Cloning and Characterization of the Full-Length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase

Li, Chi-Ming; Hong, Seungbeom; Kopal, Guido; He, Xingxing; Linke, Thomas; Hou, Wu-Shiun; Koch, J.; Gátt, Shimon; Sandhoff, Konrad; Schuchman, Edward H.

doi:10.1006/geno.1998.5334

Cited by 107 publications

(79 citation statements)

References 15 publications

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“…The acidic ceramidase is the enzyme whose inherited deficiency underlies ceramide accumulation in the lysosomal storage disease known as Farber's disease (33). The enzyme was subsequently purified from human urine, cloned from human fibroblasts (11), and recently cloned from mouse tissue (12). It is localized in the lysosome, and it appears to prefer ceramides over dihydroceramides (11).…”

Section: Discussionmentioning

confidence: 99%

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Cloning of an Alkaline Ceramidase from Saccharomyces cerevisiae

Mao¹,

Xu²,

Bielawska³

et al. 2000

Journal of Biological Chemistry

165

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Ceramide is not only a core intermediate of sphingolipids but also an important modulator of many cellular events including apoptosis, cell cycle arrest, senescence, differentiation, and stress responses. Its turnover may be tightly regulated. However, little is known about the regulation of its metabolism because most enzymes responsible for its synthesis and breakdown have yet to be cloned. Here we report the cloning and characterization of the yeast gene YPC1 (YBR183w) by screening Saccharomyces cerevisiae genes whose overexpression bestows resistance to fumonisin B1. We demonstrate that the yeast gene YPC1 encodes an alkaline ceramidase activity responsible for the breakdown of dihydroceramide and phytoceramide but not unsaturated ceramide. YPC1 ceramidase activity was confirmed by in vitro studies using an Escherichia coli expression system. Importantly, YPC1p also has reverse activity, catalyzing synthesis of phytoceramide from palmitic acid and phytosphingosine. This ceramide synthase activity is CoAindependent and is resistant to fumonisin B1, thus explaining why YPC1 was cloned as a fumonisin B1-resistant gene.

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Section: Discussionmentioning

confidence: 99%

“…Acidic ceramidase is localized in lysosomes and is responsible primarily for catabolism of ceramide (10). This enzyme has been identified, and its cDNA has been cloned from human (11) and mouse (12). On the other hand neutral and alkaline ceramidases have been implicated in signal transduction and cell regulation (13,14).…”

mentioning

confidence: 99%

Cloning of an Alkaline Ceramidase from Saccharomyces cerevisiae

Mao¹,

Xu²,

Bielawska³

et al. 2000

Journal of Biological Chemistry

165

View full text Add to dashboard Cite

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“…Although no changes in alkaline or neutral ceramidase activity could be detected, we found that acidic ceramidase activity was rapidly and transiently increased in Jurkat cells after Fas ligation, which correlated closely with the time course for sphingosine formation. It seems unlikely that this acidic ceramidase activity is the previously described lysosomal form (74), because fibroblasts from patients with Farber disease, which accumulate ceramide as the result of genetic deficiency of acid ceramidase, do not show defects in apoptosis (75). Moreover, endogenous long chain ceramide cannot exit from lysosomes, making the ceramides produced in the lysosomes unlikely signaling molecules (76).…”

Section: Potential Involvement Of Ceramide and Sphingosine In Fasindumentioning

confidence: 95%

Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells

Cuvillier¹,

Edsall

Spiegel

2000

Journal of Biological Chemistry

167

140

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Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-x L had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C 2 -ceramide, induced cytochrome c release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-x L overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells.

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“…Subsequently, this ceramide can be hydrolyzed by ceramidase (CDase 2 ) to yield a free fatty acid and sphingosine (see Fig. 1 Intracellular CDase is present in eukaryotes and prokaryotes and these enzymes have acid [5][6][7][8][9], neutral [10], or alkaline [11][12][13][14][15][16][17] pH optima. In mammals, CDase plays an important role in the control of cellular ceramide content and in the regulation of intracellular signal transduction.…”

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confidence: 99%

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

Nieuwenhuizen

Leeuwen

Jack

et al. 2003

Protein Expression and Purification

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Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 lg of extracellular alkaline, Ca 2þ -dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The CDase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. CDase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular CDase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated CDase were expressed. High levels of intracellular CDase were expressed by Escherichia coli DH5a and E. coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E. coli DH5a culture, 280 lg of pure CDase was obtained after a three-step purification protocol. Under control of the T7-promotor CDase, without its signal peptide, was produced in inclusion bodies in E. coli BL21 cells. After refolding, 1.8 mg of pure active CDase was obtained from a 2.4-L culture after ammonium sulfate precipitation and gel filtration. Both the recombinant and wild-type CDases have a pH optimum of 8.5. The recombinant enzyme was partially characterized. This is the first report of a high yield CDase production system allowing detailed characterization of the enzyme at the molecular level.

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Cloning and Characterization of the Full-Length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase

Cited by 107 publications

References 15 publications

Cloning of an Alkaline Ceramidase from Saccharomyces cerevisiae

Cloning of an Alkaline Ceramidase from Saccharomyces cerevisiae

Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells

Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01

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