PUBLICATION
A novel mitochondrial sphingomyelinase in zebrafish cells
- Authors
- Yabu, T., Shimizu, A., and Yamashita, M.
- ID
- ZDB-PUB-090518-5
- Date
- 2009
- Source
- The Journal of biological chemistry 284(30): 20349-20363 (Journal)
- Registered Authors
- Yamashita, Michiaki
- Keywords
- ENZYMES/Lipid, LIPID/Phospholipid/Metabolism, LIPID/Sphingolipid, METABOLISM/Sphingolipid, ORGANISMS/Zebra fish, SUBCELLULAR ORGANELLES/Mitochondria, ceramide, sphingomyelinase
- MeSH Terms
-
- Amino Acid Sequence
- Animals
- Bacillus cereus/enzymology
- Bacillus cereus/genetics
- Bacterial Proteins/genetics
- Cell Line
- Ceramides/analysis
- Ceramides/metabolism
- Cloning, Molecular
- Gene Expression Regulation
- Humans
- Mice
- Mitochondria/chemistry
- Mitochondria/enzymology*
- Mitochondria/metabolism
- Molecular Sequence Data
- Mutation
- Sequence Alignment
- Sphingomyelin Phosphodiesterase/analysis*
- Sphingomyelin Phosphodiesterase/genetics
- Sphingomyelin Phosphodiesterase/isolation & purification
- Sphingomyelin Phosphodiesterase/metabolism*
- Sphingomyelins/analysis
- Sphingomyelins/metabolism*
- Zebrafish/embryology
- Zebrafish/metabolism*
- PubMed
- 19429680 Full text @ J. Biol. Chem.
Citation
Yabu, T., Shimizu, A., and Yamashita, M. (2009) A novel mitochondrial sphingomyelinase in zebrafish cells. The Journal of biological chemistry. 284(30):20349-20363.
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 (ZE) cells. The cloned SMase cDNA encoded a polypeptide of 545 amino acid residues (putative molecular weight, 61.3 K) containing a mitochondrial localization signal (MLS) and a predicted transmembrane domain (TD). The mature endogenous enzyme was predicted to have a molecular weight of 57 K, and MALDI-TOF-MS 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 TD 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 ZE cells. These observations indicate that SMase catalyzes the hydrolysis of sphingomyelin and generates ceramide in mitochondria in fish cells.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping