A Novel Mitochondrial Sphingomyelinase in Zebrafish Cells

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.

Identification of Novel Anionic Phospholipid Binding Domains in Neutral Sphingomyelinase 2 with Selective Binding Preference

“…S4A), suggesting that interactions with APLs are conserved among mammalian nSMase2. Interestingly, a novel member of the N-SMase family was identified in zebrafish and found to localize to mitochondria (43). Although distinct from zebrafish nSMase2, the mitochondrial SMase was strongly activated by APLs.…”

Lactosylceramide contributes to mitochondrial dysfunction in diabetes

“…In STZ-induced diabetes, the lack of insulin releases suppression of lipolysis in adipocytes ( 67,68 ), resulting in elevated levels of FFA in plasma (69)(70)(71)(72), accompanied by expression of is not in direct equilibrium with the pool of ceramide accessible to NCDase, and that the increase in SM in mitochondrial subpopulations of diabetic hearts refl ects a suppression of SM hydrolysis rather than activation of SM synthesis. In line with this notion, there is no evidence of the SM synthase presence in mitochondria, but a novel mitochondria-associated neutral SMase has been cloned and characterized ( 12,13,83 ).…”

Identification and Characterization of Murine Mitochondria-associated Neutral Sphingomyelinase (MA-nSMase), the Mammalian Sphingomyelin Phosphodiesterase 5

“…In contrast, nSMase2 localizes to the plasma membrane and Golgi, whereas nSMase3 localizes to the Golgi and ER (16,28). More recently, a zebrafish N-SMase was identified in zebrafish that localized at least partly to mitochondria on overexpression (21). Taken together, this suggests the potential existence of a mitochondria-associated N-SMase in mammals, which could be a key enzyme for sphingolipid metabolism.…”