Mammalian Sphingomyelinase Research Articles

Potent Inhibition of Acid Sphingomyelinase by Phosphoinositide Analogues

The different mammalian sphingomyelinases are involved in cell regulation, apoptosis and inflammatory events. Recent reports suggest pharmacological potential especially for inhibitors of the acid sphingomyelinase. Phosphatidyl inositol-3,5bisphosphate (PtdIns3,5P(2)) is the most potent selective acid sphingomyelinase inhibitor known to date. In the present study, we synthesized analogues of PtdIns3,5P(2) for initial structure-activity-relationship (SAR) studies. We identified an inhibitor that is easy to synthesize, that has superior chemical and biophysical properties when compared to PtdIns3,5P(2) and that should be stable against virtually all phospholipases. Last but not least, the new inhibitor partially protected cells from dexamethasone-induced cell death.

Sphingomyelin Synthase 2 Is One of the Determinants for Plasma and Liver Sphingomyelin Levels in Mice

It has been proposed that plasma sphingomyelin (SM) plays a very important role in plasma lipoprotein metabolism and atherosclerosis. Sphingomyelin synthase (SMS) is the last enzyme for SM de novo biosynthesis. Two SMS genes, SMS1 and SMS2, have been cloned and characterized. To evaluate the in vivo role of SMS2 in SM metabolism, we prepared SMS2 knockout (KO) and SMS2 liver-specific transgenic (LTg) mice and studied their plasma SM and lipoprotein metabolism. On a chow diet, SMS2 KO mice showed a significant decrease in plasma SM levels (25%, P<0.05), but no significant changes in total cholesterol, total phospholipids, or triglyceride, compared with wild-type (WT) littermates. On a high-fat diet, SMS2 KO mice showed a decrease in plasma SM levels (28%, P<0.01), whereas SMS2LTg mice showed a significant increase in those levels (29%, P<0.05), but no significant changes in other lipids, compared with WT littermates. Atherogenic lipoproteins from SMS2LTg mice displayed a significantly stronger tendency toward aggregation after mammalian sphingomyelinase treatment, compared with controls. Moreover, SMS2 deficiency significantly increased plasma apoE levels (2.0-fold, P<0.001), whereas liver-specific SMS2 overexpression significantly decreased those levels (1.8-fold, P<0.01). Finally, SMS2 KO mouse plasma promoted cholesterol efflux from macrophages, whereas SMS2LTg mouse plasma prevented it. We therefore believe that regulation of liver SMS2 activity could become a promising treatment for atherosclerosis.

Functional analysis of ISC1 by site-directed mutagenesis.

We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p [Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798]. The present study was conducted to determine specific domains in Isc1p required for catalysis. Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1. Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues. Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins. In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain". G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis. D163A and K168A lost activity completely. Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity. The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p. Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding. Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain. This study provides novel insight into a new domain found in Isc1p and related enzymes.

Functional analysis of acid and neutral sphingomyelinases in vitro and in vivo

The molecular cloning and the elucidation of the gene structures of the acid (aSMase) and a neutral sphingomyelinases (nSMase) of mouse and human facilitated the structural and functional analysis of these enzymes responsible for the catabolism of sphingomyelin present ubiquitously in the membrane lipid bilayer of mammalian cells. The protein and enzymic properties of the glycoprotein aSMase and of a non-glycosylated nSMase residing in the membranes of the endoplasmic reticulum have been analysed in the native as well as in the recombinant shingomyelinases. Important insight was gained from gene targeting experiments in which an aSMase deficient mouse line was generated which mimics the neurovisceral form of the human Niemann–Pick disease. The availability of the cloned aSMase and nSMases discovered so far led to a genetic approach to the verification of the concept that these enzymes in the ‘sphingomelin cycle’ are responsible for the generation of ceramide regarded as a lipophilic second messenger in the intracellular signal cascades activated by e.g. TNF-α, Fas ligand or cellular stress. All the available evidence derived from the aSMase deficient mouse line and several cell lines overexpressing aSMase and nSMase questions a role of ceramide released by the mammalian sphingomyelinases known so far in intracellular signal transduction.

An Mn2+-stimulated neutral-sphingomyelinase in seminiferous tubules of immature Wistar rats.

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. The seminiferous tubules of immature (19 day-old) Wistar rats have at least three types of sphingomyelinases, a lysosomal one and two microsomal ones. One of the microsomal sphingomyelinases is active at pH 6.5 and is stimulated by Mn2+ > Co2+ > Mg2+, and the other is active at pH 7.4 and is stimulated by Mn2+ > Mg2+ and inhibited by Co2+. The two microsomal enzymes are only slightly inhibited by EDTA and at pH 7.4 the stimulatory effects of Mn2+ and Mg2+ are additive. These data characterize the existence of two different membrane-bound sphingomyelinases in the seminiferous tubules of the rat.

Increased sphingomyelin content of plasma lipoproteins in apolipoprotein E knockout mice reflects combined production and catabolic defects and enhances reactivity with mammalian sphingomyelinase.

Apolipoprotein E knockout (apoE0) mice accumulate atherogenic remnant lipoproteins in plasma. We now provide evidence that these particles are enriched in sphingomyelin (SM), and explore the mechanisms and possible pathophysiological consequences of this finding. The phosphatidylcholine/sphingomyelin (PC/SM) ratio was reduced in all lipoproteins in apoE0 mice compared with wild-type (Wt) mice (2.0+/-0.2 vs. 4.7+/-0.5; 2.8+/-0.5 vs. 5.5+/-0.9; 1.9+/-0. 5 vs. 4.6+/-0.5 for VLDL, LDL, and HDL), reflecting 400 and 179% increases in plasma pools of SM and PC, respectively. Turnover studies using [14C]PC/[3H]SM VLDL or HDL showed that the fractional catabolic rate (FCR) of VLDL-SM and HDL-SM were markedly reduced in the apoE0 mice compared with Wt mice, while the FCRs of VLDL-PC and HDL-PC were similar. By contrast, the FCRs of [3H]PC ether and [14C]SM were identical in apoE0 and Wt mice. The production rates of VLDL-SM and HDL-SM in apoE0 mice were much higher than in Wt mice, while the production rates of lipoprotein PC were similar. To assess the underlying mechanisms, we also measured the PC/SM ratio in VLDL and LDL of LDL receptor knockout (LDLr0) and hepatic LDL receptor-related protein knockout/LDLr0 mice, but found no difference with Wt mice. Using S-sphingomyelinase, an enzyme secreted by macrophages and endothelial cells, we found that VLDL and LDL from apoE0, but not from Wt or LDLr0 mice, were significantly aggregated, and that aggregation was not prevented by adding back apoE. We then enriched the apoE0-VLDL and Wt-VLDL with different amounts of SM, and found that VLDL aggregation was enhanced. Thus, the increased SM content of lipoproteins in apoE0 mice is due to combined synthesis and clearance defects. Impaired SM clearance reflects resistance to intravascular enzymes and delayed removal by a non-LDLr, non-LDLr related protein pathway. The increased SM content in slowly cleared remnant lipoproteins may enhance their susceptibility to arterial wall SMase and increase their atherogenic potential.

Zn2+-stimulated Sphingomyelinase Is Secreted by Many Cell Types and Is a Product of the Acid Sphingomyelinase Gene

Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. Although several mammalian sphingomyelinases have been identified and studied, one of these, an acidic Zn2+-stimulated sphingomyelinase (Zn-SMase) originally found in fetal bovine serum, has received little attention since its first and only report 7 years ago. We now show that Zn-SMase activity is secreted by human and murine macrophages, human skin fibroblasts, microglial cells, and several other cells in culture and is markedly up-regulated during differentiation of human monocytes to macrophages. Remarkably, peritoneal macrophages from mice in which the acid SMase gene had been disrupted by homologous recombination secreted no Zn-SMase activity, indicating that this enzyme and the intracellular lysosomal SMase, which is Zn-independent, arise from the same gene. Furthermore, skin fibroblasts from patients with types A and B Niemann-Pick disease, which are known to lack lysosomal SMase activity, also lack Zn-SMase activity in their conditioned media. Chinese hamster ovary cells stably transfected with a cDNA encoding lysosomal SMase massively overexpress both cellular lysosomal SMase and secreted Zn-SMase activities. Thus, Zn-SMase arises independently of alternative splicing, suggesting a post-translational process. In summary, a wide variety of cell types secrete Zn-SMase activity, which arises from the same gene as lysosomal SMase. This secreted enzyme may play roles in physiological and pathophysiological processes involving extracellular sphingomyelin hydrolysis.