Interindividual heterogeneity of molecular weight of human brain neutral sphingomyelinase determined by radiation inactivation method

270

267

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 Zn 2؉ -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 NiemannPick 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 ZnSMase 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. Mammalian SMases1 have been implicated in many important physiological and pathophysiological processes, including lysosomal digestion of sphingomyelin, which is important for normal neuronal and vascular function (1); ceramide-mediated signal transduction, leading to cytokine-induced apoptosis, cellular differentiation, and various immune and inflammatory responses (2, 3); lipoprotein aggregation within the vessel wall, which is a key event in atherogenesis (4 -7); and intracellular cholesterol trafficking and metabolism (8 -10).2 Several different forms of mammalian SMases have been identified, including: (a) a lysosomal SMase that is present in all tissues, acts optimally at low pH, and shows no dependence on divalent cations (11). This enzyme, which is defective in types A and B Niemann-Pick disease (11), has been purified and cloned (12), and mice in which the gene encoding lysosomal SMase has been inactivated have recently been generated (13, 14); (b) a neutral, membrane-associated, Mg 2ϩ -stimulated SMase that is found predominantly in brain and kidney (15) and that is known to arise from a separate gene from lysosomal SMase (16) Although the molecular identity and functions are definitively known only for lysosomal SMase (15), two of the other forms, namely, Mg 2ϩ -stimulated SMase and cytosolic SMase, also have been studied. Both have been partially purified (17,19,20), and these two enzymes, in addition to lysosomal SMase, ...

Section: Mammalian Smasesmentioning

confidence: 99%

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

Schissel

Schuchman

Williams

et al. 1996

270

267

“…Nevertheless, S-SMase requires exogenously added Zn 2ϩ for activation in in vitro assays, whereas L-SMase isolated from cell or tissue homogenates does not (16). In fact, the lack of stimulation of L-SMase by any cations and its lack of inhibition by EDTA has led to a long-standing body of literature labeling L-SMase as a "cation-independent" enzyme (1).…”

Section: Smasesmentioning

confidence: 99%

“…1 (SM phosphodiesterase, EC 3.1.4.12) have been implicated in a wide variety of physiologic and pathophysiologic processes, including lysosomal hydrolysis of endocytosed SM (1,2), ceramide-mediated cell signaling (3,4), membrane vesiculation (5,6), alterations in intracellular cholesterol trafficking (5,(7)(8)(9), and atherogenesis (10 -13). One type of mammalian SMase is a magnesium-dependent, membrane-bound neutral SMase, and Tomiuk et al (14) have recently reported the cloning of an enzyme that has several properties in common with this SMase.…”

Section: Smasesmentioning

confidence: 99%

The Cellular Trafficking and Zinc Dependence of Secretory and Lysosomal Sphingomyelinase, Two Products of the Acid Sphingomyelinase Gene

Schissel¹,

Keesler²,

Schuchman³

et al. 1998

230

239

The acid sphingomyelinase (ASM) gene, which has been implicated in ceramide-mediated cell signaling and atherogenesis, gives rise to both lysosomal SMase (L-SMase), which is reportedly cation-independent, and secretory SMase (S-SMase), which is fully or partially dependent on Zn 2؉ for enzymatic activity. Herein we present evidence for a model to explain how a single mRNA gives rise to two forms of SMase with different cellular trafficking and apparent differences in Zn 2؉dependence. First, we show that both S-SMase and LSMase, which contain several highly conserved zincbinding motifs, are directly activated by zinc. In addition, SMase assayed from a lysosome-rich fraction of Chinese hamster ovary cells was found to be partially zinc-dependent, suggesting that intact lysosomes from these cells contain subsaturating levels of Zn 2؉. Analysis of Asn-linked oligosaccharides and of N-terminal amino acid sequence indicated that S-SMase arises by trafficking through the Golgi secretory pathway, not by cellular release of L-SMase during trafficking to lysosomes or after delivery to lysosomes. Most importantly, when Zn 2؉ -dependent S-SMase was incubated with SMasenegative cells, the enzyme was internalized, trafficked to lysosomes, and became zinc-independent. We conclude that L-SMase is exposed to cellular Zn 2؉ during trafficking to lysosomes, in lysosomes, and/or during cell homogenization. In contrast, the pathway targeting S-SMase to secretion appears to be relatively sequestered from cellular pools of Zn 2؉ ; thus S-SMase requires exogeneous Zn 2؉ for full activity. This model provides important information for understanding the enzymology and regulation of L-and S-SMase and for exploring possible roles of ASM gene products in cell signaling and atherogenesis.

“…At least seven different SMases have been identified (14). Acid lysosomal SMase is found ubiquitously in tissues and defective in types A and B Niemann-Pick disease (15). A variety of cell types present in atherosclerotic lesions secrete SMase (16).…”

mentioning

confidence: 99%

Ceramide in Lipid Particles Enhances Heparan Sulfate Proteoglycan and Low Density Lipoprotein Receptor-related Protein-mediated Uptake by Macrophages

Morita

Kawabe

Sakurai

et al. 2004

Arterial wall sphingomyelinase (SMase) has been proposed to be involved in atherogenesis. SMase modification of lipoproteins has been shown to occur in atherosclerotic lesions and to facilitate their uptake by macrophages and foam cell formation. To investigate the mechanism of macrophage uptake enhanced by SMase, we prepared lipid emulsions containing sphingomyelin (SM) or ceramide (CER) as model particles of lipoproteins. SMase remarkably increased the uptake of SM-containing emulsions by J774 macrophages without apolipoproteins. The emulsion uptake was negatively correlated with the degree of particle aggregation by pretreatment with SMase, whereas the uptake of CERcontaining emulsions was significantly larger than SMcontaining emulsions, indicating that enhancement of uptake is due to the generation of CER molecules in particles but not to the aggregation by SMase. Heparan sulfate proteoglycans (HSPGs) and low density lipoprotein receptor-related protein (LRP) were crucial for CER-enhanced emulsion uptake, because heparin or lactoferrin inhibited the emulsion uptake. Confocal microscopy also showed that SMase promoted both binding and internalization of emulsions by J774 macrophages, which were almost abolished by lactoferrin. Apolipoprotein E further increased the uptake of CERcontaining emulsions compared with SM-containing emulsions. These findings suggest the generation of CER in lipoproteins by SMase facilitates the macrophage uptake via HSPG and LRP pathways and plays a crucial role in foam cell formation. Thus, CER may act as an important atherogenic molecule.