Abstract
Sphingolipids are important signaling molecules in many biological processes, but little is known regarding their physiological roles in the mitochondrion. We focused on the biochemical characters of a novel sphingomyelinase (SMase) and its function in mitochondrial ceramide generation in zebrafish embryonic cells. The cloned SMase cDNA encoded a polypeptide of 545 amino acid residues (putative molecular weight, 61,300) containing a mitochondrial localization signal (MLS) and a predicted transmembrane domain. The mature endogenous enzyme was predicted to have a molecular weight of 57,000, and matrix-assisted laser de sorption ionization time-of-flight mass spectrometry analysis indicated that the N-terminal amino acid residue of the mature enzyme was Ala-36. The purified enzyme optimally hydrolyzed [(14)C]sphingomyelin in the presence of 10 mm Mg(2+) at pH 7.5. In HEK293 cells that overexpressed SMase cDNA, the enzyme was localized to the mitochondrial fraction, whereas mutant proteins lacking MLS or both the MLS and the transmembrane domain were absent from the mitochondrial fraction. Endogenous SMase protein co-localized with a mitochondrial cytostaining marker. Using a protease protection assay, we found that SMase was distributed throughout the intermembrane space and/or the inner membrane of the mitochondrion. Furthermore, the overexpression of SMase in HEK293 cells induced ceramide generation and sphingomyelin hydrolysis in the mitochondrial fraction. Antisense phosphorothioate oligonucleotide-induced knockdown repressed ceramide generation and sphingomyelin hydrolysis in the mitochondrial fraction in zebrafish embryonic cells. These observations indicate that SMase catalyzes the hydrolysis of sphingomyelin and generates ceramide in mitochondria in fish cells.
Highlights
A signaling molecule in cell proliferation, apoptosis, cell cycle arrest, differentiation, and the stress response in animal cells [1,2,3,4,5]
The three amino acid residues of the putative magnesium-binding site, the substrate-binding asparagine site, and the histidine residue of the active site on the zebrafish SMase (Gln-258, Asp-404, and His-529, respectively; see Refs. 43– 47), which are critical for the enzymatic regulation of mammalian neutral SMase 2, were completely conserved (Fig. 1B)
Generation of Stable HEK293 Transfectants—HEK293 cells The mammalian neutral SMase 2 had a single collagenous were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS), 5 domain between the membrane-anchoring domain and the units/ml penicillin, and 50 g/ml streptomycin
Summary
Mammalian enzymes exhibited Mg2ϩ-dependent neutral SMase activity in vitro [9], no significant biological functions in sphingomyelin and ceramide metabolism were identified in SMase 1-overexpressing cells [10] or neutral SMase 1 knockout mice [11]. Birbes et al [28] found that the selective hydrolysis of a mitochondrial pool of sphingomyelin induced apoptosis They transfected MCF-7 cells with B. cereus SMase targeted to various subcellular organelles, but they observed cytochrome c release and apoptosis induction only when the enzyme was targeted to the mitochondria. Ceramide-metabolizing enzymes, such as a bovine liver ceramide synthase [33] and human ceramidase [34], are localized to the mitochondrion These observations suggest the existence of a mitochondrial pool of sphingomyelin and the function of a sphingomyelinspecific metabolic pathway in mitochondria.
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