Cloning and Characterization of a Novel Human Alkaline Ceramidase

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Abstract

Ceramidases are enzymes involved in regulating cellular levels of ceramides, sphingoid bases, and their phosphates. Based on sequence homology to the yeast alkaline ceramidases YPC1p (Mao, C., Xu, R., Bielawska, A., and Obeid, L. M. (2000) J. Biol. Chem. 275, 6876--6884) and YDC1p (Mao, C., Xu, R., Bielawska, A., Szulc, Z. M., and Obeid, L. M. (2000) J. Biol Chem. 275, 31369--31378), we report the identification and cloning of a cDNA encoding for a novel human alkaline ceramidase (aPHC) that hydrolyzes phytoceramide selectively. Northern blot analysis showed that aPHC was ubiquitously expressed, with the highest expression in placenta. Green fluorescent protein tagging showed that it was localized in both the Golgi apparatus and endoplasmic reticulum. Overexpression of aPHC in mammalian cells elevated in vitro ceramidase activity toward N-4-nitrobenz-2-oxa-1,3-diazole-C(12)-phytoceramide. Its expression in a yeast mutant strain devoid of any ceramidase activity restored the ceramidase activity and caused an increase in the hydrolysis of phytoceramide in yeast cells, thus leading to the decreased biosynthesis of sphingolipids. These data collectively suggest that, similar to the yeast phytoceramidase YPC1p, aPHC has phytoceramidase activity both in vitro and in cells; hence, it is a functional homolog of the yeast phytoceramidase YPC1p. However, in contrast to YPC1p, aPHC exhibited no reverse activity of ceramidase either in vitro or in cells. Biochemical characterization showed that aPHC had a pH optimum of 9.5, was activated by Ca(2+), but was inhibited by Zn(2+) and sphingosine. Substrate specificity showed that aPHC hydrolyzed phytoceramide preferentially. Together, these data demonstrate that aPHC is a novel human alkaline phytoceramidase, the first mammalian alkaline ceramidase to be identified as being specific for the hydrolysis of phytoceramide.

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