Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase

Yoshimura, Yukihiro; Tani, Motohiro; Okino, Nozomu; Iida, Hiroshi; Ito, Makoto

doi:10.1074/jbc.m405598200

Cited by 47 publications

(34 citation statements)

References 34 publications

(47 reference statements)

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“…Moreover, targeted expression of neutral CDase rescued Drosophila mutants from retinal degeneration [24]. In zebrafish (Danio rerio), neutral CDase is important in embryo development [25].…”

Section: Introductionmentioning

confidence: 99%

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: Introductionmentioning

confidence: 99%

Identification of a novel amidase motif in neutral ceramidase

Galadari

Mao

et al. 2006

Biochemical Journal

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“…Orthologous genes have been identified in human (12), rat (13), zebrafish (14), Drosophila (15), and bacteria (16). The neutral ceramidase has been identified as a type II integral membrane protein that can be cleaved to yield a soluble secreted protein (17).…”

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confidence: 99%

“…The enzyme has also been demonstrated in the apical membranes of proximal and distal tubules, collecting ducts of kidney, endosome-like organelles of hepatocytes (13), and in the epithelia of the jejunum and ileum (11,18), suggesting possible diverse physiologic functions. In zebrafish, the expression of the enzyme was found to be critical for early development (14). To identify the physiologic functions of this neutral ceramidase in mice, we inactivated the Asah2 gene by homologous recombination.…”

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confidence: 99%

Neutral Ceramidase Encoded by the Asah2 Gene Is Essential for the Intestinal Degradation of Sphingolipids

Kono

Dreier

Ellis

et al. 2006

Journal of Biological Chemistry

146

134

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Complex sphingolipids are abundant as eukaryotic cell membrane components, whereas their metabolites, in particular ceramide, sphingosine, and sphingosine 1-phosphate, are involved in diverse cell signaling processes. In mammals, degradation of ceramide by ceramidase yields sphingosine, which is phosphorylated by the action of sphingosine kinase to generate sphingosine 1-phosphate. Therefore, ceramidases are key enzymes in the regulation of the cellular levels of ceramide, sphingosine, and sphingosine 1-phosphate. To explore the physiological functions of a neutral ceramidase with diverse cellular locations, we disrupted the Asah2 gene in mice. Asah2 null mice have a normal life span and do not show obvious abnormalities or major alterations in total ceramide levels in tissues. The Asah2-encoded neutral ceramidase is highly expressed in the small intestine along the brush border, suggesting that the neutral ceramidase may be involved in a pathway for the digestion of dietary sphingolipids. Indeed, Asah2 null mice were deficient in the intestinal degradation of ceramide. Thus, the results indicate that the Asah2-encoded neutral ceramidase is a key enzyme for the catabolism of dietary sphingolipids and regulates the levels of bioactive sphingolipid metabolites in the intestinal tract.Sphingolipids have diverse functional roles (1, 2). Complex sphingolipids such as sphingomyelin and glycosphingolipids are abundant plasma membrane constituents in plant and animal cells. Here they function as organizers of membrane structure, regulators of cell signaling, and mediators of cell-cell interactions. Simple sphingolipid metabolites that can be derived by the degradation of the complex sphingolipids, including ceramide, sphingosine, and sphingosine 1-phosphate, fulfill a different role as signaling molecules. These simple sphingolipids utilize both intracellular and extracellular targets and are potent regulators of cell growth and survival.In the sphingolipid degradative pathway, ceramidases (EC 3.5.1.23) catalyze the hydrolysis of the N-acyl group of ceramide to yield sphingosine and fatty acids. Sphingosine can then be phosphorylated by sphingosine kinase to yield sphingosine 1-phosphate. Thus, ceramidases are key regulators of sphingolipid signaling metabolites. Ceramidases have traditionally been classified according to the pH range (acid, neutral, or alkaline) that supports their optimal activity. Recently, the mammalian ceramidases have been defined more precisely by genetic and structural means, which has revealed at least four distinct enzymes.

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“…Based on optimal pH and primary structure, CDases are classified into three groups: acid, neutral, and alkaline enzymes (3). Because we first purified and cloned a neutral CDase from Pseudomonas aeruginosa (4,5), neutral CDases have been cloned from slime mold (6), Drosophila (7), zebrafish (8), rat (9), mouse (10), and human (11,12). Interestingly, bacterial CDases cloned from P. aeruginosa, Mycobacterium tuberculosis (5), and Dermatophilus congolensis (13), are classified as neutral CDases based on primary structure.…”

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confidence: 99%

Ceramidase Enhances Phospholipase C-induced Hemolysis by Pseudomonas aeruginosa

Okino

Ito

2007

Journal of Biological Chemistry

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We previously reported the purification, molecular cloning, and characterization of a neutral ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368 -14373; Okino, N., Ichinose, S., Omori, A., Imayama, S., Nakamura, T., and Ito, M. (1999) J. Biol. Chem. 274, 36616 -36622). Interestingly, the gene encoding the enzyme is adjacent to that encoding hemolytic phospholipase C (plcH) in the genome of Pseudomonas aeruginosa, which is a well known pathogen for opportunistic infections. We report here that simultaneous production of PlcH and ceramidase was induced by several lipids and PlcH-induced hemolysis was significantly enhanced by the action of the ceramidase. When the strain was cultured with sphingomyelin or phosphatidylcholine, production of both enzymes drastically increased, causing the increase of hemolytic activity in the cellfree culture supernatant. Ceramide and sphingosine were also effective in promoting the production of ceramidase but not that of PlcH. Furthermore, we found that the hemolytic activity of a Bacillus cereus sphingomyelinase was significantly enhanced by addition of a recombinant Pseudomonas ceramidase. TLC analysis of the erythrocytes showed that ceramide produced from sphingomyelin by the sphingomyelinase was partly converted to sphingosine by the ceramidase. A ceramidase-null mutant strain caused much less hemolysis of sheep erythrocytes than did the wild-type strain. Sphingosine was detected in the erythrocytes co-cultured with the wild-type strain but not the mutant strain. Finally, we found that the enhancement of PlcHinduced hemolysis by the ceramidase occurred in not only sheep but also human erythrocytes. These results may indicate that the ceramidase enhances the PlcH-induced cytotoxicity and provide new insights into the role of sphingolipid-degrading enzymes in the pathogenicity of P. aeruginosa.

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Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase

Cited by 47 publications

References 34 publications

Identification of a novel amidase motif in neutral ceramidase

Identification of a novel amidase motif in neutral ceramidase

Neutral Ceramidase Encoded by the Asah2 Gene Is Essential for the Intestinal Degradation of Sphingolipids

Ceramidase Enhances Phospholipase C-induced Hemolysis by Pseudomonas aeruginosa

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