Cg. Mao et al., Cloning and characterization of a novel human alkaline ceramidase - A mammalian enzyme that hydrolyzes phytoceramide, J BIOL CHEM, 276(28), 2001, pp. 26577-26588
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.