Cloning and characterization of a novel human alkaline ceramidase - A mammalian enzyme that hydrolyzes phytoceramide

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,, B ielawska, A., Szulc, Z, M,, and Obeid, L, RI. (2000) J. Biol Chem, 275, 313 69-31378), we report the identification and cloning of a cDNA encoding for a novel human alkaline ceramidase (aPHC) that hydrolyzes phytoceramide sele ctively, Northern blot analysis showed that aPHC was ubiquitously expressed , with the highest expression in placenta. Green fluorescent protein taggin g showed that it was localized in both the Golgi apparatus and endoplasmic reticulum. Overexpression of aPHC in mammalian cells elevated in vitro cera midase 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 r estored the ceramidase activity and caused an increase in the hydrolysis of phytoceramide in yeast cells, thus leading to the decreased biosynthesis o f 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 Y PC1p, 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 Ca2+, but was inhibite d by Zn2+ and sphingosine, Substrate specificity showed that aPHC hydrolyze d phytoceramide preferentially, Together, these data demonstrate that aPHC is a novel human alkaline phytoceramidase, the first mammalian alkaline cer amidase to be identified as being specific for the hydrolysis of phytoceram ide.