Elucidating the chemical structure of native 1-deoxysphingosine

The 1-deoxysphingolipids (1-deoxySLs) are formed by an alternate substrate usage of the enzyme, serine-palmitoyltransferase, and are devoid of the C1-OH-group present in canonical sphingolipids. Pathologically elevated 1-deoxySL levels are associated with the rare inherited neuropathy, HSAN1, and diabetes type 2 and might contribute to β cell failure and the diabetic sensory neuropathy. In analogy to canonical sphingolipids, it was assumed that 1-deoxySLs also bear a (4E) double bond, which is normally introduced by sphingolipid delta(4)-desaturase 1. This, however, was never confirmed. We therefore supplemented HEK293 cells with isotope-labeled D3-1-deoxysphinganine and compared the downstream formed D3-1-deoxysphingosine (1-deoxySO) to a commercial synthetic SPH m18:1(4E)(3OH) standard. Both compounds showed the same m/z, but differed in their RPLC retention time and atmospheric pressure chemical ionization in-source fragmentation, suggesting that the two compounds are structural isomers. Using dimethyl disulfide derivatization followed by MS2 as well as differential-mobility spectrometry combined with ozone-induced dissociation MS, we identified the carbon-carbon double bond in native 1-deoxySO to be located at the (Δ14) position. Comparing the chromatographic behavior of native 1-deoxySO to chemically synthesized SPH m18:1(14Z) and (14E) stereoisomers assigned the native compound to be SPH m18:1(14Z). This indicates that 1-deoxySLs are metabolized differently than canonical sphingolipids. The 1-deoxysphingolipids (1-deoxySLs) are formed by an alternate substrate usage of the enzyme, serine-palmitoyltransferase, and are devoid of the C1-OH-group present in canonical sphingolipids. Pathologically elevated 1-deoxySL levels are associated with the rare inherited neuropathy, HSAN1, and diabetes type 2 and might contribute to β cell failure and the diabetic sensory neuropathy. In analogy to canonical sphingolipids, it was assumed that 1-deoxySLs also bear a (4E) double bond, which is normally introduced by sphingolipid delta(4)-desaturase 1. This, however, was never confirmed. We therefore supplemented HEK293 cells with isotope-labeled D3-1-deoxysphinganine and compared the downstream formed D3-1-deoxysphingosine (1-deoxySO) to a commercial synthetic SPH m18:1(4E)(3OH) standard. Both compounds showed the same m/z, but differed in their RPLC retention time and atmospheric pressure chemical ionization in-source fragmentation, suggesting that the two compounds are structural isomers. Using dimethyl disulfide derivatization followed by MS2 as well as differential-mobility spectrometry combined with ozone-induced dissociation MS, we identified the carbon-carbon double bond in native 1-deoxySO to be located at the (Δ14) position. Comparing the chromatographic behavior of native 1-deoxySO to chemically synthesized SPH m18:1(14Z) and (14E) stereoisomers assigned the native compound to be SPH m18:1(14Z). This indicates that 1-deoxySLs are metabolized differently than canonical sphingolipids. Sphingolipids are typically formed by the condensation of serine and palmitoyl-CoA, a reaction catalyzed by the serine-palmitoyltransferase (SPT) enzyme. Besides these canonical substrates, SPT can use other acyl-CoAs, but also alanine or glycine, as substrates, which then form a category of atypical 1-deoxysphingolipids (1-deoxySLs) that lack the C1-OH group of canonical sphingolipids (1Merrill Jr, A.H. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics.Chem. Rev. 2011; 111: 6387-6422Crossref PubMed Scopus (504) Google Scholar, 2Zitomer N.C. Mitchell T. Voss K.A. Bondy G.S. Pruett S.T. Garnier-Amblard E.C. Liebeskind L.S. Park H. Wang E. Sullards M.C. et al.Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: a novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals.J. Biol. Chem. 2009; 284: 4786-4795Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Several missense mutations in SPT, which are associated with the rare inherited neuropathy, HSAN1, induce a permanent shift in the substrate specificity of the enzyme resulting in increased 1-deoxySL formation. HSAN1 is a rare autosomal and dominantly inherited axonopathy and is clinically characterized by a progressive loss of pain and temperature sensation, often accompanied by neuropathic pain attacks and skin ulcers (3Penno A. Reilly M.M. Houlden H. Laura M. Rentsch K. Niederkofler V. Stoeckli E.T. Nicholson G. Eichler F. Brown Jr., R.H. et al.Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids.J. Biol. Chem. 2010; 285: 11178-11187Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The 1-deoxySLs are toxic to primary sensory neurons in culture and lead to neurite retraction and the disruption of the neuronal cytoskeleton structure in a dose-dependent manner (3Penno A. Reilly M.M. Houlden H. Laura M. Rentsch K. Niederkofler V. Stoeckli E.T. Nicholson G. Eichler F. Brown Jr., R.H. et al.Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids.J. Biol. Chem. 2010; 285: 11178-11187Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 4Jun B.K. Chandra A. Kuljis D. Schmidt B.P. Eichler F.S. Substrate availability of mutant SPT alters neuronal branching and growth cone dynamics in dorsal root ganglia.J. Neurosci. 2015; 35: 13713-13719Crossref PubMed Scopus (14) Google Scholar). They also interfere with the survival and insulin secretory capacity of pancreatic β cells and 1-deoxySL plasma levels have been found to be prospective biomarkers for the risk to develop diabetes type 2 (5Zuellig R.A. Hornemann T. Othman A. Hehl A.B. Bode H. Guntert T. Ogunshola O.O. Saponara E. Grabliauskaite K. Jang J.H. et al.Deoxysphingolipids, novel biomarkers for type 2 diabetes, are cytotoxic for insulin-producing cells.Diabetes. 2014; 63: 1326-1339Crossref PubMed Scopus (82) Google Scholar, 6Othman A. Bianchi R. Alecu I. Wei Y. Porretta-Serapiglia C. Lombardi R. Chiorazzi A. Meregalli C. Oggioni N. Cavaletti G. et al.Lowering plasma 1-deoxysphingolipids improves neuropathy in diabetic rats.Diabetes. 2015; 64: 1035-1045Crossref PubMed Scopus (54) Google Scholar, 7Othman A. Rütti M.F. Ernst D. Saely C.H. Rein P. Drexel H. Porretta-Serapiglia C. Lauria G. Bianchi R. Eckardstein A.v. et al.Plasma deoxysphingolipids: a novel class of biomarkers for the metabolic syndrome?.Diabetologia. 2012; 55: 421-431Crossref PubMed Scopus (99) Google Scholar). The 1-deoxysphinganine (1-deoxySA), which is formed by SPT, can be converted to 1-deoxyceramides, but not to complex sphingolipids, because of the missing 1-OH group. During catabolism, 1-deoxyceramide is degraded by ceramidase to form 1-deoxysphingosine (1-deoxySO), but is not phosphorylated to form the catabolic intermediate, sphingosine-1-phosphate (S1P). This prevents its cleavage to hexadecenal by S1P-lyase, meaning that 1-deoxySLs cannot be degraded by the canonical catabolic pathway (2Zitomer N.C. Mitchell T. Voss K.A. Bondy G.S. Pruett S.T. Garnier-Amblard E.C. Liebeskind L.S. Park H. Wang E. Sullards M.C. et al.Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: a novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals.J. Biol. Chem. 2009; 284: 4786-4795Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Apart from that, it was assumed that 1-deoxySA is metabolized by the same set of enzymes as canonical sphingoid bases and that 1-deoxySO, like sphingosine, bears a (4E) double bond that is introduced by the sphingolipid, delta(4)-desaturase 1. However, we observed that natively formed 1-deoxySO showed a different RPLC retention time than a synthetic SPH m18:1(4E)(3OH) standard, though the m/z was identical for both compounds. This suggested that native 1-deoxySO and the synthetic SPH m18:1(4E)(3OH) are structural isomers, probably differing in position and/or configuration of the carbon-carbon double bond. To further elucidate this difference, we used a set of tandem MS methods in combination with total synthesis to elucidate the real double bond position and configuration of native 1-deoxySO. Unless stated differently, all solvents and reagents were purchased from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland) excluding methanol, which was purchased from Honeywell Specialty Chemicals Seelze GmBH, Germany. HEK293 cells were fed with 1 μM deuterium-labeled D3-1-deoxySA (Avanti Polar Lipids, Alabaster, AL) or with the unlabeled 1-deoxySA (Avanti Polar Lipids). Cells were harvested after 24 h and the whole sphingolipid extract was hydrolyzed to get the free sphingoid bases, as described previously with some modifications (7Othman A. Rütti M.F. Ernst D. Saely C.H. Rein P. Drexel H. Porretta-Serapiglia C. Lauria G. Bianchi R. Eckardstein A.v. et al.Plasma deoxysphingolipids: a novel class of biomarkers for the metabolic syndrome?.Diabetologia. 2012; 55: 421-431Crossref PubMed Scopus (99) Google Scholar, 8Othman A. Benghozi R. Alecu I. Wei Y. Niesor E. von Eckardstein A. Hornemann T. Fenofibrate lowers atypical sphingolipids in plasma of dyslipidemic patients: A novel approach for treating diabetic neuropathy?.J. Clin. Lipidol. 2015; 9: 568-575Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The cell pellet was dissolved in 100 μl of PBS. Methanol (500 μl), including D7-sphingosine and D7-sphinganine (Avanti Polar Lipids) as the internal standards, was added. Lipids were extracted for 1 h under constant agitation at 37°C. Samples were centrifuged to pellet precipitated proteins and the supernatant was transferred into a new tube. Lipids were hydrolyzed by adding 75 μl of methanolic HCl (1 N HCl and 10 M water in methanol) and incubated for 16 h at 65°C. HCl was neutralized by adding 100 μl of KOH (10 M). Then, 625 μl chloroform was added followed by 100 μl 2 N ammonium hydroxide and 0.5 ml alkaline water to complete phase separation. The sample was vortexed, centrifuged at 16,000 g for 5 min, the upper phase discarded and the lower (organic) phase washed three times with alkaline water. The organic phase was finally dried under N2 and stored at −20°C until analysis. Throughout this work, we refer to the extracted 1-deoxySO as native. A commercial 1-deoxySO standard [SPH m18:1(4E)(3OH)] was purchased from Avanti Polar Lipids. SPH m18:1(14Z)(3OH) was synthesized according to the method described below. The SPH m18:1(E)(3OH) standards (5E, 8E, 12E, 13E, and 14E) were synthesized based on an unpublished method that will be issued elsewhere. An LC-MS method described previously (8Othman A. Benghozi R. Alecu I. Wei Y. Niesor E. von Eckardstein A. Hornemann T. Fenofibrate lowers atypical sphingolipids in plasma of dyslipidemic patients: A novel approach for treating diabetic neuropathy?.J. Clin. Lipidol. 2015; 9: 568-575Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) was used to compare retention times and in-source fragmentation. Sphingoid bases were separated by RPLC on a C18-column (Uptisphere 120 Å, 5 μm, 125 × 2 mm; Interchim, Montluçon, France) and analyzed on a TSQ Quantum Ultra or a Q Exactive (Thermo, Reinach BL, Switzerland) using an atmospheric pressure chemical ionization (APCI) interface. Mobile phases consisted of ultra-pure water/methanol (1/1 v/v) with 2.6 mM ammonium acetate (mobile phase A) and methanol (mobile phase B). Gradient was set from 50% B to 100% B within 25 min followed by 5 min 100% B and 5 min of equilibration with a flow rate of 0.3 ml/min. For mass spectral detection, the following parameters were set on the APCI source: discharge current of 4 μA, vaporizer temperature of 450°C, sheath gas pressure of 20 AU, auxiliary gas pressure of 5 AU, and capillary temperature of 200°C. Dimethyl disulfide (DMDS, 100 μl) and 20 μl of I2 (in diethyl ether, 60 mg/ml) were added to whole cell extracts or 15 nmol SPH m18:1(4E)(3OH) standard. Samples were agitated for 16 h in an Eppendorf Thermo shaker at 1,400 rpm and 35°C. The reaction was quenched with 100 μl of 5% aqueous Na2S2O3, extracted with 200 μl of hexane, and dried under N2. Samples were dissolved in 200 μl isopropanol for further analysis according to the method of Dunkelblum, Tan, and Silk (9Dunkelblum E. Tan S.H. Silk P.J. Double-bond location in monounsaturated fatty acids by dimethyl disulfide derivatization and mass spectrometry: Application to analysis of fatty acids in pheromone glands of four lepidoptera.J. Chem. Ecol. 1985; 11: 265-277Crossref PubMed Scopus (271) Google Scholar). The sample was directly injected into the mass spectrometer at a flowrate of 10 μl/min. The [M+H]+ ion of the DMDS adduct of 1-deoxySO was generated by ESI on a Thermo Fisher Scientific Q-Exactive and both collision-induced dissociation (CID) and high-resolution accurate mass analysis were performed. For detection, the following parameters were set on the ESI source: spray voltage of 4.2 kV, vaporizer temperature of 30°C, sheath gas pressure of 5 AU, auxiliary gas pressure 0 AU, capillary temperature of 320°C, and, for fragmentation, in-source CID was performed at 30 eV and higher-energy CID of the ion at m/z 378.3 was performed with a setting of 25. A SelexIONTM differential-mobility spectrometer was employed with a QTRAP®5500 triple quadrupole ion-trap mass spectrometer (SCIEX, Ontario, Canada). The instrument was modified for ozone-induced dissociation (OzID) as previously described (10Maccarone A.T. Duldig J. Mitchell T.W. Blanksby S.J. Duchoslav E. Campbell J.L. Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry.J. 2014; 55: Full Text Full Text PDF PubMed Scopus Google Scholar). for analysis were in methanol with 5 mM ammonium acetate SPH standard were were to ESI in a and the voltage the was the voltage was constant at V. were from and the of mass at voltage and have been using The ESI and differential-mobility spectrometer cell were 100 and the ESI voltage was set to V. was set to 20 for of the gas in the differential-mobility spectrometer the ESI and the MS the differential-mobility spectrometer cell into the mass spectrometer were mass in quadrupole to in the cell with was by an at in from which a was into the gas to the mass spectrometer a The time for in the cell was 100 and 15 on the of a A.T. Campbell J.L. Mitchell T.W. Blanksby S.J. dissociation of reaction rate and PubMed Scopus Google Scholar, M.C. Campbell J.L. Mitchell T.W. Blanksby S.J. dissociation on a modified tandem of different for 2010; PubMed Scopus Google Scholar). were transferred to the quadrupole mass analysis was performed using a at at were 2 and 5 compounds to the SPH were synthesized as described in the A of compound in (1 at was with a of 4 M a of 10 The resulting reaction was to for 1 h at the same to and followed by analysis. for an 2 h by with the reaction was under The was dissolved in and washed with water and The organic was dried and in to a of the using acetate and isopropanol as 0 to isopropanol in the compound as with (500 high-resolution MS m/z for [M+H]+ found HEK293 cells were in the of deuterium-labeled D3-1-deoxySA or unlabeled Cells were harvested after 24 h and the of the extracted sphingoid bases analyzed by We observed the of [M+H]+ with m/z and of m/z which both at the same time from the that 1-deoxySO is a formed downstream of 1-deoxySA and time and APCI in-source was compared native 1-deoxySO and a synthetic SPH m18:1(4E)(3OH) standard. The native 1-deoxySO from the after the synthetic SPH m18:1(4E)(3OH) after min The [M+H]+ formed by APCI showed in-source and a water loss for the SPH m18:1(4E)(3OH) standard, which was for the native 1-deoxySO both showed identical m/z using high-resolution accurate MS for [M+H]+ and the observed in retention time and in-source suggested that native 1-deoxySO and the synthetic SPH m18:1(4E)(3OH) were structural isomers. the synthetic SPH m18:1(4E)(3OH) and the native 1-deoxySO showed different RPLC we to elucidate the carbon-carbon double bond position of native 1-deoxySO by derivatization with The DMDS of 1-deoxySO were analyzed by high-resolution accurate MS using and The [M+H]+ from the DMDS of 1-deoxySO were at m/z 378.3 and to The CID showed four but at m/z m/z m/z and m/z to the loss of and water as two but were observed at m/z and m/z were formed by a cleavage of the carbon-carbon bond the two The mass of the two a double bond in for native 1-deoxySO. a at the and were not previously been for to mass analysis (10Maccarone A.T. Duldig J. Mitchell T.W. Blanksby S.J. Duchoslav E. Campbell J.L. Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry.J. 2014; 55: Full Text Full Text PDF PubMed Scopus Google Scholar, G. N. and of using differential ion spectrometry.J. 2011; PubMed Scopus Google Scholar). it was combined with which the reaction and a mass spectrometer to of the of carbon-carbon double M.C. Campbell J.L. Mitchell T.W. Blanksby S.J. dissociation on a modified tandem of different for 2010; PubMed Scopus Google Scholar, M.C. Mitchell T.W. Blanksby S.J. of double bond position within Chem. PubMed Scopus Google Scholar). In these the hydrolyzed extract from cells was with a of the unlabeled commercial SPH m18:1(4E)(3OH) and synthesized SPH structural and the to DMDS for m/z and m/z in mass to the [M+H]+ from unlabeled and 1-deoxySO, The to at two chemically of m/z with a at and at 20 V. m/z in this be by the of the synthetic SPH m18:1(4E)(3OH) and SPH isomers. The to of m/z showed an of at and 20 V. be assigned to [M+H]+ from native of 1-deoxySO present in the cell To the structure of the for of the two in the were for m/z and using of and 20 V. from the synthetic m/z in and the native m/z in Both from loss of and 24 of of a carbon-carbon double bond at the on the sphingoid S.H. Mitchell T.W. Blanksby S.J. of unsaturated by ozone-induced 2011; PubMed Scopus Google Scholar) and in the at a of 20 these were from cleavage of a bond of and were observed at m/z and 120 for the unlabeled standard and this of the is and as at m/z for the the m/z is in this to the lower of the 20 in the native an water loss was observed in the in which is in the in further of the structural the two analysis was also performed on extract from 1-deoxyceramide be as [M+H]+ with the and the showed a loss of as a for 1-deoxySO with a carbon-carbon double bond at the the of the (Δ14) carbon-carbon double bond in both 1-deoxyceramide and the 1-deoxySO and the that the observed (Δ14) is formed as an of the the analysis the (Δ14) carbon-carbon double bond in native 1-deoxySO, it not the of the bond. To further the position and configuration of the double bond, a of 1-deoxySO with different double bond and (5E, 8E, 12E, 13E, and were we on the synthesis of the SPH as on the synthesis of the other will be elsewhere. The synthesis of SPH m18:1(14Z)(3OH) from of with in the of a of the in of compound to the 4 was of with and to compound 4 in the with was performed following a previously described J. Y. synthesis of and their Scopus Google Scholar) with some modifications 1 for was added to at and the in generated was with and substrate 4 to compound 5 in a of 5 was with after of reaction to the of the as a 2 for is that to to a that was with analysis of of the identified these as the and the of the group in under to the which was to reaction with to the the chain was converted into the and to the 2 from in two the for this 2 was with of followed by of the to 10 in The of the group in 10 with in at was of the double bond to the as a of the group of with 4 M the of SPH m18:1(14Z)(3OH) The was finally after in The RPLC retention times of the synthetic SPH m18:1(E)(3OH) standards with double in 8E, 12E, were We observed an retention times and the double bond position The the double bond was the the the from the The in retention time native 1-deoxySO and the synthetic standards was for SPH the time the two compounds differed by 30 To further elucidate the we compared the retention times of native 1-deoxySO with the synthetic standard in configuration The SPH m18:1(14Z) at min and therefore with the time of native 1-deoxySO. these we that native 1-deoxySO bears a double bond. The 1-deoxySO is an atypical sphingolipid that the group of canonical is a downstream of which is formed by SPT to its with of the missing 1-deoxySO cannot be phosphorylated to and, also cannot be degraded by (2Zitomer N.C. Mitchell T. Voss K.A. Bondy G.S. Pruett S.T. Garnier-Amblard E.C. Liebeskind L.S. Park H. Wang E. Sullards M.C. et al.Ceramide synthase inhibition by fumonisin B1 causes accumulation of 1-deoxysphinganine: a novel category of bioactive 1-deoxysphingoid bases and 1-deoxydihydroceramides biosynthesized by mammalian cell lines and animals.J. Biol. Chem. 2009; 284: 4786-4795Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Pathologically elevated 1-deoxySLs an in the inherited neuropathy but were also found in other like the metabolic and type 2 diabetes (3Penno A. Reilly M.M. Houlden H. Laura M. Rentsch K. Niederkofler V. Stoeckli E.T. Nicholson G. Eichler F. Brown Jr., R.H. et al.Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids.J. Biol. Chem. 2010; 285: 11178-11187Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 7Othman A. Rütti M.F. Ernst D. Saely C.H. Rein P. Drexel H. Porretta-Serapiglia C. Lauria G. Bianchi R. Eckardstein A.v. et al.Plasma deoxysphingolipids: a novel class of biomarkers for the metabolic syndrome?.Diabetologia. 2012; 55: 421-431Crossref PubMed Scopus (99) Google Scholar). However, the of 1-deoxySLs and their have not been in Comparing native 1-deoxySO to a synthetic SPH standard by we observed a in retention the m/z was identical for both the DMDS of native 1-deoxySO by MS2 two which the double bond position at (Δ14) and, to that of the canonical The was further using differential-mobility spectrometry combined with Comparing retention times of native 1-deoxySO to a set of synthetic SPH standards finally a in native 1-deoxySO. During sphingolipid the sphingolipid delta(4)-desaturase 1 normally a (4E) double bond into the sphingoid of to form The that native 1-deoxySO bears a of a (4E) indicates that the of 1-deoxySL from that of canonical sphingolipids. a downstream of T. A. of by of the 2015; Full Text Full Text PDF PubMed Scopus Google double at both the (4E) and The of a double bond in both and native 1-deoxySO that both are of the same the enzyme is of plasma Full Text PDF PubMed Google Scholar). The that added 1-deoxySA is converted to 1-deoxySO also showed that the double bond is introduced downstream of 1-deoxySA and not formed by an of an unsaturated the downstream of which is formed by the condensation with glycine, to a The m/z as well as RPLC retention time of native that of a synthetic standard this the (4E) position of canonical sphingolipids. In we from to native 1-deoxySO as it is formed in HEK293 cells to be a SPH m18:1(14Z)(3OH) This that are metabolized from canonical sphingolipids. However, are to further the pathways and metabolic in the of these The M. as in the of the and been in the at the of the The Mitchell for the with atmospheric pressure chemical ionization collision-induced dissociation voltage double bond dimethyl disulfide differential-mobility spectrometry 1-deoxysphinganine 1-deoxysphingosine ozone-induced dissociation sphingosine-1-phosphate serine-palmitoyltransferase

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