title: Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase: an enzyme that preferentially regulates metabolism of very long chain ceramides.
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 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.
authors.affiliation: Department of Medicine, Medical University of South Carolina, Charleston 29425, USA. maoc@musc.edu
authors.forename: Cungui
authors.initials: C
authors.lastname: Mao
authors.forename: Ruijuan
authors.initials: R
authors.lastname: Xu
authors.forename: Zdzislaw M
authors.initials: ZM
authors.lastname: Szulc
authors.forename: Jacek
authors.initials: J
authors.lastname: Bielawski
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Becker
authors.forename: Alicja
authors.initials: A
authors.lastname: Bielawska
authors.forename: Sehamuddin H
authors.initials: SH
authors.lastname: Galadari
authors.forename: Wei
authors.initials: W
authors.lastname: Hu
authors.forename: Lina M
authors.initials: LM
authors.lastname: Obeid
mesh_terms: D000468:Alkalies; D055574:Alkaline Ceramidase; D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002412:Cations / Q000378:metabolism; D055572:Ceramidases; D002518:Ceramides / Q000378:metabolism*; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008246:Lysophospholipids; D051379:Mice; D008969:Molecular Sequence Data; D012867:Skin / Q000201:enzymology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000468:Alkalies; D002412:Cations; D002518:Ceramides; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D000581:Amidohydrolases; C526750:ACER1 protein, human; C526755:ACER2 protein, human; C526753:Acer1 protein, mouse; D055574:Alkaline Ceramidase; D055572:Ceramidases; D013110:Sphingosine