Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase

Abstract

The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular data on the neutral sphingomyelinases (nSMases). We recently identified a mammalian nSMase, an integral membrane protein with remote similarity to bacterial sphingomyelinases. However, its ubiquitous expression pattern is in contrast to previous findings that sphingomyelinase activity is found mainly in brain tissues. By using an improved database search method, combined with phylogenetic analysis, we identified a second mammalian nSMase (nSMase2) with predominant expression in the brain. The sphingomyelinase activity of nSMase2 has a neutral pH optimum, depends on Mg(2+) ions, and is activated by unsaturated fatty acids and phosphatidylserine. Immunofluorescence reveals a neuron-specific punctate perinuclear staining, which colocalizes with a Golgi marker in a number of cell lines. The likely identity of nSMase2 with cca1, a rat protein involved in contact inhibition of 3Y1 fibroblasts, suggests a role for this enzyme in cell cycle arrest. Both mammalian nSMases are members of a superfamily of Mg(2+)-dependent phosphohydrolases, which also contains nucleases, inositol phosphatases, and bacterial toxins.

Figure 1

Sequence analysis of nSMase2. (A) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion. Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae; SA, Staphylococcus aureus; BC, Bacillus cereus; LI, Leptospira interrogans; XL, Xenopus laevis; DM, Drosophila melanogaster; AH, Aeromonas hydrophila; SS, Synechocystis sp.; MP, Mycoplasma pulmonis; BS, Bacillus subtilis; DD, Dictyostelium discoideum; EC, Escherichia coli; BM, Bombyx mori; TB, Trypanosoma brucei; HD, Haemophilus ducreyi; and CJ, Campylobacter jejuni. In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. (B) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg²⁺-complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. (C) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.

Figure 2

Enzymatic properties of murine nSMase2. (A) pH optimum and ion requirement of murine nSMase2 from membrane extracts of stably transfected HEK 293 cells. Control assay mixtures contain 5 mM MgCl₂, 0.05% Triton X-100, 5 mM DTT, 100 mM Tris⋅Cl at pH 7.4, and a protease inhibitor mixture (1× Complete without EDTA; Roche Molecular Biochemicals). Measurements with EDTA and metal ions were done with 5 mM EDTA and 10 mM of the respective metal ion. (B) Influence of phospholipids on nSMase2 activity. Phospholipids were dissolved in 0.05% Triton X-100 by sonication and premixed with the assay components before adding the enzyme preparation. Results represent mean values (±SD) from three independent measurements. PS, phosphatidylserine; PS(C18), dioleoyl-PS; PE, phosphatidylethanolamine. (C) Activation of nSMase2 activity by unsaturated fatty acids. Fatty acids, dissolved in EtOH at various concentrations, were added directly to the assay mixture. ⋄, Stearic acid; ▵, linolenic acid; ○, linoleic acid; and □, arachidonic acid.

Figure 3

Expression of nSMase2 mRNA and protein. (A) Northern blot analysis of poly(A)⁺ mRNA derived from various human tissues, using a fragment containing the coding region of the murine nSMase2 cDNA as hybridization probe. The membrane was rehybridized with a β-actin probe for standardization. (B) Western blot analysis with protein extracts from different murine tissues, using the purified anti-mouse nSMase2 antibody in a concentration of 1 μg/ml. (C) Western blot analysis of murine embryonal fibroblasts, rat PC-12 cells, mock-transfected and nSMase2-transfected HEK 293 cells, and mouse brain extract, using the anti-mouse nSMase2 antibody at 1 μg/ml. The only specific signal is a single or double band at 70 kDa.

Figure 4

Immunofluorescence analysis. (A–D) Localization of nSMase2 protein in the Golgi apparatus of rat PC-12 cells. Cells were costained with anti-mnSMase2 and anti-Golgi 58-kDa protein antibodies and the corresponding Cy2- and Cy3-conjugated secondary antibodies and analyzed by confocal microscopy. (A) Anti-mnSMase2-Cy3. (B) Anti-58-kDa-Cy2. (C) Superposition of the Cy2 and Cy3 channels. Yellow indicates colocalization. (D) Corresponding light-microscopic image. (E) Confocal microscopic image of SH-SY5Y cells costained with anti-mnSMase2 (red) and anti-58-kDa (green) antibodies. Note the distinct punctate staining of the nSMase2 protein (indicated by arrows) within the Golgi apparatus. (F–H) Immunofluorescence of nSMase2 in coronal slices of total mouse brain is restricted to neurons. (F) Cerebellar neurons stained with anti-mnSMase2 antibody. (G) nSMase2 staining pattern in the cerebellum. Labels: p, Purkinje cell layer; g, granular layer; m, molecular layer. (H) nSMase2 staining of pyramidal cells (py) and dentate gyrus granular layer (dg).