein concentration from 0.05 to 0.5 mg/ml resulted in a constant increase of sphingomyelin hydrolysis at pH 5.1. Kinetic parameters of both neutral and acid activities have been determined and were similar in magnitude to values reported previously for neural sphingomyelinases. This work demonstrates the occurrence of a neutral sphingomyelinase activity in purified rat brain microvessels, an observation raising the question of its role at the level of the blood-brain interface.     

Comparative Hydrolysis of Sphingomyelin and 2-N-(Hexadecanoyl)-Amino-4-Nitrophenyl-Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH

Hydrolysis of sphingomyelin and 2-N-(hexade-canoyl)-amino-4-nitrophenyl-phosphorylcholine (HDA-PC), a synthetic analogue of sphingomyelin, by acid and Mg-dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl₂ nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA-PC to sphingomyelin is ?2). In contrast, Mg-dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl₂, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA-PC to sphingomyelin is ?0-0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg-dependent neutral sphingomyelinase.     

Structural analysis of the Leptospiral sphingomyelinases: in silico and experimental evaluation of Sph2 as an Mg-dependent sphingomyelinase

BACKGROUND: Leptospiral sphingomyelinases are candidate virulence factors present only in pathogenic Leptospira spp. Leptospira interrogans serovar Lai encodes Sph1, Sph2, Sph3, Sph4 and SphH. Except for Sph4, they all possess the exo-endo-phosphatase domain that groups them under the DNase I superfamily. METHODS, RESULTS AND CONCLUSIONS: Modeling of exo-endo-phosphatase domains reveals high-level structural similarity of Sph2 with the crystal structure of SmcL and BC SMase sphingomyelinases from Listeria ivanovii and Bacillus cereus, respectively. A β-hairpin loop, essential for host cell membrane interaction, is absent in leptospiral sphingomyelinases. Instead, several aromatic amino acids were oriented outward from the surface of these molecules and formed clusters of hydrophobic regions that possibly enables the anchoring of these molecules into the host cell membrane, as demonstrated in Sph2 and Sph3. Sph2 is unique and possesses the Mg(++)-binding Glu53 residue in the metal-binding site and two His residues (His151 and His286) in the catalytic site. We demonstrate experimentally the Mg(++)-dependent hemolysis of erythrocytes by rSph2 and its ability to cleave sphingomyelin to ceramide. Anti-Sph2 antibodies neutralized the hemolytic activity of Sph2. In conclusion, we provide evidence showing that Sph2 is a Mg(++)-dependent hemolysin with both sphingomyelinase and hemolytic activities.     

Phosphatidylinositol-3,5-Bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P2 is 0.53 microM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 microM, PtdIns3,5P2 neither decreased plasma membrane-associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.     

Sphingolipid-mediated apoptotic signaling pathways

Various sphingolipids are being viewed as bioactive molecules and/or second messengers. Among them, ceramide (or N-acylsphingosine) and sphingosine generally behave as pro-apoptotic mediators. Indeed, ceramide mediates the death signal initiated by numerous stress agents which either stimulate its de novo synthesis or activate sphingomyelinases that release ceramide from sphingomyelin. For instance, the early generation of ceramide promoted by TNF is mediated by a neutral sphingomyelinase the activity of which is regulated by the FAN adaptor protein, thereby controlling caspase activation and the cell death programme. In addition, the activity of this neutral sphingomyelinase is negatively modulated by caveolin, a major constituent of some membrane microdomains. The enzyme sphingosine kinase also plays a crucial role in apoptosis signalling by regulating the intracellular levels of two sphingolipids having opposite effects, namely the pro-apoptotic sphingosine and the anti-apoptotic sphingosine 1-phosphate molecule. Ceramide and sphingosine metabolism therefore appears as a pivotal regulatory pathway in the determination of cell fate.