Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes
2007; Elsevier BV; Volume: 49; Issue: 3 Linguagem: Inglês
10.1194/jlr.m700414-jlr200
ISSN1539-7262
AutoresHenrik Fyrst, Xinyi Zhang, Deron R. Herr, Hoe Sup Byun, Robert Bittman, Van Hung Phan, Greg L. Harris, Julie D. Saba,
Tópico(s)Lipid metabolism and biosynthesis
ResumoSphingolipids comprise a complex group of lipids concentrated in membrane rafts and whose metabolites function as signaling molecules. Sphingolipids are conserved in Drosophila, in which their tight regulation is required for proper development and tissue integrity. In this study, we identified a new family of Drosophila sphingolipids containing two double bonds in the long chain base (LCB). The lipids were found at low levels in wild-type flies and accumulated markedly in Drosophila Sply mutants, which do not express sphingosine-1-phosphate lyase and are defective in sphingolipid catabolism. To determine the identity of the unknown lipids, purified whole fly lipid extracts were separated on a C18-HPLC column and analyzed using electrospray mass spectrometry. The lipids contain a LCB of either 14 or 16 carbons with conjugated double bonds at C4,6. The Δ4,6-sphingadienes were found as free LCBs, as phosphorylated LCBs, and as the sphingoid base in ceramides. The temporal and spatial accumulation of Δ4,6-sphingadienes in Sply mutants suggests that these lipids may contribute to the muscle degeneration observed in these flies. Sphingolipids comprise a complex group of lipids concentrated in membrane rafts and whose metabolites function as signaling molecules. Sphingolipids are conserved in Drosophila, in which their tight regulation is required for proper development and tissue integrity. In this study, we identified a new family of Drosophila sphingolipids containing two double bonds in the long chain base (LCB). The lipids were found at low levels in wild-type flies and accumulated markedly in Drosophila Sply mutants, which do not express sphingosine-1-phosphate lyase and are defective in sphingolipid catabolism. To determine the identity of the unknown lipids, purified whole fly lipid extracts were separated on a C18-HPLC column and analyzed using electrospray mass spectrometry. The lipids contain a LCB of either 14 or 16 carbons with conjugated double bonds at C4,6. The Δ4,6-sphingadienes were found as free LCBs, as phosphorylated LCBs, and as the sphingoid base in ceramides. The temporal and spatial accumulation of Δ4,6-sphingadienes in Sply mutants suggests that these lipids may contribute to the muscle degeneration observed in these flies. double-stranded RNA long chain base long chain base phosphate 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium RNA interference sphingosine-1-phosphate lyase Sphingolipids are a diverse group of membrane lipids that are highly conserved throughout evolution (1.Oskouian B. Saba J.D. Death and taxis: what non-mammalian models tell us about sphingosine-1-phosphate.Semin. Cell Dev. Biol. 2004; 15: 529-540Crossref PubMed Scopus (38) Google Scholar, 2.Merrill Jr., A.H. Sullards M.C. Wang E. Voss K.A. Riley R.T. 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In this species, tight regulation of sphingolipid levels is required for proper development, reproduction, and the maintenance of tissue integrity, as demonstrated by the severe phenotypes observed in mutants with disrupted sphingolipid metabolism (13.Adachi-Yamada T. Gotoh T. Sugimura I. Tateno M. Nishida Y. Onuki T. Date H. De novo synthesis of sphingolipids is required for cell survival by down-regulating c-Jun N-terminal kinase in Drosophila imaginal discs.Mol. Cell. Biol. 1999; 19: 7276-7286Crossref PubMed Scopus (86) Google Scholar, 14.Herr D.R. Fyrst H. Creason M.B. Phan V.H. Saba J.D. Harris G.L. Characterization of the Drosophila sphingosine kinases and requirement for Sk2 in normal reproductive function.J. Biol. Chem. 2004; 279: 12685-12694Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15.Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.Development. 2003; 130: 2443-2453Crossref PubMed Scopus (98) Google Scholar, 16.Kohyama-Koganeya A. Sasamura T. Oshima E. Suzuki E. Nishihara S. Ueda R. Hirabayashi Y. Drosophila glucosylceramide synthase: a negative regulator of cell death mediated by proapoptotic factors.J. Biol. Chem. 2004; 279: 35995-36002Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 17.Phan V. Herr D. Panton D. Fyrst H. Saba J. Harris G. Disruption of sphingolipid metabolism elicits apoptosis-associated reproductive defects in Drosophila.Dev. Biol. 2007; 309: 329-341Crossref PubMed Scopus (40) Google Scholar). However, a clear understanding of the role of sphingolipid metabolism and, in particular, the mechanisms by which sphingolipid metabolites influence physiological processes in this organism has been hampered by an incomplete knowledge of the chemical structures of endogenous Drosophila sphingolipids and their metabolic products. Previously, we found that the Drosophila free sphingoid bases or long chain bases (LCBs) are composed largely of C14- and C16-sphingosine and dihydrosphingosine (18.Fyrst H. Herr D.R. Harris G.L. Saba J.D. Characterization of free endogenous C14 and C16 sphingoid bases from Drosophila melanogaster.J. Lipid Res. 2004; 45: 54-62Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In the current study, we have identified a second family of sphingolipids recognized by their differential separation on HPLC compared with known Drosophila sphingolipid species. Mass spectrometry approaches were used to characterize the structures of these unknown lipids as C14- and C16-sphingadienes with Δ-4,6 conjugated double bonds and to further identify a family of related lipids built upon the same Δ4,6-sphingadiene LCB. The presence of sphingolipids containing a Δ4,6-sphingadiene LCB has been reported in other insect species. A study of sphingomyelins from the tobacco hornworm Manduca sexta revealed the presence of Δ4,6-sphingadiene LCBs in sphingomyelin, ceramide-phosphoethanolamine, and ceramide (19.Abeytunga D. Glick J. Gibson N. Oland L. Somogyi A. Wysocki V. Polt R. Presence of unsaturated sphingomyelins and changes in their composition during the life cycle of the moth Manduca sexta.J. Lipid Res. 2004; 45: 1221-1231Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Moreover, a ceramide compound purified from larvae of the silk moth Bombyx mori was shown to contain a Δ4,6-sphingadiene LCB (20.Kwon H.C. Lee K.C. Cho O.R. Jung I.Y. Cho S.Y. Kim S.Y. Lee K.R. Sphingolipids from Bombycis corpus 101A and their neurotrophic effects.J. Nat. Prod. 2003; 66: 466-469Crossref PubMed Scopus (29) Google Scholar). In both studies, the Δ4,6-sphingadiene-containing ceramides were found to have potent although diverse biological effects. The ceramide compound from M. sexta was found to increase apoptosis in an embryonic M. sexta cell line (19.Abeytunga D. Glick J. Gibson N. Oland L. Somogyi A. Wysocki V. Polt R. Presence of unsaturated sphingomyelins and changes in their composition during the life cycle of the moth Manduca sexta.J. Lipid Res. 2004; 45: 1221-1231Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), whereas the compound from B. mori was shown to promote neurite outgrowth in the rat pheochromocytoma cell line PC12 (20.Kwon H.C. Lee K.C. Cho O.R. Jung I.Y. Cho S.Y. Kim S.Y. Lee K.R. Sphingolipids from Bombycis corpus 101A and their neurotrophic effects.J. Nat. Prod. 2003; 66: 466-469Crossref PubMed Scopus (29) Google Scholar). Sphingosine-1-phosphate lyase (SPL) is an important enzyme in the sphingolipid degradation pathway, as it catalyzes the cleavage of a long chain base phosphate (LCBP) to yield a long chain aldehyde and ethanolamine phosphate, thereby irreversibly removing LCBPs from the sphingolipid pool. We previously reported the severe reproductive organ and muscle phenotypes of the Drosophila SPL mutant fly Sply and showed increased levels of sphingosine, dihydrosphingosine, and the corresponding LCBPs in the adult Sply fly (15.Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.Development. 2003; 130: 2443-2453Crossref PubMed Scopus (98) Google Scholar). In this study, we demonstrate the presence of Drosophila sphingolipids containing Δ4,6-sphingadiene LCBs. These lipids are found in wild-type flies from mid embryogenesis to adulthood and accumulate markedly in the Sply mutant. Although we found a general accumulation of total LCBs and ceramides in all of the tissues of Sply mutant flies, a greater accumulation of Δ4,6-sphingadienes was found in the thorax, which contains the flight muscles that undergo spontaneous degeneration. Moreover, we found that Δ4,6-C14-sphingadienes and the corresponding C2-sphingadiene-ceramide decreased cell proliferation in the Drosophila wing disc cell line Cl.8. These findings suggest that Δ4,6-sphingadiene-containing sphingolipids likely contribute to flight muscle tissue degeneration in the Sply mutant. Wild-type Canton-S (BL-1), SPL knockout strain Sply05091 (BL-11393), and lace2/k05305 (BL-3156 and BL-12176) were obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN). Flies were reared on standard fly medium at room temperature. Flies carrying the double knockout mutation Sply/lace and the Sply revertant Sply14a were generated as described previously (15.Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.Development. 2003; 130: 2443-2453Crossref PubMed Scopus (98) Google Scholar). Sk2KG05894 was a gift of the P-element Screen/Gene Disruption project of the Bellen/Rubin/Spradling laboratories (14.Herr D.R. Fyrst H. Creason M.B. Phan V.H. Saba J.D. Harris G.L. Characterization of the Drosophila sphingosine kinases and requirement for Sk2 in normal reproductive function.J. Biol. Chem. 2004; 279: 12685-12694Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In all cases, control and mutant flies were reared in parallel under identical conditions. For developmental analysis, adult flies were allowed to deposit embryos on grape juice agar plates, and embryos were collected, staged, and prepared as described (18.Fyrst H. Herr D.R. Harris G.L. Saba J.D. Characterization of free endogenous C14 and C16 sphingoid bases from Drosophila melanogaster.J. Lipid Res. 2004; 45: 54-62Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For the isolation of tissue, 100 female flies, 4 to 6 days old, were euthanized, and abdomen, head, thorax, and ovaries were collected. Drosophila S2 cells and Cl.8 cells were obtained from the Drosophila Genomics Resource Center at Indiana University. S2 cells were cultured at 28°C in Schneider's medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were routinely passaged every second day to a density of ∼2 × 106 cells/ml. Cl.8 wing disc cells were cultured at 28°C in Shields and Sang Medium 3 (Sigma-Aldrich, St. Louis, MO) supplemented with 2% heat-inactivated FBS, 12.5 IU/100 ml insulin, and 2.5% extract from adult wild-type flies. A DNA fragment containing a 720 bp segment of the Sply open reading frame containing T7 promoter sequences at the 5′ and 3′ ends was generated by PCR using Vent DNA polymerase (New England Biolabs, Beverly, MA). As a control, a DNA fragment containing the full-length open reading frame of an unrelated Caenorhabditis elegans gene, elt2, was generated using similar methods. The preparation of double-stranded RNA (dsRNA) in vitro was performed in a reaction mixture of diethylpyrocarbonate-treated water containing 2 μg of DNA template, 10 mM ribonucleotide triphosphate, 150 units of T7 polymerase enzyme (New England Biolabs), and 8 μl of 10× T7 polymerase buffer in a total volume of 80 μl. Samples were incubated at 37°C for 8 h, the reaction was stopped, and the RNA was precipitated by adding 8 μl of 3 M sodium acetate, pH 5.2, and 200 μl of 2-propanol. Samples were stored at −20°C overnight, and the precipitate containing the RNA was isolated by centrifugation at 14,000 g for 30 min. The supernatant was discarded, and the pellet was washed twice with 200 μl of 70% ethanol and then allowed to air-dry for 10 min. The pellet was then dissolved in 50 μl of diethylpyrocarbonate-treated water, and annealing of the two RNA strands was performed by heating the sample for 5 min at 75°C followed by slow cooling to room temperature. The status of the synthesized dsRNA was evaluated on a 1% agarose gel. S2 cells were harvested and resuspended in serum free Schneider's Drosophila medium to a density of 1 × 106 cells/ml. The Sply or control dsRNA was then added directly to the cell suspension to a final concentration of 15 μg dsRNA/ml, and cells were incubated at room temperature for 30 min. After incubation, 2 volumes of Schneider's Drosophila medium containing 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin was added. For each RNA interference (RNAi) experiment performed, the dsRNA treatment was repeated on 2 consecutive days. The Δ4,6-sphingadiene backbone was prepared according to a procedure described previously (21.Chun J. Li G. Byun H. Bittman R. Synthesis of new trans double bond sphingolipid analogues: D4,6 and D6 ceramides.J. Org. Chem. 2002; 67: 2600-2605Crossref PubMed Scopus (44) Google Scholar). N-Acetylation of sphingadiene to generate (2S,3R,4E,6E)-2-acetamidotetradecadiene-1,3-diol (C2-sphingadiene ceramide) was carried out with p-nitrophenyl acetate followed by purification using flash chromatography (gradient of chloroform-methanol, 15:1 to 9:1). The purity and fragmentation patterns of the final products were verified by electrospray ionization mass spectrometry. Samples were scanned from m/z 100 to 350 in positive mode on a Micromass Quattro LCZ (Waters Corp., Milford, MA). Samples containing 50 mg of frozen intact fly material or isolated fly tissues were placed in a glass Potter Elvehjem homogenizer. Twenty microliters of an internal standard mixture containing 200 pmol of each C17-sphingosine (Avanti Polar Lipids, Alabaster, AL) and C17-sphingosine-1-phosphate (Matreya, Inc., Pleasant Gap, PA) was then added, and fly materials were homogenized in 0.5 ml of methanol until the pestle moved smoothly. An equal volume of water was then added, and homogenization was continued with another 10 strokes. Fly homogenates were transferred to a glass tube, and a two-phase separation was obtained after the addition of 1 ml of chloroform and 0.75 ml of 1 M ammonium hydroxide followed by vortexing and a 10 min spin at 1,000 g. For the analysis of LCBPs, the water phase was recovered, dried down, and resuspended in 0.1 ml of methanol-water (1:1, v/v). For the analysis of LCBs, a portion of the organic phase was recovered, dried down, and resuspended in 0.1 ml of methanol. For the analysis of ceramides, a portion of the organic phase was recovered, dried down, and resuspended in 0.5 ml of 1 M potassium hydroxide in methanol, and ceramides were hydrolyzed by incubating for 1 h at 90°C. After hydrolysis, a two-phase separation was obtained as described above and the organic phase was recovered. Lipid extracts were purified on a Strata C18-E solid-phase extraction column, and LCBs were derivatized with ortho-phthalaldehyde and separated by HPLC as described (18.Fyrst H. Herr D.R. Harris G.L. Saba J.D. Characterization of free endogenous C14 and C16 sphingoid bases from Drosophila melanogaster.J. Lipid Res. 2004; 45: 54-62Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For the identification of the structure of novel Drosophila LCBs, the Strata C18-E purified lipid extract was separated on a C18-HPLC column (2.0 × 50 mm; S-5 120 Å) (Waters Corp.) at a flow rate of 0.4 ml/min. The gradient used was from 30% to 80% methanol containing 0.1% formic acid in 10 min and 80–95% methanol containing 0.1% formic acid in 2 min. The data were acquired in positive mode on an Esquire3000plus ion trap mass spectrometer (Bruker Daltonics, Billerica, MA). Data acquisitions with up to AutoMS4 mode were applied for the characterization of unknowns. Crude Drosophila lipid extracts prepared as described above were used for the quantitation of LCBs, LCBPs, and ceramides. Lipids were separated on a C18-HPLC column (2.0 × 75 mm; Luna) (Phenomenex, Torrance, CA) at a flow rate of 0.25 ml/min. The gradient used was from 50% to 80% methanol containing 0.1% formic acid in 4 min and 80% methanol containing 0.1% formic acid for 6 min. Intact ceramides were analyzed after direct injection of 10 μl of the organic phase from the lipid extraction described above. The mobile phase was 95% methanol containing 0.1% formic acid. The flow rate was 0.05 ml/min. The data were acquired in positive mode on a Micromass Quattro LCZ (Waters Corp.). Lipids were identified based on their specific precursor and product ion pair and quantitated using multiple reaction monitoring as described (22.Sullards M.C. Merrill A.H.J. Analysis of sphingosine-1-phosphate, ceramides, and other bioactive sphingolipids by high-performance liquid chromatography.Sci. STKE. 2001; 67: 1-11Google Scholar). Drosophila Cl.8 cells were treated for 6 h with 20 μM Δ4,6-sphingadiene-containing lipids in the presence of 2% heat-inactivated FBS. The viability of the cells was determined by measuring their ability to hydrolyze the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) into formazan according to a standard procedure using the CellTiter 96 Aqueous NonRadioactive Cell Proliferation Assay (Promega, Madison, WI). Cl.8 cells were seated on a 96-well plate and lipid-treated in place. Twenty microliters of MTS substrate was added to each well, and the 96-well plate was incubated at 28°C. SPL catalyzes the final step in sphingolipid degradation, namely the cleavage at C2-3 of LCBPs. The Drosophila Sply mutant contains a P-element transposon insertion within the open reading frame of the SPL gene, resulting in a complete lack of expression of the SPL homolog SPLY. Flies homozygous for this mutation undergo proper embryogenesis and emerge from pupation. However, female homozygotes demonstrate diminished fertility, which is associated with a progressive loss of ovarian tissue as a result of apoptotic cell death (17.Phan V. Herr D. Panton D. Fyrst H. Saba J. Harris G. Disruption of sphingolipid metabolism elicits apoptosis-associated reproductive defects in Drosophila.Dev. Biol. 2007; 309: 329-341Crossref PubMed Scopus (40) Google Scholar). In addition, adult homozygotes exhibit a flightless phenotype, attributable to a myopathy resulting from degeneration of the dorsal longitudinal muscles that power the wings (15.Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.Development. 2003; 130: 2443-2453Crossref PubMed Scopus (98) Google Scholar). Previously, we demonstrated that the ovarian degeneration and muscle wasting were not observed in homozygous Sply revertants Sply14a (15.Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.Development. 2003; 130: 2443-2453Crossref PubMed Scopus (98) Google Scholar, 17.Phan V. Herr D. Panton D. Fyrst H. Saba J. Harris G. Disruption of sphingolipid metabolism elicits apoptosis-associated reproductive defects in Drosophila.Dev. Biol. 2007; 309: 329-341Crossref PubMed Scopus (40) Google Scholar). This finding confirms that the loss of Sply expression is responsible for the observed phenotypes. The Sply phenotypes were also ameliorated by the introduction of a second mutation in the lace gene, which encodes one subunit of serine palmitoyltransferase, the first enzyme in the sphingolipid biosynthetic pathway (15.Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.Development. 2003; 130: 2443-2453Crossref PubMed Scopus (98) Google Scholar). This suggested that the phenotypes of the Sply mutant are caused by abnormal accumulation of sphingolipid intermediates, a state that is corrected by reducing the synthesis of these lipids. In support of this possibility, we found that the predominant LCBs in the fly, which we had previously determined to be saturated dihydrosphingosine and Δ-4 monounsaturated sphingosines with 14 and 16 carbon chains (15.Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Sply regulation of sphingolipid signaling molecules is essential for Drosophila development.Development. 2003; 130: 2443-2453Crossref PubMed Scopus (98) Google Scholar, 18.Fyrst H. Herr D.R. Harris G.L. Saba J.D. Characterization of free endogenous C14 and C16 sphingoid bases from Drosophila melanogaster.J. Lipid Res. 2004; 45: 54-62Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), were markedly increased in Sply mutants but normalized in the double mutant cross. However, in analyzing the sphingolipids in our fly extracts, we noticed the presence of an unknown lipid, which was also increased in the Sply mutant and normalized in the double mutant cross. This unknown lipid was not initially recognizable and was thus referred to as "lipid X." Lipid X was readily detected in our HPLC system after derivatization with ortho-phthalaldehyde, which modifies free amino groups. Lipid X eluted from the HPLC system with a different retention time than C14-sphingosine and C14-sphingosine-1-phosphate standards when tested under different running conditions (Table 1). By changing the pH of the potassium phosphate buffer from 7.2 to 5.5, a 3.3 min shift in the retention time of the C14-sphingosine-1-phosphate standard was observed. This change in pH had no effect on the retention time of lipid X, indicating that this lipid is not phosphorylated.TABLE 1.Retention time of ortho-phthalaldehyde-derivatized LCBs and LCBPs using different HPLC mobile phasesMobile PhaseC14-Sphingosine-1-Phosphate StandardC14-Sphingosine StandardC14-Sphingosine from Fly ExtractLipid X from Fly ExtractminA Methanol/water/1 M TBAP (82:17:1)14.819.119.014.8 Methanol/water/1 M TBAP (79:20:1)22.5 (7.7)27.3 (8.2)27.1 (8.1)22.1 (7.3)B Methanol/10 mM potassium phosphate/1 M TBAP (81:18:1), pH 7.215.021.421.817.1 Methanol/10 mM potassium phosphate/1 M TBAP (81:18:1), pH 5.518.3 (3.3)21.9 (0.5)22.0 (0.2)17.2 (0.1)LCB, long chain base; LCBP, long chain base phosphate; TBAP, tetrabutylammonium dihydrogen phosphate. Values shown are averages of at least three independent measurements. Numbers in parentheses represent the minute change in retention time obtained by changing the percentage of methanol (mobile phase A) or the pH of the potassium phosphate buffer (mobile phase B). Open table in a new tab LCB, long chain base; LCBP, long chain base phosphate; TBAP, tetrabutylammonium dihydrogen phosphate. Values shown are averages of at least three independent measurements. Numbers in parentheses represent the minute change in retention time obtained by changing the percentage of methanol (mobile phase A) or the pH of the potassium phosphate buffer (mobile phase B). As shown in Table 2, lipid X was significantly increased in the Sply mutant compared with wild-type controls. To a lesser degree, lipid X was increased in a null Sk2 transposon insertion mutant that has reduced capacity to phosphorylate LCBs as a result of the loss of expression of one of two sphingosine kinases present in this organism. The Sk2 mutant demonstrates impaired flight performance and reproductive defects that are less severe than those observed in the Sply mutant. In contrast, lipid X was barely detectable in the lace mutant, which is defective in sphingolipid synthesis. Lipid X levels were reduced to wild-type levels in the Sply revertant line Sply14a, which shows minimal if any of the defects observed in the Sply mutant. Lipid X levels in the Sply/lace double mutant were intermediate between those of the Sply mutant and the wild type. These double mutants demonstrate correction of the Sply mutant muscle dropout but exhibit impaired flight performance, similar to the Sk2 mutant. Based on these findings, we hypothesized that lipid X was a sphingolipid and most likely a free LCB and that the level of lipid X accumulation correlates with the presence and severity of flight impairment, reproductive defects, and tissue degeneration.TABLE 2.HPLC analysis of endogenous lipid X in adult flies from different Drosophila strainsLipidCanton-SSply05091lace2/k05305Sply14aSply05091/lace2/k05305Sk2KG05894Lipid X (pmol/mg protein)9.04 ± 2.9563.83 ± 4.410.82 ± 0.419.92 ± 2.3440.63 ± 6.7443.41 ± 4.38Values shown are means ± SD for three independent measurements. Canton-S is the wild type, Sply indicates the homozygous Sply-null mutant, lace is the recessive lethal allele of serine palmitoyltransferase, Sply14a is the homozygous Sply revertant, and Sk2 is the homozygous sphingosine kinase 2 null mutant. Open table in a new tab Values shown are means ± SD for three independent measurements. Canton-S is the wild type, Sply indicates the homozygous Sply-null mutant, lace is the recessive lethal allele of serine palmitoyltransferase, Sply14a is the homozygous Sply revertant, and Sk2 is the homozygous sphingosine kinase 2 null mutant. By LC-MS analysis, lipid X was found to have a molecular weight of 241.1. Because the molecular weight found for C14-sphingosine is 243.1 and the molecular weight found for C14-dihydrosphingosine is 245.1, we suspected that lipid X was a C14-LCB with two double bonds. Figure 1 shows a LC-MS-ESI+ run of lipids extracted from adult flies, identifying LCBs in wild-type and Sply mutant flies. In addition to lipid X at m/z 242.1, a peak at m/z 270.1 was identified, suggesting the presence of the C16 species. To verify the structure of lipid X and map the location of the double bonds, an LC-MS spectrum of lipid X was obtained and compared with that of C14-sphingosine. As shown in Fig. 2A, the peak intensity of intact lipid X (m/z 242.1) was much lower than that of its water-loss product ion (m/z 224.0). In comparison, the peak intensity of the C14-sphingosine (m/z 244.1) was only slightly lower than that of its water-loss product ion (m/z 226.1) (Fig. 2B). The stability of lipid X's water-loss product suggested that the second double bond was conjugated with the first double bond. The fragmentation pattern of the carbon chain of the water-loss component of lipid X (m/z 224.0) was further investigated by AutoMSn up to 3 using an LC-ESI ion trap MS system (Fig. 3). As shown in Fig. 3A, the product ion at m/z 67 was barely detected, whereas that at m/z 93 was the most abundant product i
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