Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines.

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.

Mass Spectrometric Analysis of Sphingomyelin with N-α-Hydroxy Fatty Acyl Residue in Mouse Tissues.

Sphingomyelin (SM) with N-α-hydroxy fatty acyl residues (hSM) has been shown to occur in mammalian skin and digestive epithelia. However, the metabolism and physiological relevance of this characteristic SM species have not been fully elucidated yet. Here, we show methods for mass spectrometric characterization and quantification of hSM. The hSM in mouse skin was isolated by TLC. The hydroxy hexadecanoyl residue was confirmed by electron impact ionization-induced fragmentation in gas chromatography-mass spectrometry. Mass shift analysis of acetylated hSM by time of flight mass spectrometry revealed the number of hydroxyl groups in the molecule. After correcting the difference in detection efficacy, hSM in mouse skin and intestinal mucosa were quantified by liquid chromatography-tandem mass spectrometry, and found to be 16.5 ± 2.0 and 0.8 ± 0.4 nmol/µmol phospholipid, respectively. The methods described here are applicable to biological experiments on hSM in epithelia of the body surface and digestive tract.

A Cleanup Method for Mass Spectrometric Analysis of Sphingosine- and Ceramide-1-Phosphate in Blood and Solid Tissue Using a Phosphate Capture Molecule.

Cleanup technology and mass spectrometric determination of sphingosine-1-phosphate (S1P) using a phosphate capture molecule are shown. The protocol is rapid, requires neither thin-layer chromatography nor liquid chromatography, and is applicable to both blood and solid tissue samples. The mass spectrometric method is also applicable to ceramide-1-phosphate.

Calcium-dependent generation of N-acylethanolamines and lysophosphatidic acids by glycerophosphodiesterase GDE7.

N-Acylethanolamines form a class of lipid mediators and include an endocannabinoid arachidonoylethanolamide (anandamide), analgesic and anti-inflammatory palmitoylethanolamide, and appetite-suppressing oleoylethanolamide. In animal tissues, N-acylethanolamines are synthesized from N-acylated ethanolamine phospholipids directly by N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or through multi-step pathways via N-acylethanolamine lysophospholipids. We previously reported that glycerophosphodiesterase (GDE) 4, a member of the GDE family, has lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines. Recently, GDE7 was shown to have lysoPLD activity toward lysophosphatidylcholine to produce lysophosphatidic acid (LPA). Here, we examined the reactivity of GDE7 with N-acylethanolamine lysophospholipids as well as the requirement of divalent cations for its catalytic activity. When overexpressed in HEK293 cells, recombinant GDE7 proteins of human and mouse showed lysoPLD activity toward N-palmitoyl, N-oleoyl, and N-arachidonoyl-lysophosphatidylethanolamines and N-palmitoyl-lysoplasmenylethanolamine to generate their corresponding N-acylethanolamines and LPAs. However, GDE7 hardly hydrolyzed glycerophospho-N-palmitoylethanolamine. Overexpression of GDE7 in HEK293 cells increased endogenous levels of N-acylethanolamines and LPAs. Interestingly, GDE7 was stimulated by micromolar concentrations of Ca2+ but not by millimolar concentrations of Mg2+, while GDE4 was stimulated by Mg2+ but was insensitive to Ca2+. GDE7 was widely distributed in various tissues of humans and mice with the highest levels in their kidney tissues. These results suggested that GDE7 is a novel Ca2+-dependent lysoPLD, which is involved in the generation of both N-acylethanolamines and LPAs.

Analysis of Molecular Species Profiles of Ceramide-1-phosphate and Sphingomyelin Using MALDI-TOF Mass Spectrometry.

Ceramide-1-phosphate (C1P) is a potential signaling molecule that modulates various cellular functions in animals. It has been known that C1P with different N-acyl lengths induce biological responses differently. However, molecular species profiles of the C1P in animal tissues have not been extensively examined yet. Here, we developed a method for determination of the molecular species of a C1P using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with Phos-tag, a phosphate capture molecule. The amounts of total C1P in skin, brain, liver, kidney and small intestine of mice were determined to be 344, 151, 198, 96 and 90 pmol/g wet weight, respectively. We found a C1P species having an α-hydroxypalmitoyl residue (h-C1P, 44 pmol/g wet weight) in mouse skin. The h-C1P was detected only in the skin, and not other tissues of mice. The same analysis was applied to sphingomyelin after conversion of sphingomyelin to C1P by Streptomyces chromofuscus phospholipase D. We found that molecular species profiles of sphingomyelin in skin, kidney and small intestine of mice were similar to those of C1P in corresponding tissues. In contrast, molecular species profiles of sphingomyelin in liver and brain were quite different from those of C1P in these tissues, indicating selective synthesis or degradation of C1P in these tissues. The method described here will be useful for detection of changes in molecular species profiles of C1P and sphingomyelin.

Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves.

The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage (Brassica oleracea) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI-TOF MS as an adduct with Phos-tag, a phosphate-capture zinc complex. Various α-hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC-MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4-hydroxysphingenine (dehydrophytosphingosine)-1-phosphate by MALDI-TOF MS with Phos-tag. From these results, lipid X was determined to be phytoceramide-1-phosphate (PC1P) with an α-hydroxy fatty acid. When cabbage homogenates were incubated, PC1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (GIPC). The formation of PC1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin. Arabidopsis thaliana also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC1P in brassicaceous plants.