Publications

• Purification and characterization of human intestinal neutral ceramidase.

  Ohlsson L., Palmberg C., Duan R.D., Olsson M., Bergman T., Nilsson A.

  Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of ... >> More

  Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[(14)C]sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu(2+) and Zn(2+) ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut. << Less

  Biochimie 89:950-960(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Role of alkaline ceramidases in the generation of sphingosine and its phosphate in erythrocytes.

  Xu R., Sun W., Jin J., Obeid L.M., Mao C.

  Plasma sphingosine-1-phosphate (S1P) has been suggested to mainly originate from erythrocytes; however, within the erythrocyte, how sphingosine (SPH) generation--the precursor to S1P--is controlled is unknown. SPH is only generated from the hydrolysis of ceramides via ceramidases. Five human ceram ... >> More

  Plasma sphingosine-1-phosphate (S1P) has been suggested to mainly originate from erythrocytes; however, within the erythrocyte, how sphingosine (SPH) generation--the precursor to S1P--is controlled is unknown. SPH is only generated from the hydrolysis of ceramides via ceramidases. Five human ceramidases have been identified: 1 acid, 1 neutral, and 3 alkaline ceramidases (ACER1, ACER2, and ACER3). Here, we demonstrate that only alkaline ceramidase activity is expressed in erythrocytes and that it is instrumental for SPH generation. Erythrocytes have alkaline but not acid or neutral ceramidase activity on D-e-C(18:1)-ceramide, a common substrate of ceramidases. Not only alkaline ceramidase activity but also the generation of SPH and S1P are increased during erythroid differentiation in K562 erythroleukemic cells. Such SPH and S1P increases were inhibited by the alkaline ceramidase inhibitor D-e-MAPP, suggesting that alkaline ceramidases have a role in the generation of SPH and S1P in erythroid cells. Alkaline ceramidase activity is highly expressed in mouse erythrocytes, and intravenous administration of D-e-MAPP decreased both SPH and S1P in erythrocytes and plasma. Collectively, these results suggest that alkaline ceramidase activity is important for the generation of SPH, the S1P precursor in erythrocytes. << Less

  FASEB J. 24:2507-2515(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The Arabidopsis alkaline ceramidase TOD1 is a key turgor pressure regulator in plant cells.

  Chen L.-Y., Shi D.-Q., Zhang W.-J., Tang Z.-S., Liu J., Yang W.-C.

  Turgor pressure plays pivotal roles in the growth and movement of walled cells that make up plants and fungi. However, the molecular mechanisms regulating turgor pressure and the coordination between turgor pressure and cell wall remodelling for cell growth remain poorly understood. Here, we repor ... >> More

  Turgor pressure plays pivotal roles in the growth and movement of walled cells that make up plants and fungi. However, the molecular mechanisms regulating turgor pressure and the coordination between turgor pressure and cell wall remodelling for cell growth remain poorly understood. Here, we report the characterization of Arabidopsis TurgOr regulation Defect 1 (TOD1), which is preferentially expressed in pollen tubes and silique guard cells. We demonstrate that TOD1 is a Golgi-localized alkaline ceramidase. tod1 mutant pollen tubes have higher turgor than wild type and show growth retardation both in pistils and in agarose medium. In addition, tod1 guard cells are insensitive to abscisic acid (ABA)-induced stomatal closure, whereas sphingosine-1-phosphate, a putative downstream component of ABA signalling and product of alkaline ceramidases, promotes closure in both wild type and tod1. Our data suggest that TOD1 acts in turgor pressure regulation in both guard cells and pollen tubes. << Less

  Nat. Commun. 6:6030-6030(2015) [PubMed] [EuropePMC]

• Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase: an enzyme that preferentially regulates metabolism of very long chain ceramides.

  Mao C., Xu R., Szulc Z.M., Bielawski J., Becker K.P., Bielawska A., Galadari S.H., Hu W., Obeid L.M.

  Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids. In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity. maCER1 consists of 287 amino acids, and it has ... >> More

  Ceramidases deacylate ceramides, important intermediates in the metabolic pathway of sphingolipids. In this study, we report the cloning and characterization of a novel mouse alkaline ceramidase (maCER1) with a highly restricted substrate specificity. maCER1 consists of 287 amino acids, and it has a 28 and 32% identity to the Saccharomyces alkaline ceramidases (YPC1p and YDC1p) and the human alkaline phytoceramidase, respectively. Reverse transcriptase-PCR analysis demonstrated that maCER1 was predominantly expressed in skin. maCER1 was localized to the endoplasmic reticulum as revealed by immunocytochemistry. In vitro biochemical characterization determined that maCER1 hydrolyzed D-erythro-ceramide exclusively but not D-erythro-dihydroceramide or D-ribo-phytoceramide. Similar to other alkaline ceramidases, maCER1 had an alkaline pH optimum of 8.0, and it was activated by Ca2+ but inhibited by Zn2+,Cu2+, and Mn2+. maCER1 was also inhibited by sphingosine, one of its products. Metabolic labeling studies showed that overexpression of maCER1 caused a decrease in the incorporation of radiolabeled dihydrosphingosine into ceramide and complex sphingolipids but led to a concomitant increase in sphingosine-1-P (S1P) in HeLa cells. Mass measurement showed that overexpression of maCER1 selectively lowered the cellular levels of D-erythro-C24:1-ceramide, but not other ceramide species and caused an increase in the levels of S1P. Taken together, these data suggest that maCER1 is a novel alkaline ceramidase with a stringent substrate specificity and that maCER1 is selectively expressed in skin and may have a role in regulating the levels of bioactive lipids ceramide and S1P, as well as complex sphingolipids. << Less

  J. Biol. Chem. 278:31184-31191(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 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.