Molecular Cloning and Characterization of a Human Mitochondrial Ceramidase

Bawab, Samer El; Roddy, Patrick; Qian, Ting; Bielawska, Alicja; Lemasters, John J.; Hannun, Yusuf A.

doi:10.1074/jbc.m002522200

Cited by 233 publications

(217 citation statements)

References 14 publications

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“…Using the advanced BLAST program, a genomic DNA sequence encoding the human mito-CDase (mitochondrial CDase) was identified [12]. Another ATG codon upstream of the translational initiation site proposed for mito-CDase was found in this genomic DNA sequence which was not detected in the original sequencing of the cDNA.…”

Section: Cloning Of the Human Neutral Cdasementioning

confidence: 99%

“…In order to generate the construct pYES2-CDase that expresses the human neutral CDase under the control of the Gal1 promoter, the open reading frame of the full-length human neutral CDase gene was amplified by PCR using previously described conditions [12]. The forward primer:…”

Section: Expression Of the Human Neutral Cdase In Yeastmentioning

confidence: 99%

“…Using peptide sequences obtained from the purified rat brain enzyme, the human neutral CDase was cloned [12]. A homologous gene was independently purified and cloned from bacteria [13].…”

Section: Introductionmentioning

confidence: 99%

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Identification of a novel amidase motif in neutral ceramidase

Galadari

Mao

et al. 2006

Biochemical Journal

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Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast doubleknockout strain ∆ypc1∆ydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na + and Ca 2+ . Mg 2+ and Mn 2+ were somewhat stimulatory, but Zn 2+ , Cu 2+ and Fe 2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser 354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXG P / X XC. Moreover, mutations of Asp 352 and Cys 362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.

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Section: Cloning Of the Human Neutral Cdasementioning

confidence: 99%

Section: Expression Of the Human Neutral Cdase In Yeastmentioning

confidence: 99%

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Identification of a novel amidase motif in neutral ceramidase

Galadari

Mao

et al. 2006

Biochemical Journal

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“…Interestingly, a recent study revealed that the three isoforms can also be distinguished by their primary structure, suggesting that they are derived from different ancestral genes (7). Neutral CDase, which shows an optimum pH of 6.5-8.5, has been cloned from bacteria (8), Drosophila (9), mouse (7), rat (10), and human (11). Recently, we reported the molecular cloning of the neutral CDase homologue of slime mold, which exhibits maximal activity at around pH 3 (12).…”

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confidence: 99%

Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase

Yoshimura¹,

Tani²,

Okino³

et al. 2004

Journal of Biological Chemistry

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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.

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“…Three categories of CDases: acid, neutral, and alkaline, are clearly distinguished not only by their catalytic pH optima, but also their primary structures (5). Neutral CDase, which shows an optimum pH of 6.5-8.5, has been cloned from bacteria (6), Drosophila (7), mouse (8), rat (9), and human (10). Accumulating evidence suggests that neutral CDase regulates the intracellular content of Cer and thereby Cer-mediated signaling.…”

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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

Journal of Biological Chemistry

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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.

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Molecular Cloning and Characterization of a Human Mitochondrial Ceramidase

Cited by 233 publications

References 14 publications

Identification of a novel amidase motif in neutral ceramidase

Identification of a novel amidase motif in neutral ceramidase

Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase

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

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