On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid
2016; Elsevier BV; Volume: 57; Issue: 10 Linguagem: Inglês
10.1194/jlr.m067637
ISSN1539-7262
AutoresPhilippe Pierre Robichaud, Samuel Poirier, Luc H. Boudreau, Jérémie A. Doiron, David A. Barnett, Éric Boilard, Marc E. Surette,
Tópico(s)RNA Interference and Gene Delivery
ResumoAlkyne and azide analogs of natural compounds that can be coupled to sensitive tags by click chemistry are powerful tools to study biological processes. Arachidonic acid (AA) is a FA precursor to biologically active compounds. 19-Alkyne-AA (AA-alk) is a sensitive clickable AA analog; however, its use as a surrogate to study AA metabolism requires further evaluation. In this study, AA-alk metabolism was compared with that of AA in human cells. Jurkat cell uptake of AA was 2-fold greater than that of AA-alk, but significantly more AA-Alk was elongated to 22:4. AA and AA-alk incorporation into and remodeling between phospholipid (PL) classes was identical indicating equivalent CoA-independent AA-PL remodeling. Platelets stimulated in the presence of AA-alk synthesized significantly less 12-lipoxygenase (12-LOX) and cyclooxygenase products than in the presence of AA. Ionophore-stimulated neutrophils produced significantly more 5-LOX products in the presence of AA-alk than AA. Neutrophils stimulated with only exogenous AA-alk produced significantly less 5-LOX products compared with AA, and leukotriene B4 (LTB4)-alk was 12-fold less potent at stimulating neutrophil migration than LTB4, collectively indicative of weaker leukotriene B4 receptor 1 agonist activity of LTB4-alk. Overall, these results suggest that the use of AA-alk as a surrogate for the study of AA metabolism should be carried out with caution. Alkyne and azide analogs of natural compounds that can be coupled to sensitive tags by click chemistry are powerful tools to study biological processes. Arachidonic acid (AA) is a FA precursor to biologically active compounds. 19-Alkyne-AA (AA-alk) is a sensitive clickable AA analog; however, its use as a surrogate to study AA metabolism requires further evaluation. In this study, AA-alk metabolism was compared with that of AA in human cells. Jurkat cell uptake of AA was 2-fold greater than that of AA-alk, but significantly more AA-Alk was elongated to 22:4. AA and AA-alk incorporation into and remodeling between phospholipid (PL) classes was identical indicating equivalent CoA-independent AA-PL remodeling. Platelets stimulated in the presence of AA-alk synthesized significantly less 12-lipoxygenase (12-LOX) and cyclooxygenase products than in the presence of AA. Ionophore-stimulated neutrophils produced significantly more 5-LOX products in the presence of AA-alk than AA. Neutrophils stimulated with only exogenous AA-alk produced significantly less 5-LOX products compared with AA, and leukotriene B4 (LTB4)-alk was 12-fold less potent at stimulating neutrophil migration than LTB4, collectively indicative of weaker leukotriene B4 receptor 1 agonist activity of LTB4-alk. Overall, these results suggest that the use of AA-alk as a surrogate for the study of AA metabolism should be carried out with caution. 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Porter N.A. omega-Alkynyl lipid surrogates for polyunsaturated fatty acids: free radical and enzymatic oxidations.J. Am. Chem. Soc. 2014; 136: 11529-11539Crossref PubMed Scopus (21) Google Scholar). AA is the PUFA precursor to a number of potent biologically active molecules such as prostaglandins and leukotrienes; thus this probe may serve as a suitable tracker for cellular AA metabolism that is under tight regulation (Fig. 2). However, prior to utilizing any new analog as a surrogate in metabolic studies, it is important to make sure that its metabolism and regulation resembles that of AA itself. Mammalian cells cannot synthesize AA de novo and must obtain this essential FA from exogenous sources as intact AA or as one of its precursors. Cells mainly store AA in the sn-2 position of membrane PLs, although AA can also be elongated to 22:4 n-6 prior to its incorporation into PLs. AA undergoes a specific pattern of incorporation into PLs where initial acylation is primarily in PC and PI resulting from reactions catalyzed by ACSs and lyso-PLATs. Once AA is incorporated into PC species, it is then transferred primarily into 1-radyl PE species by a CoA-IT-catalyzed reaction (19Yamashita A. Hayashi Y. Nemoto-Sasaki Y. Ito M. Oka S. Tanikawa T. Waku K. Sugiura T. Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms.Prog. Lipid Res. 2014; 53: 18-81Crossref PubMed Scopus (163) Google Scholar, 20Kramer R.M. Deykin D. Arachidonoyl transacylase in human platelets. Coenzyme A-independent transfer of arachidonate from phosphatidylcholine to lysoplasmenylethanolamine.J. Biol. Chem. 1983; 258: 13806-13811Abstract Full Text PDF PubMed Google Scholar, 21Fonteh A.N. Chilton F.H. 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Opin. Endocrinol. Diabetes Obes. 2015; 22: 112-118Crossref PubMed Scopus (21) Google Scholar).Fig. 2Schematic representation of cellular AA incorporation and metabolism. Once incorporated into cells, free AA is activated by acyl-CoA synthetases (ACSs) to produce the AA-CoA required for its incorporation into phospholipids (PLs) by the action of lysophospholipid acyl-CoA acyltransferases (lyso-PLATs). The action of phospholipase A2 (PLA2) is required to generate the 2-lyso-PL substrate. AA-CoA can also be elongated to 22:4 n-6 following the action of elongases of very long chain FAs (ELOVL). Once incorporated into PLs, AA can also be directly transferred between PL species by CoA-independent transacylase (CoA-IT)-catalyzed reactions. Upon cell stimulation, PLA2 catalyzes the hydrolysis of AA from PLs, which can be converted into various lipid mediators (eicosanoids) by the action of cyclooxygenases (COXs) and lipoxygenases (LOXs). 12-HHTrE, 12-hydroxyheptadecatrienoic acid; LTA4H, leukotriene A4 hydrolase; LTB4, leukotriene B4; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PGH2, prostaglandin H2; PI, phosphatidylinositol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Upon appropriate cell stimulation, AA can be hydrolyzed from PL by PLA2 and can be converted by LOXs and COXs into many different bioactive lipid mediators, called eicosanoids, which include HETEs, leukotrienes, prostaglandins, and lipoxins (24Robichaud P.P. Surette M.E. Polyunsaturated fatty acid-phospholipid remodeling and inflammation.Curr. Opin. Endocrinol. Diabetes Obes. 2015; 22: 112-118Crossref PubMed Scopus (21) Google Scholar, 25Rådmark O. Samuelsson B. 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The 1,2-diheptadecanoyl-PC was from Biolynx (Brockville, ON, Canada). FA methyl esters (FAMEs) and FFAs were obtained from Nu-check Prep (Elysian, MN). AA-alk was purchased from NuChem Thérapeutiques Inc. (Montreal, QC, Canada). Jurkat cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 10 mM HEPES, d-glucose (to 25 mM) and 1 mM sodium pyruvate at 37°C in a 5% CO2 atmosphere. For FA incorporation studies, Jurkat cells were incubated in the presence of 20 μM AA or 20 μM AA-alk for 2 h at 37°C. Cells were then washed twice with culture medium, and cellular lipids were extracted in chloroform using heptadecanoyl-PC as an internal standard (30Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar). For pulse-labeling experiments, Jurkat cells (6 × 107) were pulse labeled in 3 ml of culture medium (2% FBS) containing 20 μM [3H]AA or 20 μM AA-alk for 2 h at 37°C. Cells were then washed twice with culture medium and incubated for another 0, 4, or 24 h before cellular lipid extraction. Cellular lipids were extracted in chloroform (30Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42689) Google Scholar), PL classes were separated by reverse phase-HPLC (RP-HPLC) (31Chilton F.H. Separation and characterization of arachidonate-containing phosphoglycerides.Methods Enzymol. 1990; 187: 157-167Crossref PubMed Scopus (9) Google Scholar), and fractions containing neutral lipids, PE, PI, phosphatidylserine (PS), and PC were collected using elution times determined with PL standards. The internal standard heptadecanoyl-PC was added to each fraction prior to further analyses. Cellular lipid extracts or HPLC fractions were dried and saponified with 400 µl of 0.5 M KOH in methanol at 100°C for 15 min, and FAMEs were then prepared by adding 1 ml of 14% BF3 in methanol and heating at 100°C for 10 min. FAMEs were extracted in hexane and quantified by gas chromatography with flame ionization detection (GC/FID) using a 30 m trace-FAME column on a Thermo Trace gas chromatograph (Thermo Electron Corporation, Mississauga, ON, Canada) (32Surette M.E. Koumenis I.L. Edens M.B. Tramposch K.M. Chilton F.H. Inhibition of leukotriene synthesis, pharmacokinetics, and tolerability of a novel dietary fatty acid formulation in healthy adult subjects.Clin. Ther. 2003; 25: 948-971Abstract Full Text PDF PubMed Scopus (41) Google Scholar). Authentic FAME standards were used for the identification of FA peak retention times and for standard curve quantification. For pulse-label studies, the radioactivity was also measured in each fraction by liquid scintillation counting (Beckman Instruments LS 5000 CE). To confirm the identity of the 22:4-alkyne, FAMEs were analyzed and measured by positive ion chemical ionization GC/MS using a Polaris Q mass spectrometer (Thermo). The positive chemical ionization ion trap scan was 300–350 u, the reagent gas was methane (0.6 ml/min, 180°C), and helium was the damping gas (0.3 ml/min). Platelets were isolated from heparinized blood obtained from healthy donors as previously described (33Cloutier N. Tan S. Boudreau L.H. Cramb C. Subbaiah R. Lahey L. Albert A. Shnayder R. Gobezie R. Nigrovic P.A. et al.The exposure of autoantigens by microparticles underlies the formation of potent inflammatory components: the microparticle-associated immune complexes.EMBO Mol. Med. 2013; 5: 235-249Crossref PubMed Scopus (186) Google Scholar). Briefly, blood was centrifuged at 200 g for 10 min at room temperature. The platelet-rich plasma fraction (upper phase) was collected and centrifuged at 400 g for 2 min to remove remaining erythrocytes. Platelets were then pelleted following centrifugation at 1,300 g for 10 min, and platelets (300 × 106 cells/ml) were resuspended in Tyrode buffer (134 mM NaCl, 2.9 mM KCl, 20 mM HEPES, 5 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 0.34 mM Na2HPO4, 12 mM NaHCO3, and 0.5 mg/ml BSA, pH 7.4). Stimulation of platelets was initiated with the addition of 10 μM calcium ionophore A23187 (Sigma-Aldrich) in the presence of 10 μM AA or 10 μM AA-alk for 15 min at 37°C. Stimulations were stopped with the addition of 2 vol of cold methanol containing 50 ng of 19-OH-prostaglandin B2 (PGB2; internal standard), and samples were stored at −20°C prior to analysis by RP-HPLC. Neutrophils were isolated from heparinized blood obtained from healthy donors as previously described (34Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g.Scand. J. Clin. Lab. Invest. Suppl. 1968; 97: 77-89PubMed Google Scholar). Briefly, blood was centrifuged at 200 g for 10 min at room temperature, the platelet-rich plasma was discarded, and erythrocytes were removed following dextran sedimentation. Following centrifugation on a lymphocyte separation medium cushion (density, 1.077 g/ml) (Wisent, St. Bruno, QC, Canada) at 900 g for 20 min at room temperature, mononuclear cells were eliminated, and neutrophils (>96%) were obtained from the pellet after hypotonic lysis in purified water to eliminate contaminating erythrocytes. Neutrophils suspended in HBSS (1 × 107 cells/ml) containing 1.6 mM CaCl2 and 0.4 U/ml of adenosine deaminase were stimulated with 10 μM calcium ionophore A23187 in the presence of 10 μM AA or 10 μM AA-alk for 5 min at 37°C. For autocrine loop experiments, neutrophils were stimulated with varying concentrations of AA or AA-alk for 5 min at 37°C (35Surette M.E. Krump E. Picard S. Borgeat P. Activation of leukotriene synthesis in human neutrophils by exogenous arachidonic acid: inhibition by adenosine A(2a) receptor agonists and crucial role of autocrine activation by leukotriene B(4).Mol. Pharmacol. 1999; 56: 1055-1062Crossref PubMed Scopus (65) Google Scholar). All neutrophil stimulations were stopped with the addition of 0.5 vol of cold methanol containing 25 ng of 19-OH-PGB2, and samples were stored at −20°C prior to analysis. HEK293 cells that were stably transfected to express human 5-LOX and human 5-LOX activating protein (FLAP) (36Boudreau L.H. Bertin J. Robichaud P.P. Laflamme M. Ouellette R.J. Flamand N. Surette M.E. Novel 5-lipoxygenase isoforms affect the biosynthesis of 5-lipoxygenase products.FASEB J. 2011; 25: 1097-1105Crossref PubMed Scopus (20) Google Scholar, 37Allain E.P. Boudreau L.H. Flamand N. Surette M.E. The intracellular localisation and phosphorylation profile of the human 5-lipoxygenase delta13 isoform differs from that of its full length counterpart.PLoS One. 2015; 10: e0132607Crossref PubMed Scopus (16) Google Scholar) were cultured in DMEM medium supplemented with 10% FBS at 37°C in a humidified 5% CO2 environment. Cells were washed, suspended in HBSS (1 × 107 cells/ml) containing 1.6 mM CaCl2, and stimulated with 10 μM calcium ionophore A23187 in the presence of 10 μM AA or 10 μM AA-alk for 15 min at 37°C. Stimulations were stopped with the addition of 0.5 vol of cold methanol containing 25 ng of 19-OH-PGB2, and samples were stored at −20°C prior to analysis by RP-HPLC. Samples were centrifuged at 300 g to remove precipitated proteins, and the supernatants were diluted with water to obtain a final methanol content of 20% (v/v). Samples were then subjected to in-line solid phase extraction and RP-HPLC analysis with UV detection optimized to separate LOX products as previously described (38Borgeat P. Picard S. Vallerand P. Bourgoin S. Odeimat A. Sirois P. Poubelle P.E. Automated on-line extraction and profiling of lipoxygenase products of arachidonic acid by high-performance liquid chromatography.Methods Enzymol. 1990; 187: 98-116Crossref PubMed Scopus (62) Google Scholar) with some variations. Briefly, samples were injected onto an Agilent 1100 HPLC equipped with an Oasis HLB online cartridge column (3.9 × 20 mm, 15 μm particle size; Waters, Milford, MA) for in-line extraction using 0.1% acetic acid as mobile phase at a flow rate of 3 ml/min for 3 min. The solvent was then changed over 0.1 min to solvent A (54% water:23% methanol:23% acetonitrile:0.0025% H3PO4), and a Rheodyne® valve was switched to direct the flow to a Chromolith® HighResolution RP-18 end-capped column (100 × 4.6 mm) (EMD Millipore, Etobicoke, ON, Canada) at a flow rate of 2.2 ml/min. After 5.11 min, the mobile phase was then changed to 85% solvent A and 15% solvent B (5% water:32% methanol:63% acetonitrile:0.01% H3PO4) for 1 min, followed by a linear gradient to 55% solvent A and 45% solvent B over the next 0.3 min, and held at that proportion for an additional 1.3 min. The gradient was then changed in a linear fashion to 30% solvent A and 70% solvent B over a 1.3 min period and held for an additional 1.3 min at which time the mobile phase was changed to 100% solvent B over 0.2 min and held for 3.5 min. Peaks were quantified by absorbance at 236 nm and 270 nm using a diode array detector. Selected peaks eluting from the above-mentioned HPLC analyses were collected for further characterization by MS. LC/MS/MS analysis was performed using a Dionex Ultimate 3000 liquid chromatograph coupled to a Thermo-Fisher Scientific Linear Ion Trap (LTQ-XL) using a Hypersil Gold C18 column (150 mm × 2.1 mm inner diameter) with a solvent gradient of 50% to 100% methanol (solvent B) over 40 min at a flow rate of 100 μl/min. Solvent A consisted of water, and both solvents were of HPLC grade with no buffer additives. The sample injection volume was constant for all samples at 5 μl. The mass spectrometer was operated in negative ion mode with LC/MS spectra collected in full scan mode over an m/z range of 200–800 and LC/MS/MS spectra collected from 90 to 400. The MS/MS collision energy was set to 35% with an isolation mass width of 3. Interface parameters for the mass spectrometer were as follows: sheath gas (15, arbitrary units), auxiliary gas (1, arbitrary units), capillary temperature (250°C), capillary voltage (−45 volts), and tube lens voltage (−150 volts). Full scan MS and MS/MS spectra were collected for separate LC injections of 5 μl. In order to produce LTB4 and LTB4-alk for functional studies, HEK293 cells that were stably transfected to express 5-LOX and FLAP (36Boudreau L.H. Bertin J. Robichaud P.P. Laflamme M. Ouellette R.J. Flamand N. Surette M.E. Novel 5-lipoxygenase isoforms affect the biosynthesis of 5-lipoxygenase products.FASEB J. 2011; 25: 1097-1105Crossref PubMed Scopus (20) Google Scholar, 37Allain E.P. Boudreau L.H. Flamand N. Surette M.E. The intracellular localisation and phosphorylation profile of the human 5-lipoxygenase delta13 isoform differs from that of its full length counterpart.PLoS One. 2015; 10: e0132607Crossref PubMed Scopus (16) Google Scholar) were stimulated with A23187 in the presence of 40 μM of AA or AA-alk, respectively. 5-LOX products were separated by RP-HPLC as described above, and the LTB4 and LTB4-alk peaks were collected, dried, and resuspended in ethanol. Control experiments were performed with nontransfected HEK293 cells that do not express 5-LOX and FLAP and do not produce 5-LOX products (36Boudreau L.H. Bertin J. Robichaud P.P. Laflamme M. Ouellette R.J. Flamand N. Surette M.E. Novel 5-lipoxygenase isoforms affect the biosynthesis of 5-lipoxygenase products.FASEB J. 2011; 25: 1097-1105Crossref PubMed Scopus (20) Google Scholar). To measure the chemoattractant activity of LTB4 and LTB4-alk, 200 µl of neutrophil suspension (2.5 × 106 cells/ml HBSS containing 1.6 mM CaCl2 and 5% FBS) preincubated with 0.3 U/ml adenosine deaminase were transferred to cell culture inserts (3.0 µm pore size; Falcon). Neutrophils were allowed to migrate (2 h, 37°C, 5% CO2) to the lower chamber containing 700 µl HBSS/1.6 mM CaCl2 along with 10 nM of LTB4-alk, LTB4, or diluent as negative control. Inserts were then discarded, and cells that had migrated to the lower chamber were counted using the MOXI Z Mini automated cell counter (Orflo Technologies, Ketchum, ID). Calculations were performed as previously described (39Provost V. Larose M.C. Langlois A. Rola-Pleszczynski M. Flamand N. Laviolette M. CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.J. Leukoc. Biol. 2013; 94: 213-222Crossref PubMed Scopus (81) Google Scholar). Statistical analyses were performed using Prism software (GraphPad Software Inc., La Jolla, CA) as described in the figure legends. Data show the means ± SEM for n = 3 to n = 6 independent experiments. This study was approved by the Université de Moncton institutional Review Committee for Research involving human subjects. All subjects provided informed consent prior to their participation in the study. FAMEs were prepared from pure AA and AA-alk and were separated by GC. It was determined that AA-alk-methyl ester eluted at a retention time of ∼17.5 min, thus about 6 min later than that of AA-methyl ester (not shown). To compare the ability of cells to take up exogenous AA-alk and AA, Jurkat cells were incubated with 20 μM of each FA for 2 h, cells were washed, lipids were extracted, FAMEs were prepared, and cellular FAs were measured. In cells incubated with AA-alk, a peak corresponding to AA-alk was observed on chromatograms, as well as second peak eluting at 21 min (supplemental Figure S1). Based on the relative elution profiles of AA-alk, AA, and 22:4n-6, this second peak eluted at a retention time where the elongation product 22:4-alk would be expected. The identity of the putative 22:4-alk was confirmed by MS where the molecular mass of the FAME was determined to be m/z 343.3, which is the expected mass of the protonated methyl-22:4-alk. The quantities of AA-alk and 22:4-alk were approximately equivalent suggesting a very efficient elongation of AA-alk during this 2 h incubation period (Fig. 3). In cells incubated with exogenous AA, both the cellular AA and 22:4 content increased following the 2 h incubation period, though the increase in cellular AA was ∼4-fold greater than that of 22:4 (Fig. 3). When the total uptake of exogenous AA and AA-alk were measured, including their elongation products, cells incorporated nearly two times more AA than AA-alk during this 2 h incubation period (Fig. 3). Overall this indicates that while the capacity to incorporate AA into cells was greater than that of AA-alk, a greater proportion of the AA-alk was elongated compared with AA. The uptake of exogenous AA into glycerophospholipids is known to follow a distinct pattern where the initial incorporation of AA is primarily into PC species, followed by a CoA-IT-driven remodeling into PE species (Fig. 2) (20Kramer R.M. Deykin D. Arachidonoyl transacylase in human platelets. Coenzyme A-independent transfer of arachidonate from phosphatidylcholine to lysoplasmenylethanolamine.J. Biol. Chem. 1983; 258: 138
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