methods A rapid ceramide synthase activity using NBD- sphinganine and solid phase extraction † † 1, Rotem Tidhar , * Kacee Sims , Eden Rosenfeld-Gur , * Walter Shaw , and Anthony H. Futerman * Department of Biological Chemistry,* Weizmann Institute of Science , Rehovot 76100, Israel ; and Avanti Polar Lipids Inc. , † Alabaster, AL 35007-9105 Abstract Ceramides are synthesized by six mammalian ce- For many years, CerS activity was assayed using radioac- ramide synthases (CerSs), each of which uses fatty acyl- tive substrates [either 3 H-Sph ( 7 ) or 14 C-labeled acyl-CoAs CoAs of different chain lengths for N -acylation of the ( 20 )], with separation of the substrate and products per- sphingoid long-chain base. We now describe a rapid and reli- formed by TLC. Recently, a fl uorescent Sph analog has able CerS assay that uses a fl uorescent N-[6-[(7-nitrobenzo- become available, ␻ (7-nitro-2-1,3-benzoxadiazol-4-yl) 2-oxa-1,3-diazol-4-yl) (NBD ) sphinganine substrate followed by separation of the NBD-lipid substrate and products (2 S ,3 R )-2-aminooctadecane-1,3-diol (NBD-Sph), which is using solid phase extraction (SPE) C18 chromatography. a substrate for CerS ( 18, 21 ). Clearly, the use of fl uores- SPE chromatography is a quick and reliable alternative cent substrates is preferable to use of radioactive substrates to TLC, and moreover, there is no degradation of either as it avoids radiation hazards ( 21 ). NBD-sphinganine or NBD-ceramide. We have optimized the We now describe an assay using NBD-Sph, in which the assay for use with minimal amounts of protein in a minimal fl uorescent lipid products are separated by solid phase ex- volume. This assay will prove useful for the analysis of traction (SPE) C18 chromatography using a 96-well plate. CerS activity, which is of particular importance in light of This assay has a number of advantages over other assays, the growing involvement of CerS in cell regulation and in the pathology of human diseases. —Tidhar, R., K. Sims, E. including short assay times, the use of small reaction vol- Rosenfeld-Gur, W. Shaw, and A. H. Futerman. A rapid umes (20 µl) and small amounts of protein. It alleviates ceramide synthase activity using NBD-sphinganine and solid the use of TLC for lipid separation, which often results in phase extraction. J. Lipid Res . 2015. 56: 193–199. degradation of Sph on the TLC plate. We suggest that this assay will permit the rapid assay of CerS activity in large Supplementary key words c eramides • sphingolipids • in vitro assay • numbers of samples and will prove particularly useful for chromatography • enzymology • long-chain base • fatty acyl-CoA • laboratories that do not have access to facilities available in N -acylation more lipid-oriented laboratories. Ceramide, the backbone of all sphingolipids ( 1 ), is synthesized by the N -acylation of sphingoid long-chain MATERIALS AND METHODS bases by ceramide synthases (CerSs). Mammals contain six distinct CerSs, with each using a subset of fatty acyl- Materials CoAs for the N -acylation of sphinganine (Sph) to gener- Methanol gradient grade for liquid chromatography, water ate dihydroceramides or of sphingosine to generate for chromatography, and silica gel 60 TLC plates were from ceramides ( 2, 3 ). CerSs have become the focus of great Merck (Darmstadt, Germany). Formic acid (purity >98%) was interest due to the realization that ceramides with differ- from Sigma-Aldrich (St. Louis, MO). Chloroform for spectro- ent acyl chain lengths play distinct roles in cell physiol- photometry was from J. T. Baker (Center Valley, PA). Ammo- ogy ( 4–6 ), with studies focusing on biochemical analysis nium acetate was from Macron Chemicals (Center Valley, PA). Strata end-capped silica-based C18-E (15 mg/well) 96-well of the mode of CerS regulation (7–13 ), on biological ap- plates and the vacuum manifold for SPE with vacuum gauge proaches using CerS-null mice ( 4, 6, 14 ), and on patho- and cover mat were from Phenomenex (Torrance, CA). The physiological studies using tissues from patients in which vacuum pump was an Alcatel 2002 from Ideal Vacuum Products CerS activity changes during the development and pro- (Albuquerque, NM). gression of pathology ( 15–19 ). Abbreviations: CerS, ceramide synthase; NBD-C18-ceramide, A. H. Futerman is the Joseph Meyerhoff Professor of Biochemistry at the Weizmann ␻ Institute of Science . (7-nitro-2-1,3-benzoxadiazol-4-yl)- N -stearoyl- D - erythro -sphingosine; NBD-sphinganine, ␻ (7-nitro-2-1,3-benzoxadiazol-4-yl)(2 S ,3 R )-2- Manuscript received 15 June 2014 and in revised form 3 November 2014. aminooctadecane-1,3-diol; Sph, sphinganine; SPE, solid phase extraction . 1 Published, JLR Papers in Press, November 3, 2014 To whom correspondence should be addressed. DOI 10.1194/jlr.D052001 e-mail: [email protected] Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 56, 2015 193 Cell culture, transfection, and preparation of cell homogenates HEK 293T cells were cultured in Dulbecco’s modifi ed Eagle’s medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 110 µg /ml sodium py- ruvate. Transfection was performed with the polyethylenimine reagent (Sigma-Aldrich) using 10 µg of plasmid per 10 cm cul- ture dish. Thirty-six to 48 h after transfection, cells were removed from culture dishes and washed twice with phosphate-buffered saline. Cell homogenates were prepared in 20 mM HEPES-KOH, pH 7.2, 25 mM KCl, 250 mM sucrose, and 2 mM MgCl 2 contain- ing a protease inhibitor cocktail (Sigma-Aldrich). Protein was determined using the Bradford reagent (Bio-Rad, Hercules, CA). CerS assay and lipid separation by TLC Cell homogenates were incubated at 37°C with 15 µM NBD- Sph, 20 µM defatted BSA, and 50 µM acyl-CoA, based on condi- tions used previously with [4,5- 3 H]Sph as substrate ( 22 ), but using a 20 ␮ l reaction volume. Reactions were terminated by the addition of chloroform-methanol (1:2, v/v), and lipids were ex- tracted ( 23 ). Lipids were then dried under N2 , resuspended in chloroform-methanol (9:1, v/v), and separated by TLC using chloroform-methanol-2M NH4OH (40:10:1, v/v/v) as the devel- oping solvent. NBD-labeled lipids were visualized using a Typhoon 9410 variable mode imager and quantifi ed by ImageQuantTL (GE Healthcare, Chalfont St. Giles, UK). NBD-Sph stability NBD-Sph (400 µl from a stock solution of 3.7 mg/ml) was separated by preparative TLC using chloroform-methanol-water (65:25:4, v/v/v) as the developing solvent. Bands were excised from the TLC plate and extracted twice using methanol-chloroform (75:25, v/v). NBD-lipids were analyzed using negative ion mode electrospray tandem mass spectrometry with an ABI 4000 QTrap mass spectrometer and an Agilent 1100 series HPLC. A Fig. 1. Structure of NBD-lipids and optimization of CerS assay 5 µl injection volume and a mobile phase consisting of methanol using NBD-Sph. A: Structure of NBD-lipids. B: Homogenates from containing 1% formic acid were used for the analysis. NBD-Sph cells overexpressing CerS5 or CerS4 were assayed in a 20 ␮ l reaction and ␻ (7-nitro-2-1,3-benzoxadiazol-4-yl)-N -stearoyl- D - erythro - volume with increasing amounts of NBD-Sph with 50 ␮ M C16-CoA sphingosine (NBD-C18-ceramide) were also analyzed before and or C20-CoA, respectively, and 20 ␮ M defatted BSA, at 37°C. Results after elution from the SPE columns. are means ± SEM of two to three independent experiments. for CerS4. Both these results compare favorably with Km 3 RESULTS AND DISCUSSION values obtained previously using [4,5- H]Sph ( 22 ) and NBD-Sph ( 21 ) (which were obtained using higher amounts Optimizing conditions for use of NBD-Sph in of protein and/or longer reaction times). The need for the CerS assay lower amounts of protein could be particularly useful While NBD-Sph ( Fig. 1 ) has been used previously to as- when only small amounts of tissue or cells are available, say CerS ( 17, 18, 21 ), no attempts have been made to mini- such as might be the case when using clinical samples. mize reaction volumes to allow the use of small amounts of Interestingly, whereas NBD-Sph was a good substrate biological material or to improve the mode of separating for CerS, { N -[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] NBD-lipid substrates and products. We have now been amino} palmitoyl coenzyme A (NBD-palmitoyl-CoA) did able to decrease the reaction volume to a minimum of not act as a CerS5 substrate ( Fig. 2A ), nor did it act as a 20 µl, rather than the 100–250 µl used previously with [4,5- competitive inhibitor of CerS5 (data not shown), strength- 3 H]Sph ( 22 ) or with NBD-Sph ( 21 ), permitting the use ening the notion ( 4 ) that CerSs are highly specifi c for the of small amounts of protein for the assay. Thus, we could acyl-CoA donor but less specifi c for the sphingoid long- assay CerS5 activity using as little as 1 µg of protein and chain base. Degradation products of NBD-Sph are readily 5 min reaction time, and CerS4 activity using 10 (Fig. 1B ) apparent upon separation by TLC, which is of concern if or 20 µg (not shown) of protein and 20 min reaction time; low levels of CerS activity are assayed because some of the note that each CerS displays different specifi c activities, degradation products have Rf values similar to that of NBD- necessitating optimization of assay conditions for each ceramide ( Fig.