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J. Biol. Chem., Vol. 278, Issue 33, 31184-31191, August 15, 2003

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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. Becker ¶, Alicja Bielawska ¶, Sehamuddin H. Galadari ||, Wei Hu  ¶ and Lina M. Obeid  ¶ ** 

From the Departments of Medicine and ^¶Biochemistry and Molecular Biology and ^**Ralph H. Johnson Veterans Administration Hospital, Medical University of South Carolina, Charleston, South Carolina, 29425 and ^||Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates

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-C_24:1-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.

Received for publication, April 14, 2003 , and in revised form, May 29, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF347023.

^* This work was supported in part by a Veterans Affairs merit award and by National Institutes of Health Grants GM62887-01 and 1P20RR017677-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence may be addressed: Division of General Internal Medicine, 114 Doughty St., Rm. 605 STB, P. O. Box 250779, Charleston, SC 29425. Tel.: 843-876-5173; Fax: 843-876-5172; E-mail: maoc{at}musc.edu.

To whom correspondence may be addressed: Division of General Internal Medicine, 114 Doughty St., Rm. 605 STB, P. O. Box 250779, Charleston, SC 29425. Tel.: 843-876-5173; Fax: 843-876-5172; E-mail: obeidl{at}musc.edu.

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