The Reverse Activity of Human Acid Ceramidase

Abstract

An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [ 14 C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was ϳ5.5, as compared with ϳ4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The K m and V max values toward sphingosine were 23.75 M and 208.3 pmol/g/h, respectively, whereas for lauric acid they were 73.76 M and 232.5 pmol/g/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced ϳ30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.

Sphingosine 1-Phosphate Lyase Deficiency Disrupts Lipid Homeostasis in Liver

“…In the recycling pathway, S1P is dephosphorylated by S1P phosphatases, which reside in the endoplasmic reticulum, to produce sphingosine. Ceramide is then synthesized, via acylation of sphingosine, by either ceramide synthases (CerS), encoded by Lass genes (16), or by the reverse ceramide synthase activity intrinsic to ceramidases, encoded by Asah genes (17).…”

The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

“…Ceramide is generated by (i) sphingomyelinases [30, 64, 65], (ii) by a de novo synthase pathway [30, 65], (iii) by a retrograde activity of the enzyme ceramidase that mediates the synthesis of ceramide from sphingosine [66], (iv) by the hydrolysis of complex glycosylated lipids [67] and (v) by hydrolysis of ceramide 1-phosphate [68]. Sphingomyelinases hydrolyse sphingomyelin to release ceramide.…”

Multi-system disorders of glycosphingolipid and ganglioside metabolism

“…Abbreviations: 3-KSR (3-ketosphinganine reductase), Sk (sphingosine kinase), Sa1P (sphinganine 1-phosphate), S1P (sphingosine 1-phosphate), S1-P Pase (sphingosine 1-phosphate phosphatase), (Sa) N -ACT (sphinganine N -acyltransferase), DHCerS (dihydroceramide desaturase), CerS (ceramide synthase, also called longevity assurance genes), SMS (sphingomyelin synthase), GT3 synthase ( ␣ -N-acetyl-neuraminide ␣ -2,8-sialyltransferase). The chemical structures are adapted from ( 3,36,100,(313)(314)(315) and http://www.cybercolloids.net/library/sugars/hexoses.php. Nomenclature of the enzymes and protein are from IUBMB (http://www.chem.qmul.ac.uk/iubmb/enzyme) ( 37,43,315 ).…”