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.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla

Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition.     

Characterization and subcellular localization of human neutral class II alpha-mannosidase [corrected

A glycosyl hydrolase family 38 enzyme, neutral alpha-mannosidase, has been proposed to be involved in hydrolysis of cytosolic free oligosaccharides originating either from ER-misfolded glycoproteins or the N-glycosylation process. Although this enzyme has been isolated from the cytosol, it has also been linked to the ER by subcellular fractionations. We have studied the subcellular localization of neutral alpha-mannosidase by immunofluorescence microscopy and characterized the human recombinant enzyme with natural substrates to elucidate the biological function of this enzyme. Immunofluorescence microscopy showed neutral alpha-mannosidase to be absent from the ER, lysosomes, and autophagosomes, and being granularly distributed in the cytosol. In experiments with fluorescent recovery after photo bleaching, neutral alpha-mannosidase had slower than expected two-phased diffusion in the cytosol. This result together with the granular appearance in immunostaining suggests that portion of the neutral alpha-mannosidase pool is somehow complexed. The purified recombinant enzyme is a tetramer and has a neutral pH optimum for activity. It hydrolyzed Man(9)GlcNAc to Man(5)GlcNAc in the presence of Fe(2+), Co(2+), and Mn(2+), and uniquely to neutral alpha-mannosidases from other organisms, the human enzyme was more activated by Fe(2+) than Co(2+). Without activating cations the main reaction product was Man(8)GlcNAc, and Cu(2+) completely inhibited neutral alpha-mannosidase. Our findings from enzyme-substrate characterizations and subcellular localization studies support the suggested role for neutral alpha-mannosidase in hydrolysis of soluble cytosolic oligomannosides.     

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3

Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mM SNP. SNP also decreased the sphingomyelin level to approximately 70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling.     

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.     

Irreversible inactivation of magnesium-dependent neutral sphingomyelinase 1 (NSM1) by peroxynitrite,a nitric oxide-derived oxidant

Previous results have indicated that the generation of ceramide by hydrolysis of sphingomyelin by magnesium-dependent neutral sphingomyelinase 1 (NSM1) is reversibly inhibited by hydrogen peroxide (H2O2) and oxidized glutathione (GSSG). This redox-dependent reversible regulation of NSM1 activity has been shown to involve the reversible formation and breakage of disulfide bonds. In this paper, we show that peroxynitrite, a nitric oxide-derived oxidant generated by SIN1, inactivates dose-dependently the NSM1 activity in an irreversible manner. In addition, we show that, in contrast to the reversible inhibition of NSM1 by H2O2 or GSSG which involves the formation of disulfide bonds, irreversible inactivation of this enzyme by peroxynitrite generated from SIN1 is likely due to definitive oxidative thiol modification. These results suggest that depending on the nature of the oxidative stress, the enzymatic activity of NSM1 could be reversibly or irreversibly inactivated.     

Characterization and subcellular localization of chlorophyllase from Ginkgo biloba

Chlorophyllase (Chlase) catalyzes the initial step of chlorophyll (Chl)-degradation, but the physiological significance of this reaction is still ambiguous. Common understanding of its role is that Chlase is involved in de-greening processes such as fruit ripening, leaf senescence, and flowering. But there is a possibility that Chlase is also involved in turnover and homeostasis of Chls. Among the de-greening processes, autumnal coloration is one of the most striking natural phenomena, but the involvement of Chlase during autumnal coloration is not clear. Previously, it was shown that Chlase activity and expression level of the Chlase gene were not increased during autumnal coloration in Ginkgo biloba, indicating that Chlase does not work specially in the de-greening processes in G. biloba. In this study, we characterized the recombinant Chlase and analyzed its subcellular localization to understand the role of the cloned Chlase of G. biloba (GbCLH). GbCLH exhibited its highest activity at pH 7.5, 40 degrees C. Kinetic analysis revealed that GbCLH hydrolyzes pheophytin (Pheo) a and Chl a more rapidly than Pheo b and Chl b. Transient expression analysis of 40 N-terminus amino acids of GbCLH fused with GFP (green fluorescent protein) and subcellular fractionation showed that GbCLH localizes within chloroplasts. Together with our previous results, property of GbCLH and its location within the chloroplasts suggest that GbCLH plays a role in the turnover and homeostasis of Chls in green leaves of G. biloba.     

The Kindlins: subcellular localization and expression during murine development

The three Kindlins are a novel family of focal adhesion proteins. The Kindlin-1 (URP1) gene is mutated in Kindler syndrome, the first skin blistering disease affecting actin attachment in basal keratinocytes. Kindlin-2 (Mig-2), the best studied member of this family, binds ILK and Migfilin, which links Kindlin-2 to the actin cytoskeleton. Kindlin-3 is expressed in hematopoietic cells. Here we describe the genomic organization, gene expression and subcellular localization of murine Kindlins-1 to -3. In situ hybridizations showed that Kindlin-1 is preferentially expressed in epithelia, and Kindlin-2 in striated and smooth muscle cells. Kindlins-1 and -2 are both expressed in the epidermis. While both localize to integrin-mediated adhesion sites in cultured keratinocytes Kindlin-2, but not Kindlin-1, colocalizes with E-cadherin to cell-cell contacts in differentiated keratinocytes. Using a Kindlin-3-specific antiserum and an EGFP-tagged Kindlin-3 construct, we could show that Kindlin-3 is present in the F-actin surrounding ring structure of podosomes, which are specialized adhesion structures of hematopoietic cells.     

Identification,partial purification,and localization of a neutral sphingomyelinase in rabbit skeletal muscle: Neutral sphingomyelinase in skeletal muscle

Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic -cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).     

Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells

The enzymatic activities of acidic and neutral sphingomyelinases (aSMase and nSMase) in crude extracts of HL-60 cells prepared by short ultrasonic irradiation (sonicates) were characterized. It was found that although both have similar Km and Vmax (approximately 0.2 mM and approximately 3.5 nmol/mg per h, respectively), the two activities differ in many other aspects, including the following: (1) the aSMase activity has higher stability at 37 degrees C; (2) the aSMase is much less sensitive to Triton X-100 ( > 5 mM), compared with < or = 0.4 mM for the nSMase; (3) the nSMase, but not the aSMase, can discriminate between the natural bovine sphingomyelin substrate and the fluorescent substrate lissamine rhodamine dodecanoyl sphingosyl phosphocholine, suggesting that nSMase has higher substrate specificity. TNFalpha, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect Km or Vmax of the nSMase in HL-60 sonicates. This suggests that TNFalpha may operate through translocation of either the enzyme or the substrate, thereby enhancing substrate availability and rate of hydrolysis, and not through enzyme activation. The relevance of these studies to the sphingomyelin cycle is discussed.     

Characterization and subcellular localization of Tektin 3 in rat spermatozoa

Mammalian sperm flagella have filament‐forming Tektin proteins (Tektin 1–5) reported to be involved in the stability and structural complexity of flagella. Male mice null for Tektin3 produce spermatozoa with reduced forward progression and increased flagellar structural bending defects. The subcellular localization of Tektin3 (TEKT3) in spermatozoa, however, has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT3 in flagella of rat spermatozoa, we performed extraction studies followed by immunoblot analysis, immunofluorescence microscopy, and immunogold electron microscopy. Extraction of sperm flagella from the cauda epididymis resulted in complete removal of axonemal tubulins, while TEKT3 was resistant to extraction with the same S‐EDTA (1% SDS, 75 mM NaCl, 24 mM EDTA, pH 7.6) solution, suggesting that TEKT3 might be present in the peri‐axonemal component and not directly associated with axonemal tubulins. Resistance to S‐EDTA extraction might be due to disulfide bond formation during epididymal maturation since concentrations of DTT greater than 5 mM drastically promoted release of TEKT3 from flagella. Immunofluorescence microscopy and pre‐embedding immunoelectron microscopy revealed that TEKT3 was predominantly associated with the surface of mitochondria and outer dense fibers in the middle piece. In addition, TEKT3 was found to be present at the equatorial segment region of the acrosome membrane in sperm heads. TEKT3 might not only work as a flagellar constituent required for flagellar stability and sperm motility but also may be involved in acrosome‐related events, such as the acrosome reaction or sperm–egg fusion. Mol. Reprod. Dev. 78:611–620, 2011. © 2011 Wiley‐Liss, Inc.     

Purification,localization, and expression of human intestinal alkaline sphingomyelinase

Sphingomyelin (SM) metabolism in the gut may have an impact on colon cancer development. In this study, we purified alkaline sphingomyelinase (alk-SMase) from human intestinal content, and studied its location in the mucosa, expression in colon cancer, and function on colon cancer cells. The enzyme was purified by a series of chromatographies. The molecular mass of the enzyme is 60 kDa, optimal pH is 8.5, and isoelectric point is 6.6. Under optimal conditions, 1 mg of the enzyme hydrolyzed 11 mM SM per hour. The properties of the enzyme are similar to those of rat intestinal alk-SMase but not to those of bacterial neutral SMase. Immunogold electronmicroscopy identified the enzyme on the microvillar membrane in endosome-like structures and in the Golgi complexes of human enterocytes. The expression and the activity of the enzyme were decreased in parallel in human colon cancer tissues compared with the adjacent normal tissue. The enzyme inhibited DNA biosynthesis and cell proliferation dose dependently and caused a reduction of SM in HT29 cells. Intestinal alk-SMase is localized in the enterocytes, down-regulated in human colon cancer, and may have antiproliferative effects on colon cancer cells.     

Neutral sphingomyelinase 2 is palmitoylated on multiple cysteine residues. Role of palmitoylation in subcellular localization

The neutral sphingomyelinases (nSMases) are considered major candidates for mediating the stress-induced production of ceramide. nSMase2, which has two hydrophobic segments near the NH(2)-terminal region, has been reported to be located at the plasma membrane and play important roles in ceramide-mediated signaling. In this study, we found that nSMase2 is palmitoylated on multiple cysteine residues via thioester bonds. Site-directed mutagenesis of cysteine residues to alanine indicated that two cysteine clusters of the enzyme are multiply palmitoylated; one cluster is located between the two hydrophobic segments, and the second one is located in the middle of the catalytic region of the protein. When overexpressed in the confluent phase of MCF-7 cells, wild-type nSMase2 was strictly localized in the plasma membranes, and the cysteine mutants of each palmitoylated cysteine cluster were seen not only at the plasma membrane but also in some punctate structures. Furthermore, mutation of all potential palmitoylation sites resulted in a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures. The palmitoylation-deficient mutant was directed to lysosomes and rapidly degraded. Palmitoylation had no effect on enzyme activity but affected membrane-association properties of the protein. Finally, the catalytic region of nSMase2 where palmitoylation occurs was found to be localized at the inner leaflet of the plasma membrane. In summary, the results from this study reveal for the first time the palmitoylation of nSMase2 via thioester bonds and its importance in the subcellular localization and stability of this protein.     

Characterization and subcellular localization of GnRH analog binding in rat brain

The binding of a degradation-resistant analog of gonadotropin-releasing hormone, [D-Phe6]GnRH, to rat brain crude particulate preparation was studied. The binding of this analog at 0 degrees C was saturable and Scatchard analysis revealed the presence of 2 binding sites: one with KD = 1.39 x 10(-7) M and Bmax = 265 pmole/mg protein, and another of lower affinity but higher capacity with KD = 5.58 X 10(-6) M and Bmax = 1734 pmoles/mg protein. The binding at 0 degrees C was substantially higher than that obtained at 37 degrees C, due to binding site-inactivation processes occurring at 37 degrees C. The binding sites exhibited a considerable degree of specificity for GnRH as unrelated peptides (with the exception of ACTH) display a much weaker affinity than GnRH and GnRH analogs. Subcellular fractionation demonstrated that most of the binding was associated with the mitochondrial fraction.