LASS5 Is a Bona Fide Dihydroceramide Synthase That Selectively Utilizes Palmitoyl-CoA as Acyl Donor

Abstract

We demonstrated recently (Riebeling, C., Allegood, J.C., Wang, E., Merrill, A. H. Jr., and Futerman, A. H. (2003) J. Biol. Chem. 278, 43452–43459) that upon over-expression in human embryonic kidney cells, longevity assurance gene homolog 5 (LASS5, previously named TRH4) elevates the synthesis of (dihydro)ceramides selectively enriched in palmitic acid. To determine whether LASS5 is a bona fide dihydroceramide synthase or, alternatively, whether it modifies an endogenous dihydroceramide synthase, we over-expressed LASS5 with a hemagglutinin (HA) tag at the C terminus, solubilized it using digitonin, and purified it by immunoprecipitation. Solubilized LASS5-HA displays the same fatty acid selectivity as the membrane-bound enzyme. After elution from agarose beads, only one band could be detected by SDS-PAGE, and its identity was confirmed to be LASS5 by mass spectrometry. Dihydroceramide synthase activity of the eluted LASS5-HA protein was totally dependent on exogenously added phospholipids. Moreover, eluted LASS5-HA was highly selective toward palmitoyl-CoA as acyl donor and was inhibited by the (dihydro)ceramide synthase inhibitor, fumonisin B1. This study identifies LASS5 as a genuine dihydroceramide synthase and demonstrates that mammalian dihydroceramide synthases do not require additional subunits for their activity. We demonstrated recently (Riebeling, C., Allegood, J.C., Wang, E., Merrill, A. H. Jr., and Futerman, A. H. (2003) J. Biol. Chem. 278, 43452–43459) that upon over-expression in human embryonic kidney cells, longevity assurance gene homolog 5 (LASS5, previously named TRH4) elevates the synthesis of (dihydro)ceramides selectively enriched in palmitic acid. To determine whether LASS5 is a bona fide dihydroceramide synthase or, alternatively, whether it modifies an endogenous dihydroceramide synthase, we over-expressed LASS5 with a hemagglutinin (HA) tag at the C terminus, solubilized it using digitonin, and purified it by immunoprecipitation. Solubilized LASS5-HA displays the same fatty acid selectivity as the membrane-bound enzyme. After elution from agarose beads, only one band could be detected by SDS-PAGE, and its identity was confirmed to be LASS5 by mass spectrometry. Dihydroceramide synthase activity of the eluted LASS5-HA protein was totally dependent on exogenously added phospholipids. Moreover, eluted LASS5-HA was highly selective toward palmitoyl-CoA as acyl donor and was inhibited by the (dihydro)ceramide synthase inhibitor, fumonisin B1. This study identifies LASS5 as a genuine dihydroceramide synthase and demonstrates that mammalian dihydroceramide synthases do not require additional subunits for their activity. Ceramide, an important lipid second messenger (1Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 25847-25850Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar, 2Futerman A.H. Hannun Y.A. EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (521) Google Scholar, 3Gulbins E. Kolesnick R. Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (343) Google Scholar), consists of a sphingoid long chain base to which a fatty acid is attached at carbon-2 via an amide bond. Ceramide is also a key metabolite in the pathway of sphingolipid (SL) 2The abbreviations used are: SL, sphingolipid; DOPC, dioleoylphosphatidylcholine;DOPS, dioleoylphosphatidylserine; HA, hemagglutinin; LASS5, longevity assurance gene 5; MALDI-TOF, matrix-assisted laser desorption/time of flight ionization.2The abbreviations used are: SL, sphingolipid; DOPC, dioleoylphosphatidylcholine;DOPS, dioleoylphosphatidylserine; HA, hemagglutinin; LASS5, longevity assurance gene 5; MALDI-TOF, matrix-assisted laser desorption/time of flight ionization. biosynthesis (4Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar), and within the past 2–3 years the molecular identities of most of the enzymes in this pathway have been identified (5Futerman A.H. Riezman H. Trends Cell Biol. 2005; 15: 312-318Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Among these, a family of mammalian genes that regulates ceramide synthesis has been discovered (6Venkataraman K. Futerman A. FEBS Lett. 2002; 528: 3-4Crossref PubMed Scopus (56) Google Scholar, 7Winter E. Ponting C.P. Trends Biochem. Sci. 2002; 27: 381-383Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Surprisingly, over-expression of each of these genes in various mammalian cells leads to an increase in ceramides containing different fatty acids (8Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 9Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 10Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar). Thus, over-expression of LASS1 leads to an increase in the synthesis of ceramides containing stearic acid (8Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), whereas over-expression of LASS5 leads to an increase of ceramide containing palmitic acid (9Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). However, it is not known whether these genes modify an endogenous ceramide synthase activity, and thereby confer fatty acid selectivity, or whether the LASS proteins themselves are bona fide dihydroceramide synthases. We have now purified, by immunoprecipitation, LASS5 with an HA tag at the C terminus and demonstrate here that it is a genuine dihydroceramide synthase that displays the same fatty acid selectivity as seen upon its over-expression in mammalian cells. This is the first biochemical isolation of a mammalian dihydroceramide synthase and paves the way for studying the role of LASS proteins in regulating ceramide production both for SL biosynthesis and for the regulation of the defined aspects of cell physiology in which it plays vital roles. Materials—1-[14C]Palmitoyl-CoA (specific activity, 60 mCi/mmol) was from Amersham Biosciences, and 1-[14C]stearoyl-CoA (specific activity, 55 mCi/mmol) was from American Radiolabeled Chemicals (St. Louis, MO). Fumonisin B1, defatted bovine serum albumin, phenylmethylsulfonyl fluoride, leupeptin, antipain, and aprotinin were from Sigma. Sphinganine, sphingosine, palmitoylsphingosine, and stearoylsphingosine were from Matreya (Pleasant Gap, PA). Digitonin was from Sigma or Calbiochem. A monoclonal anti-HA-agarose conjugate (clone HA-7) was from Sigma, a rabbit polyclonal anti-HA antibody (Y-11) and protein A-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA), and peroxidase-conjugated AffiniPure goat anti-mouse IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA). Dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) were from Avanti Polar Lipids (Alabaster, AL). Silica gel 60 TLC plates were from Merck. All solvents were of analytical grade and were purchased from Biolab (Jerusalem, Israel). Cell Culture and Transfection—Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Transfections using LASS5-HA (9Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) were performed by the calcium phosphate method. Transfections were also performed with pcDNA-HA as a control. Solubilization of LASS5-HA—293T cells, at ∼90% confluency, were washed with phosphate-buffered saline and removed from culture dishes using trypsin (0.05%, w/v). After centrifugation (4 min, 150 × gav) cell pellets were washed twice with phosphate-buffered saline by centrifugation (4 min, 150 × gav) and then homogenized in 20 mm HEPES-KOH, pH 7.4, 25 mm KCl, 250 mm sucrose, and 2 mm MgCl2 containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml antipain, and 100 kallikrein-inactivating units/ml aprotinin). Digitonin was added to the homogenate to give a final concentration of 1% (w/v), and after 1 h at 4°C, the homogenate was centrifuged at 100,000 × gav for 30 min. The resulting supernatant was used for subsequent purification steps. Immunoprecipitation of LASS5-HA—The digitonin-solubilized supernatant was concentrated using an iCON concentrator (Pierce) and then incubated with protein A-agarose for 1 h at 4 °C to reduce nonspecific binding. After removal of protein A-agarose by centrifugation, the supernatant was incubated with an anti-HA agarose conjugate overnight at 4 °C. The conjugate was then pelleted by centrifugation and washed with the same HEPES buffer used for homogenization followed by a wash with 1 m NaCl. LASS5-HA was eluted from the beads using 100 mm glycine, pH 2.5, and immediately neutralized using 1 m Trizma (Tris base), pH 11. The eluate was concentrated using an iCON concentrator, and in some cases, DOPC or DOPS liposomes prepared as described (11Bodennec J. Pelled D. Riebeling C. Trajkovic S. Futerman A.H. FASEB J. 2002; 16: 1814-1816Crossref PubMed Scopus (69) Google Scholar), were added to the concentrated eluate prior to analysis of dihydroceramide synthase activity. The composition of the eluate was analyzed by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gel) and Western blotting using a rabbit polyclonal anti-HA antibody. Control immunoprecipitations were performed using cells that had been transfected with pcDNA-HA. Mass Spectrometry—Protein bands were excised from the SDS gel and subsequently reduced, alkylated, and in-gel-digested for 18 h with bovine trypsin (sequencing grade, Roche Diagnostics) at a concentration of 12.5 ng/μl in 50 mm ammonium bicarbonate at 37 °C. An extracted peptide solution was dried for subsequent matrix-assisted laser desorption/time of flight ionization (MALDI-TOF) and electrospray ionization mass spectrometric analyses. Aliquots of the extracted peptide mixture and electroeluted proteins, dissolved in 0.1% trifluoroacetic acid or a mixture of formic acid/isopropanol/H20 (1/3/2; v/v/v), were used for MALDI-TOF mass spectrometry. Peptide fingerprinting was performed on a Bruker Reflex IIITM MALDI-TOF mass spectrometer (Bruker, Bremen, Germany) equipped with a delayed extraction ion source, a reflector, and a 337-nm nitrogen laser. Dihydroceramide Synthase Assay—For cell fractions, 500 μg of protein (determined using BCA reagent (Pierce)) was incubated with 15 μm sphinganine and 20 μm defatted bovine serum albumin (12Hirschberg K. Rodger J. Futerman A.H. Biochem. J. 1993; 290: 751-757Crossref PubMed Scopus (160) Google Scholar) for 5 min at 37 °C with or without fumonisin B1 (20 μm), and the reaction was initiated by the addition of either 0.12 μCi of 1-[14C]palmitoyl-CoA or 0.12 μCi of 1-[14C]stearoyl-CoA for a further 20 min (9Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). When immunoprecipitated LASS5-HA was assayed, either sphinganine or sphingosine was used as long chain base, and the reaction time was 1 h. Lipids were extracted and levels of dihydroceramide synthesis analyzed by thin layer chromatography using chloroform/methanol/2 m ammonium hydroxide (40/10/1; v/v/v) as the developing solvent and using palmitoylsphingosine and stearoylsphingosine as standards. Lipids were visualized using a phosphorimaging screen (Fuji, Tokyo, Japan), recovered from TLC plates by scraping the silica directly into scintillation vials, and quantified by liquid scintillation counting. Initial experiments were performed to establish a means to solubilize membrane-bound LASS5-HA. Digitonin, which was previously used to solubilize Lag1p (13Vallee B. Riezman H. EMBO J. 2005; 24: 730-741Crossref PubMed Scopus (116) Google Scholar), a yeast LASS homolog (14Guillas I. Kirchman P.A. Chuard R. Pfefferli M. Jiang J.C. Jazwinski S.M. Conzelmann A. EMBO J. 2001; 20: 2655-2665Crossref PubMed Scopus (217) Google Scholar, 15Schorling S. Vallee B. Barz W.P. Riezman H. Oesterhelt D. Mol. Biol. Cell. 2001; 12: 3417-3427Crossref PubMed Scopus (218) Google Scholar), was able to solubilize LASS5-HA and preserve dihydroceramide synthase activity. Most of the dihydroceramide synthase activity was recovered in the digitoninsolubilized supernatant, which correlated with levels of LASS5-HA detected by Western blotting (Fig. 1A). Digitonin-solubilized LASS5-HA displayed the same fatty acid specificity as membranebound LASS5, showing an ∼10-fold higher activity toward palmitoyl-CoA than toward stearoyl-CoA (Fig. 1B), and was inhibited by the (dihydro)ceramide synthase inhibitor fumonisin B1 (16Merrill A.H. Liotta D.C. Riley R. Trends Cell Biol. 1996; 6: 218-223Abstract Full Text PDF PubMed Scopus (132) Google Scholar) to a similar extent as observed in the homogenate (Fig. 1B). Digitonin-solubilized LASS5-HA was subsequently immunoprecipitated using an anti-HA agarose conjugate and eluted using 100 mm glycine. A significant fraction of the immunoprecipitated LASS5-HA could be eluted from the beads (Fig. 2A), and analysis by SDS-polyacrylamide gel electrophoresis revealed only one band, with a molecular mass of ∼48 kDa (Fig. 2B), similar to the predicted molecular mass of LASS5-HA. No other bands could be detected reproducibly either by silver staining of the gel (Fig. 2B) or by Coomassie staining (not shown). The identity of the band as LASS5-HA was confirmed by Western blotting (Fig. 2C) and by MALDI-TOF mass spectrometry (TABLE ONE). In addition, no other peptides co-migrated with LASS5 (TABLE ONE), which was confirmed by nano-liquid chromatography electrospray ionization tandem mass spectrometry (not shown).TABLE ONEMALDI-TOF MS identification of LASS5Measured massTheoretical massMass accuracyPosition in the sequencePeptide sequenceDaDappm694.342694.347-50062-66MLFER710.342710.342062-66MoxLFER902.462902.461+1114-120QLDWSVR908.432908.4320122-127IQCWFR914.472914.4720398-405ATGNLHFR1030.4621030.556-94114-121QLDWSVRK1014.4021014.405-3141-147FCESMoxWR1036.5221036.527-5121-127KIQCWFR1074.5921074.600-867-75FIAKPCALR1140.5821140.613-31131-140NQDKPPTLTK1296.6721296.714-42130-140RNQDKPPTLTK1410.6521410.739-8795-106VFVSVTKYPDEK1566.7421566.840-9895-107VFVSVTKYPDEKR1683.7621683.831-6980-94DSPVNKVEPNDTLEK1773.7221773.793-71173-185CWYNYPYQPLSR Open table in a new tab Eluted LASS5-HA did not show any dihydroceramide synthase activity unless phospholipids were added to the eluate. Of the two phospholipids tested, DOPC was more efficient than DOPS at preserving dihydroceramide synthase activity (Fig. 3A). Moreover, eluted LASS5-HA was highly selective toward palmitoyl-CoA compared with stearoyl-CoA (Fig. 3B), could be inhibited by fumonisin B1, and was able to acylate both sphinganine and sphingosine (Fig. 3C). 3We attempted to determine the extent of recovery and -fold purification of LASS5 from the digitonin-solubilized supernatant. However, because of the low amounts of purified LASS5 protein and the inherent difficulties in estimating the amount of protein after silver staining of the SDS-gel, we can only provide estimates of these values. In three separate experiments, the -fold increase in specific activity of the immunoprecipitated protein compared with the digitonin-solubilized supernatant varied between ∼5,000 and 12,000, and the recovery of LASS5 activity (toward 1-[14C]palmitoyl-CoA as substrate) varied between 26 and 49%. These values are probably underestimates because we have not systematically determined the conditions for optimal activity of purified LASS5. In the current study we have demonstrated that LASS5 is a bona fide (dihydro)ceramide synthase. This is the first time that a mammalian dihydroceramide synthase has been purified, representing one of the last recalcitrant enzymes in the SL biosynthetic pathway (5Futerman A.H. Riezman H. Trends Cell Biol. 2005; 15: 312-318Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In contrast to yeast, in which SLs contain only one kind of fatty acid, namely C26, mammalian (dihydro)ceramides contain a wide fatty acid spectrum (2Futerman A.H. Hannun Y.A. EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (521) Google Scholar, 4Merrill Jr., A.H. J. Biol. Chem. 2002; 277: 25843-25846Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). It was formerly assumed that this was due to a lack of specificity of dihydroceramide synthase with respect to the use of fattyacyl-CoAs. However, the discovery of a family of mammalian LASS genes that each synthesizes dihydroceramides containing different fatty acids (at least those characterized to date (8Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 9Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 10Mizutani Y. Kihara A. Igarashi Y. Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (297) Google Scholar)) demonstrates that this is not the case. In contrast, yeast have only two highly homologous ceramide synthase genes, LAG1 and LAC1, which together are responsible for the synthesis of C26-ceramides. Evidence is accumulating that ceramides containing specific fatty acids are involved in defined cell functions in mammalian cells (2Futerman A.H. Hannun Y.A. EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (521) Google Scholar, 17Koybasi S. Senkal C.E. Sundararaj K. Spassieva S. Bielawski J. Osta W. Day T.A. Jiang J.C. Jazwinski S.M. Hannun Y.A. Obeid L.M. Ogretmen B. J. Biol. Chem. 2004; 279: 44311-44319Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). This being the case, it might be expected that individual LASS genes would be expressed in either a tissue-specific (9Riebeling C. Allegood J.C. Wang E. Merrill Jr., A.H. Futerman A.H. J. Biol. Chem. 2003; 278: 43452-43459Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 18Xu Z. Zhou J. McCoy D.M. Mallampalli R.K. J. Lipid Res. 2005; 46: 1229-1238Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) or temporal manner so as to supply specific ceramides for the distinct events in which they are involved. Our demonstration that LASS5 is a bona fide dihydroceramide synthase that selectively utilizes palmitoyl-CoA supports this likelihood and strengthens the hypothesis that specific LASS proteins play distinct roles either in ceramide signaling or in SL metabolism. Evidence for the latter has already been obtained by the observation that C18-ceramide formed by LASS1 is selectively channeled into neutral glyco-SLs but not gangliosides. The availability of purified LASS5 will permit systematic characterization of the reaction mechanism and modes of regulation of a mammalian dihydroceramide synthase (19Kok J.W. Nikolova-Karakashian M. Klappe K. Alexander C. Merrill A.H. J. Biol. Chem. 1997; 272: 21128-21136Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The yeast ceramide synthase, Lag1p/Lac1p, was recently purified, and was shown to co-immunoprecipitate with an additional subunit, Lip1p (13Vallee B. Riezman H. EMBO J. 2005; 24: 730-741Crossref PubMed Scopus (116) Google Scholar), which was absolutely required for yeast ceramide synthesis, at least using C26-fatty acids. No mammalian homologs of Lip1p are found by data base searches, and our demonstration that no other protein is required for the dihydroceramide synthase activity of LASS5 indicates that LASS5 by itself is sufficient for catalytic activity in mammalian cells. However, the dihydroceramide synthase activity of LASS5 is totally dependent on exogenously added phospholipids, whereas no such requirement was observed for yeast Lag1p/Lac1p/Lip1p. Together, these data suggest a unique role for Lip1p in yeast that is not required in mammalian cells, and suggest that the regulation of mammalian (dihydro)ceramide synthesis may differ significantly from that in yeast. We thank Christian Riebeling for the initial cloning of LASS5-HA, Howard Riezman and Beatrice Vallee for sharing unpublished data about the purification of yeast Lag1p/Lac1p/Lip1p, and Dr. Alla Shainskaya from the Mass Spectrometry Unit of the Weizmann Institute of Science for the MALDI-TOF analyses.