Publications

• Purification, characterization, and biosynthesis of human acid ceramidase.

  Bernardo K., Hurwitz R., Zenk T., Desnick R.J., Ferlinz K., Schuchman E.H., Sandhoff K.

  Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty acid. Its inherited deficiency causes ceramide accumulation in Farber's disease. The enzyme was purified to apparent homogeneity from human urine b ... >> More

  Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty acid. Its inherited deficiency causes ceramide accumulation in Farber's disease. The enzyme was purified to apparent homogeneity from human urine by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue-Sepharose, and DEAE-cellulose. The final preparation, which was enriched approximately 4450-fold over the starting material, resulted in a polypeptide of approximately 50 kDa and could be reduced into two subunits of approximately 13 (alpha) and approximately 40 (beta) kDa. Treatment of the purified enzyme with endoglycosidase H or peptido-N-glycanase F reduced the molecular mass of the beta subunit to approximately 30-35 and approximately 27 kDa, respectively. In contrast, the molecular mass of the alpha subunit was unchanged. The purified enzyme had an apparent Km of 149 microM and a Vmax of 136 nmol/mg/h using N-lauroylsphingosine as substrate. Polyclonal antibodies were raised against the purified urinary enzyme and used to investigate the biosynthesis of acid ceramidase. Immunoprecipitation studies on metabolically labeled skin fibroblasts indicated that both subunits arose from a single precursor of approximately 55 kDa. A minor portion of newly synthesized acid ceramidase was secreted into the medium as a monomeric 47-kDa protein, indicating that generation of the mature heterodimeric enzyme occurred in endosomal and/or lysosomal compartments. << Less

  J. Biol. Chem. 270:11098-11102(1995) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Substrate specificity, membrane topology, and activity regulation of human alkaline ceramidase 2 (ACER2).

  Sun W., Jin J., Xu R., Hu W., Szulc Z.M., Bielawski J., Obeid L.M., Mao C.

  Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltayd ... >> More

  Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation. << Less

  J. Biol. Chem. 285:8995-9007(2010) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase.

  El Bawab S., Birbes H., Roddy P., Szulc Z.M., Bielawska A., Hannun Y.A.

  We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this stu ... >> More

  We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid. << Less

  J. Biol. Chem. 276:16758-16766(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Expression, purification, and characterization of a recombinant neutral ceramidase from Mycobacterium tuberculosis.

  Okino N., Ikeda R., Ito M.

  Ceramidase (CDase) catalyzes the hydrolysis of ceramide (Cer) to sphingosine (Sph) and fatty acid. We have reported the molecular cloning and preliminary characterization of the Mycobacterium CDase (MtCDase) (J. Biol. Chem., 274, 36616-36622 (1999)). To determine its function further, MtCDase was ... >> More

  Ceramidase (CDase) catalyzes the hydrolysis of ceramide (Cer) to sphingosine (Sph) and fatty acid. We have reported the molecular cloning and preliminary characterization of the Mycobacterium CDase (MtCDase) (J. Biol. Chem., 274, 36616-36622 (1999)). To determine its function further, MtCDase was expressed in Escherichia coli and purified by Ni-Sepharose and gelfiltration. The purified recombinant enzyme showed a single band and a molecular weight estimated to be 71 kDa on SDS-PAGE. It had a pH optimum at 8.0-9.0 and quite broad specificity for various Cers. Of the Cers of different fatty acid moieties tested, those composed of C6-C24 fatty acids were well hydrolyzed, and Cers with mono unsaturated fatty acids were much more hydrolyzed than those with saturated fatty acids. Using N-dodecanoyl-7-nitrobenz-2-oxa-1,3-4-diazole (NBD)-D-erythro-sphingosine (C12-NBD-Cer) as substrates, the reaction followed normal Michaelis-Menten kinetics. The apparent Km and Vmax values for C12-NBD-Cer were 98.7 muM and 21.1 pmol/min respectively. The purified enzyme also catalyzed the synthesis of Cer in vitro, using NBD-labeled dodecanoic acid and Sph as substrates. << Less

  Biosci. Biotechnol. Biochem. 74:316-321(2010) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• Alkaline ceramidase 3 (ACER3) hydrolyzes unsaturated long-chain ceramides, and its down-regulation inhibits both cell proliferation and apoptosis.

  Hu W., Xu R., Sun W., Szulc Z.M., Bielawski J., Obeid L.M., Mao C.

  Ceramides with different fatty acyl chains may vary in their physiological or pathological roles; however, it remains unclear how cellular levels of individual ceramide species are regulated. Here, we demonstrate that our previously cloned human alkaline ceramidase 3 (ACER3) specifically controls ... >> More

  Ceramides with different fatty acyl chains may vary in their physiological or pathological roles; however, it remains unclear how cellular levels of individual ceramide species are regulated. Here, we demonstrate that our previously cloned human alkaline ceramidase 3 (ACER3) specifically controls the hydrolysis of ceramides carrying unsaturated long acyl chains, unsaturated long-chain (ULC) ceramides. In vitro, ACER3 only hydrolyzed C(18:1)-, C(20:1)-, C(20:4)-ceramides, dihydroceramides, and phytoceramides. In cells, ACER3 overexpression decreased C(18:1)- and C(20:1)-ceramides and dihydroceramides, whereas ACER3 knockdown by RNA interference had the opposite effect, suggesting that ACER3 controls the catabolism of ULC ceramides and dihydroceramides. ACER3 knockdown inhibited cell proliferation and up-regulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1). Blocking p21(CIP1/WAF1) up-regulation attenuated the inhibitory effect of ACER3 knockdown on cell proliferation, suggesting that ACER3 knockdown inhibits cell proliferation because of p21(CIP1/WAF1) up-regulation. ACER3 knockdown inhibited cell apoptosis in response to serum deprivation. ACER3 knockdown up-regulated the expression of the alkaline ceramidase 2 (ACER2), and the ACER2 up-regulation decreased non-ULC ceramide species while increasing both sphingosine and its phosphate. Collectively, these data suggest that ACER3 catalyzes the hydrolysis of ULC ceramides and dihydroceramides and that ACER3 coordinates with ACER2 to regulate cell proliferation and survival. << Less

  J. Biol. Chem. 285:7964-7976(2010) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.