Purification and Characterization of a Neutral Ceramidase from Mouse Liver

Self Cite

105

We report here the molecular cloning, sequencing, and expression of the gene encoding the mouse neutral ceramidase, which has been proposed to function in sphingolipid signaling. A full-length cDNA encoding the neutral ceramidase was cloned from a cDNA library of mouse liver using the partial amino acid sequences of the purified mouse liver ceramidase. The open reading frame of 2,268 nucleotides encoded a polypeptide of 756 amino acids having nine putative N-glycosylation sites. Northern blot analysis revealed that the mRNA of the ceramidase was expressed widely in mouse tissues, with especially strong signals found in the liver and kidney. The ceramidase activity of lysates of CHOP cells increased more than 900-fold when the cells were transformed with a plasmid containing the cDNA encoding ceramidase. We also cloned the ceramidase homologue from the cDNA library of mouse brain and found that the sequence of the open reading frame, but not the 5-noncoding region, was identical to that of the liver.

mentioning

confidence: 99%

“…The final preparation showed a single protein band corresponding to a molecular mass of 94 kDa on SDS-polyacrylamide gel electrophoresis. This CDase seems to be a glycoprotein with N-glycans (24). Nonlysosomal CDase with a neutral to alkaline pH optimum was also purified from the rat brain (25).…”

mentioning

confidence: 99%

Molecular Cloning of the Full-length cDNA Encoding Mouse Neutral Ceramidase

Tani

Okino

Mori

et al. 2000

Self Cite

105

“…3, B and D). These results suggest that the subcellular localization of the znCD is almost the same as that of mouse and rat neutral CDases and that znCD is likely to present itself as a type II integral protein with its N-terminal end anchored to the plasma membrane and Cterminal end exposed to the extracellular space (10,19).…”

Section: Resultsmentioning

confidence: 55%

“…Interestingly, neutral CDases of bacteria (18) and Drosophila (9) were solely detached from the cells as a soluble form, whereas those of mammalian origins were mainly recovered in membrane fractions (19). Recently, we clarified the reason for this discrepancy at the molecular level, i.e.…”

mentioning

confidence: 97%

Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase

Yoshimura¹,

Tani²,

Okino³

et al. 2004

Self Cite

Sphingolipids and their metabolites are multifunctional components of eukaryotic cell membranes that are involved in a variety of biological processes. Ceramide (Cer) 1 has been implicated as a novel lipid modulator in signal transduction pathways involved in cell growth, differentiation, and apoptosis (1, 2). The metabolite of Cer, sphingosine-1-phosphate (S1P), which is produced through the phosphorylation of sphingosine (Sph) by sphingosine kinase, promotes the proliferation and migration of endothelial cells through the activation of G protein-coupled receptors from the S1P (Edg) family (3-5).Ceramidase (CDase, EC 3.5.1.23), which catalyzes the hydrolysis of the N-acyl linkage of Cer, serves to control the intracellular levels of Sph/Cer and possibly S1P and then regulate the cell functions.

“…CDase Assay-The hydrolysis and reverse hydrolysis activities of neutral CDase were measured using C12-NBD-Cer (for the hydrolysis reaction) and NBD-dodecanoic acid and Sph (for the reverse hydrolysis reaction) as substrates (18).…”

Section: Methodsmentioning

confidence: 99%

Conserved Amino Acid Residues in the COOH-terminal Tail Are Indispensable for the Correct Folding and Localization and Enzyme Activity of Neutral Ceramidase

Tani

Okino

Sueyoshi

et al. 2004

Self Cite

Several lines of evidence suggest that neutral ceramidase is involved in the regulation of ceramide-mediated signaling. Recently, the enzymes from mouse and rat were found to be localized at plasma membranes as a type II integral membrane protein, occasionally being detached from the cells after proteolytic processing of the NH 2 -terminal anchoring region (Tani, M., Iida, H., and Ito, M. (2003) J. Biol. Chem. 278, 10523-10530). We report here that conserved hydrophobic amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral ceramidase. Truncation of four, but not three, amino acid residues from the COOH terminus of rat neutral ceramidase resulted in a complete loss of enzyme activity as well as cell surface expression in HEK293 cells. Point mutation analysis revealed that Ile 758 , the 4 th amino acid residue from the COOH terminus, and Phe 756 are essential for the enzyme to function. The truncated and mutated enzymes were found to be retained in the endoplasmic reticulum (ER) and rapidly degraded without transportation to the Golgi apparatus. Treatment of the cells expressing the aberrant COOH-terminal enzyme with MG-132, a specific inhibitor for the proteasome, increased the accumulation of the enzyme in the ER, indicating that the misfolded enzyme was degraded by the proteasome. It was also found that the COOH-terminal tail was indispensable for the enzyme activity and correct folding of the prokaryote ceramidase from Pseudomonas aeruginosa, indicating that the importance of the COOH-terminal tail of the enzyme has been preserved through evolution.