Identification and characterization of murine mitochondria-associated neutral sphingomyelinase (MA-nSMase), the mammalian sphingomyelin phosphodiesterase 5

Abstract

Sphingolipids play important roles in regulating cellular responses. Although mitochondria contain sphingolipids, direct regulation of their levels in mitochondria or mitochondria-associated membranes is mostly unclear. Neutral SMase (N-SMase) isoforms, which catalyze hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine, have been found in the mitochondria of yeast and zebrafish, yet their existence in mammalian mitochondria remains unknown. Here, we have identified and cloned a cDNA based on nSMase homologous sequences. This cDNA encodes a novel protein of 483 amino acids that displays significant homology to nSMase2 and possesses the same catalytic core residues as members of the extended N-SMase family. A transiently expressed V5-tagged protein co-localized with both mitochondria and endoplasmic reticulum markers in MCF-7 and HEK293 cells; accordingly, the enzyme is referred to as mitochondria-associated nSMase (MA-nSMase). MA-nSMase was highly expressed in testis, pancreas, epididymis, and brain. MA-nSMase had an absolute requirement for cations such as Mg(2+) and Mn(2+) and activation by the anionic phospholipids, especially phosphatidylserine and the mitochondrial cardiolipin. Importantly, overexpression of MA-nSMase in HEK293 cells significantly increased in vitro N-SMase activity and also modulated the levels of SM and ceramide, indicating that the identified cDNA encodes a functional SMase. Thus, these studies identify and characterize, for the first time, a mammalian MA-nSMase. The characterization of MA-nSMase described here will contribute to our understanding of pathways regulated by sphingolipid metabolites, particularly with reference to the mitochondria and associated organelles.

FIGURE 1.

Sequence analysis of mouse MA-nSMase. A, alignment of the deduced amino acid sequences of mouse MA-nSMase, mouse nSMase2, and zebrafish mitochondrial SMase (Z-MTSMASE). The sequences were aligned by the GCG Pileup program. Identical residues in all the three sequences are indicated by bold characters. The mitochondrial signal peptide in zebrafish mitochondrial SMase is highlighted. The predicted transmembrane domain and p-loop like domain are underlined. B, hydrophobicity profile of mouse MA-nSMase. The deduced amino acid sequence of MA-nSMase was analyzed by the method of Kyte-Doolittle (dark line) and by the Goldman method (light line) for hydrophobicity plotting. The predicted transmembrane (TM) domain is indicated. C, evolutionary relationships between MA-nSMase and other nSMases. A phylogenetic tree of various nSMases was plotted by the GCG program using the Kimura protein distance correction. The length of each horizontal line in the tree is proportional to the difference of the amino acid sequences. The nSMase sequences are from human (h), mouse (m), Bacillus cereus (B.c) and Listeria ivanovii (L.i). The ISC1 and CSS1 sequences are from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

FIGURE 2.

Distribution of MA-nSMase mRNA in mouse tissues. Total RNAs from various mouse tissues were isolated, and cDNA was synthesized from 1 μg of total RNA. Real time RT-PCR was performed using primers specific to MA-nSMase. β-Actin was used as an internal reference control to normalize relative levels of gene expression. Real time PCR results were expressed as mean normalized expression. The values are expressed as the mean ± S.D. (n = 3).

FIGURE 3.

Overexpression of the MA-nSMase in mammalian cell lines. HEK293 and MCF-7 cells were transfected with an expression construct of mouse MA-nSMase. A, the expression of MA-nSMase was detected by Western blot analysis with a polyclonal antibody against V5. CTL, control. The N-SMase activity of the overexpressed enzyme was detected in both HEK293 (B) and MCF-7 cells (C). The values are expressed as the mean ± S.D. (n = 3).

FIGURE 4.

Subcellular localization of mouse MA-nSMase. A and B, MCF-7 (A) and HEK293 (B) cells were transfected with MA-nSMase expression vector. After 24 h, the cells were fixed and co-stained with an antibody against V5 (green) for MA-nSMase signal and antibodies against various subcellular markers (red), including HSP60 and Tom20 (mitochondrial markers), calreticulin (ER marker), and giantin (Golgi marker) and then subjected to confocal microscopic observation. The co-localization signals were observed as yellow or orange.

FIGURE 5.

Characterization of MA-nSMase expressed in HEK293 cells. A, pH dependence of MA-nSMase activity. The SMase activity MA-nSMase was measured using 25 μg of protein from cells transfected with the MA-nSMase expression construct or with empty vector. The pH was adjusted by the addition of the indicated buffers at a final concentration of 100 mm. The following buffers were used: acetate buffer (pH 4.0–5.5), MES buffer (pH 5.7–6.3), Tris buffer (pH 7.0–8.5), and glycine-NaOH buffer (pH 9.0–10.0). B, N-SMase activity toward increasing concentrations of SM substrate. C, cation effects were assayed using 6.5 mol % (100 μm) of SM and 6.5 mol % of PS. N-SMase activity was measured at various concentrations of MgCl₂, MnCl₂, CaCl₂, ZnCl₂, FeCl₂, or CuCl₂. B, the results are averages of triplicates. A and C, the data are the averages of duplicates. The values are expressed as the mean ± S.D. The data are representative of at least two independent experiments.

FIGURE 6.

Effects of phospholipids on N-SMase activity. The N-SMase activity was measured using 6.5 mol % of SM. A, the indicated dioleoyl-phospholipids were delivered at a final concentration of 6.5 mol % (100 μm), and enzyme activity was assayed in the presence of PS or other phospholipids (PA, PC, PE, PG, PI, or CL). B, the activity of MA-nSMase on SM was measured at various concentrations of PS or CL. The data are the averages of duplicates from at least two independent experiments. The values are expressed as the mean ± S.D.

FIGURE 7.

Modulation of ceramide levels by overexpression of MA-nSMase in HEK293 cells. Changes in ceramide species were analyzed 18 and 36 h after cells were transfected by control (CTL) pcDNA3 or MA-nSMase vector. A, total ceramide. B, ceramide (Cer) profiles at 18 h after transfection. C, ceramide profiles at 36 h after transfection. D, -fold change of ceramide profiles in MA-nSMase-transfected cells compared with control cells. Each sample was normalized to its respective total protein levels. The values are expressed as the mean ± S.D. (n = 3). Statistical significance was calculated with respect to control (*, p < 0.05). The data are representative of two independent experiments.

FIGURE 8.

Modulation of SM levels by overexpression of MA-nSMase using HEK293 cells. Changes in SM species were analyzed 18 and 36 h after cells were transfected with control (CTL) pcDNA3 or MA-nSMase vectors. A, total SM. B, SM profiles at 18 h after transfection. C, SM profiles at 36 h after transfection. D, -fold change of SM profiles in MA-nSMase-transfected cells of control cells. Each sample was normalized to its respective total protein levels. The values are expressed as the mean ± S.D. (n = 3). Statistical significance was calculated with respect to control (*, p < 0.05). The data are representative of two independent experiments.