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

Cungui Mao, Ruijuan Xu, Zdzislaw M. Szulc, Jacek Bielawski, Kevin P. Beeker, Alicja Bielawska, Sehamuddin Galadari, Wei Hu, Lina M. Obeid

    Research output: Contribution to journalArticle

    Abstract

    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 Mn 2+. 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.

    Original languageEnglish (US)
    Pages (from-to)31184-31191
    Number of pages8
    JournalJournal of Biological Chemistry
    Volume278
    Issue number33
    DOIs
    StatePublished - Aug 15 2003

    Fingerprint

    Alkaline Ceramidase
    Ceramides
    Cloning
    Metabolism
    Endoplasmic Reticulum
    Organism Cloning
    Sphingosine
    Sphingolipids
    Enzymes
    Substrate Specificity
    Skin
    Ceramidases
    Saccharomyces
    RNA-Directed DNA Polymerase
    Substrates
    Metabolic Networks and Pathways
    Reverse Transcriptase Polymerase Chain Reaction
    HeLa Cells
    Labeling
    Immunohistochemistry

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase. An enzyme that preferentially regulates metabolism of very long chain ceramides. / Mao, Cungui; Xu, Ruijuan; Szulc, Zdzislaw M.; Bielawski, Jacek; Beeker, Kevin P.; Bielawska, Alicja; Galadari, Sehamuddin; Hu, Wei; Obeid, Lina M.

    In: Journal of Biological Chemistry, Vol. 278, No. 33, 15.08.2003, p. 31184-31191.

    Research output: Contribution to journalArticle

    Mao, Cungui ; Xu, Ruijuan ; Szulc, Zdzislaw M. ; Bielawski, Jacek ; Beeker, Kevin P. ; Bielawska, Alicja ; Galadari, Sehamuddin ; Hu, Wei ; Obeid, Lina M. / Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase. An enzyme that preferentially regulates metabolism of very long chain ceramides. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 33. pp. 31184-31191.
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    abstract = "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 Mn 2+. 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.",
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    AU - Xu, Ruijuan

    AU - Szulc, Zdzislaw M.

    AU - Bielawski, Jacek

    AU - Beeker, Kevin P.

    AU - Bielawska, Alicja

    AU - Galadari, Sehamuddin

    AU - Hu, Wei

    AU - Obeid, Lina M.

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    AB - 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 Mn 2+. 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.

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