Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays. Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays. In vitro enzymatic assays are a major source of information about the characteristics of enzymes. In particular the determination of functional parameters relies on data in well-defined in vitro systems. Enzymes of lipid metabolism have been investigated with radiolabeled substrates for decades (1Weiss S.B. Kennedy E.P. Kiyasu J.Y. The enzymatic synthesis of triglycerides.J. Biol. Chem. 1960; 235: 40-44Abstract Full Text PDF PubMed Google Scholar). They represent ideal tracers that display all the characteristics of the unlabeled compound, but their synthesis or use can be laborious, requires special equipment, is often subject to special regulations, and the quantification of signal can be tedious. In recent times, fluorescently tagged lipids have been developed as an alternative to radioactive labeling. They are convenient and sensitive reporters, but in general contain spacious tags of high impact on the structure, likely to influence specificity and kinetic parameters of enzymatic reactions. Although there are reports of assays with unchanged affinity of the enzyme to fluorescently labeled substrates, notably nitrobenzoxadiazole (NBD)-sphingolipids [e.g., NBD-sphinganine (2Kim H.J. Qiao Q. Toop H.D. Morris J.C. Don A.S. A fluorescent assay for ceramide synthase activity.J. Lipid Res. 2012; 53: 1701-1707Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), NBD-sphingosine-1-phosphate (3Bandhuvula P. Fyrst H. Saba J.D. A rapid fluorescence assay for sphingosine-1-phosphate lyase enzyme activity.J. Lipid Res. 2007; 48: 2769-2778Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar)] the applicability of a substrate has to be validated thoroughly. For instance, when the boron dipyrromethene (BDP) analog, BDP-sphingosine-1-phosphate, was developed as a more photostable and hydrophobic alternative to NBD-sphingosine-1-phosphate, the BDP-, but not the NBD-derivate displayed a higher Km value than the natural substrate (4Bandhuvula P. Li Z. Bittman R. Saba J.D. Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate.Biochem. Biophys. Res. Commun. 2009; 380: 366-370Crossref PubMed Scopus (27) Google Scholar). A smaller nonradioactive universally applicable tag with less impact on the substrate structure would hence be beneficial. Click chemistry (5Kolb H.C. Finn M.G. Sharpless K.B. Click chemistry: diverse chemical function from a few good reactions.Angew. Chem. Int. Ed. Engl. 2001; 40: 2004-2021Crossref PubMed Scopus (11509) Google Scholar), and especially the copper-catalyzed Huisgen 1,3-dipolar cycloaddition between a terminal alkyne group and an azide, provide new possibilities for the bioorthogonal labeling of molecules like amino acids (6Dieterich D.C. Link A.J. Graumann J. Tirrell D.A. Schuman E.M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).Proc. Natl. Acad. Sci. USA. 2006; 103: 9482-9487Crossref PubMed Scopus (551) Google Scholar), sugars (7Saxon E. Bertozzi C.R. Cell surface engineering by a modified Staudinger reaction.Science. 2000; 287: 2007-2010Crossref PubMed Scopus (1845) Google Scholar), and nucleotides (8Salic A. Mitchison T.J. A chemical method for fast and sensitive detection of DNA synthesis in vivo.Proc. Natl. Acad. Sci. USA. 2008; 105: 2415-2420Crossref PubMed Scopus (1323) Google Scholar), which have already triggered many applications in life sciences (9Baskin J.M. Bertozzi C.R. Bioorthogonal click chemistry: covalent labeling in living systems.QSAR Comb. Sci. 2007; 26: 1211-1219Crossref Scopus (251) Google Scholar, 10Best M.D. Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules.Biochemistry. 2009; 48: 6571-6584Crossref PubMed Scopus (509) Google Scholar). In lipid biology, alkyne and azide analogs and precursors are already applied broadly, e.g., in protein lipidation analyses [reviewed in (11Charron G. Wilson J. Hang H.C. Chemical tools for understanding protein lipidation in eukaryotes.Curr. Opin. Chem. Biol. 2009; 13: 382-391Crossref PubMed Scopus (56) Google Scholar)], receptor binding studies (12Smith M.D. Sudhahar C.G. Gong D. Stahelin R.V. Best M.D. Modular synthesis of biologically active phosphatidic acid probes using click chemistry.Mol. Biosyst. 2009; 5: 962-972Crossref PubMed Scopus (20) Google Scholar), tracing of fatty acid metabolism (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google Scholar), and the labeling of lipids in vivo followed by fluorescence imaging (14Jao C.Y. Roth M. Welti R. Salic A. Metabolic labeling and direct imaging of choline phospholipids in vivo.Proc. Natl. Acad. Sci. USA. 2009; 106: 15332-15337Crossref PubMed Scopus (159) Google Scholar, 15Neef A.B. Schultz C. Selective fluorescence labeling of lipids in living cells.Angew. Chem. Int. Ed. Engl. 2009; 48: 1498-1500Crossref PubMed Scopus (203) Google Scholar). In addition to these applications, it is especially worthwhile to test the feasibility of click-based in vitro assays in lipid metabolism, for which other labeled substrates often are not readily at hand. As the alkyne moiety is a small, neither overly hydrophobic nor hydrophilic label, a good affinity of enzymes to alkyne-modified substrates can be expected. For the first time this study addresses the usage of alkyne lipids in enzymatic assays through the determination of Michaelis-Menten kinetics for these substrates. Here we describe a click-based in vitro enzymatic assay to study enzymes of lipid metabolism, notably from the families of lysophosphatidic acid acyltransferases (LPAATs), lysophosphatidylcholine acyltransferases (LPCATs), and ceramide synthases (CerSs). The chemical syntheses of the alkyne lipids are available in the supplementary material to this study, or were described previously (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google Scholar). E. coli (strain RosettaTM 2 pLysS, Merck Millipore) cultured in Luria Broth at 37°C to an OD600 of 1.6 were harvested by centrifugation at 3,000 g for 10 min. Microsomal fractions were prepared according to Lewin, Wang, and Coleman (16Lewin T.M. Wang P. Coleman R.A. Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction.Biochemistry. 1999; 38: 5764-5771Crossref PubMed Scopus (233) Google Scholar), frozen in liquid nitrogen and stored at −80°C. The protein concentration in the microsomes was determined with the BioRad Bradford assay kit using BSA as a standard. HuH7 cells were grown in RPMI 1640 (PAN Biotech P04-17500) supplemented with 10 mM HEPES, 0.1 mM nonessential amino acids, 2 mM l-glutamine, 10% FBS, at 5% CO2. Cells were washed, scraped into ice-cold buffer (20 mM HEPES/NaOH, pH 7.0, 200 mM sucrose) and homogenized in a cooled EMBL cell cracker (HGM, Heidelberg, Germany) with five double strokes and a maximum clearance of 18 μm. The lysate was centrifuged at 500 g for 5 min at 4°C, the supernatant frozen in liquid nitrogen and stored at −80°C. The protein content of the lysate was determined with the bicinchoninic acid assay kit (Pierce) using BSA as a standard. Membrane fraction samples were prepared of the brain, liver, and kidneys of C57BL/6 wild-type (+/+) and CerS2-deficient mice (−/−) (one individual each, littermates, 13–14 weeks old) according to the protocol described by Imgrund et al. (17Imgrund S. Hartmann D. Farwanah H. Eckhardt M. R. J. K. K. ceramide synthase 2 mice and Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus (203) Google Scholar), frozen in liquid nitrogen and stored at −80°C. A lysate of wild-type was The protein content of the and of lysate was determined with the bicinchoninic assay kit (Pierce) using BSA as a standard. assays were in in a of The was by addition of the mix to the enzyme and the were in a and the of enzyme were for assay not to substrate and of the with to time and enzyme For the kinetic of and or various of acid and were with E. coli microsomes in buffer C. Spandl J. A. C. lysophosphatidylcholine acyltransferases and 2 are in lipid the of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google mM pH mM BSA for 5 min at For the of the specificity 5 min and the displayed in the 10 min and were For the kinetic of or and were with 5 HuH7 lysate for min at in buffer mM pH mM For the that the of the assay and 5 HuH7 lysate were for min. The kinetic were with of and 5 wild-type The assay was for min at 37°C in buffer (20 mM pH mM mM 2 mM (17Imgrund S. Hartmann D. Farwanah H. Eckhardt M. R. J. K. K. ceramide synthase 2 mice and Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus (203) Google Scholar). For the that the of the assay or and wild-type microsomes were For various for 10 brain, or microsomes of wild-type or mice were with and of was in buffer for min at with or and microsomes of was with of lysate and in buffer mM pH 7.0, for min at or were in buffer at with or the addition of microsomes for assay were by the addition of 500 to the assay were for 5 min in an and 500 acid were followed by and centrifugation at g for 2 min. of the were to a new The was with 200 The were in a and the click as described previously (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google the were to lipids. click mix of 2 3-azido-7-hydroxycoumarin, of 10 mM in were and the in a at all was the After addition of and 5 min in an the samples were applied were developed in the for all assays first acid for for 18 with between the or for the (2Kim H.J. Qiao Q. Toop H.D. Morris J.C. Don A.S. A fluorescent assay for ceramide synthase activity.J. Lipid Res. 2012; 53: 1701-1707Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). of and in were prepared or of this to a the and the lipids in the assay buffer After the addition of enzyme the mix was according to the assay protocol and to click and was as using the of as for the lipids. fluorescence detection, the was for 5 in in and was to in a The for the imaging of the and quantification was described (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google Scholar). through a were for were with a with a for detection of the and for detection and of all of that the high sensitivity of the is of detection is not to the which can be already by but is for We use a with a with good were to the lipid by of in the to the click and to in the of a were the signal of the all data are as Michaelis-Menten kinetics were in a is For the kinetic constants Km and were from the data using in G. S. data in 5: PubMed Scopus Google Scholar), and the value and of these are The general as in of the enzymatic using alkyne lipid and of the alkyne with the fluorogenic dye in a After separation by lipids are by fluorescence The high sensitivity and of this the click and imaging were (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google and are to using lipids. For an quantification of the assay a and is the of the lipid of alkyne lipids that represent of the or assay were to the vitro enzymatic click but using enzyme The was by quantification of fluorescent signal that of of that not been to assay and were for all tested and with good to good the applicability of the protocol been for for the assays in this study, and of the quantification an of fluorescent signal to the of lipid in the assay is In E. the of lysophosphatidic acid to phosphatidic acid is by the of acyltransferase J. of the coli for acyltransferase PubMed Scopus Google Scholar). has been with J. of coli cells in acyltransferase activity.J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of kinetic constants have not been determined for this We a assay with the labeled of acid using E. coli microsomes and determined the constants and Km for this substrate by as an chain we and Km for The Km for was than for was for with but the not to between enzyme In other Km previously been determined for enzymes using radiolabeled substrates. A similar chain specificity in the was in M. of the acyltransferase from Scopus Google with as the a Km of was for acid and of for For the a Km of with as the chain A. H. H. R. K. K. of acyltransferase motifs and substrate specificity and in acyltransferase Biophys. 2007; PubMed Scopus Google Scholar), and of with S. K. D. S. J.D. A. and and to in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), was For Km in the were with the chain D. E. H. specificity of lysophosphatidic acid acyltransferase from and cell Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google and S. K. D. S. J.D. A. and and to in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google For a of as the was as S. K. D. S. J.D. A. and and to in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. E. H. specificity of lysophosphatidic acid acyltransferase from and cell Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). with and is by the E. coli enzyme as the for the at the in with D. J. M. acyltransferase of coli of acid into the of Scopus Google constants by and from chain and assay are in the and from chain and assay are in the to the of substrates in the enzymatic from to the of substrates in the enzymatic from and from chain and assay are in the to the of substrates in the enzymatic from in a new that and are good substrates for the E. coli They that the enzyme have a Km for lysophosphatidic acids than the or mammalian enzymes investigated The alkyne substrates described represent the that are available for an acyltransferase assay with alkyne labeling of the chain or the chain the is the more general the of all acyltransferase with a small of labeled it from the of and the of of labeled from enzymatic and of the In the use of to a few fluorescent from of the the to The labeled on the other high specificity for the enzymatic of They or the fluorescent which be for In alkyne-labeled for assay using in was applied in a assay using microsomes from of the assay with microsomes from a C57BL/6 wild-type were using of the For assay and Michaelis-Menten kinetics for The the with the Michaelis-Menten using the for and Km by are of the and was in microsomes from brain, and from wild-type (+/+) or (−/−) The is for the to the of The allows the fast detection of the for various in is to by lysophosphatidylcholine In are HuH7 cells and but not C. Spandl J. A. C. lysophosphatidylcholine acyltransferases and 2 are in lipid the of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.M. Li S. M. G. and of a major lysophosphatidylcholine Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). We synthesized alkyne-labeled and that because is by enzymatic from it a of fatty acids at by M. of fatty acids in 2006; 13: Scopus Google Scholar). the of the substrates in a assay on HuH7 lysate using as the chain The Km value we for was similar to that determined for with for H. H. D. R. A. R. and of acyltransferase in cells and in Biol. 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(17Imgrund S. Hartmann D. Farwanah H. Eckhardt M. R. J. K. K. ceramide synthase 2 mice and Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus (203) Google a CerS2-deficient and the and various and in and that can be as a convenient alternative substrate for applications, we a similar small with brain, liver, and microsomes and several and fluorescent of the of this pilot Imgrund et al. (17Imgrund S. Hartmann D. Farwanah H. Eckhardt M. R. J. K. K. ceramide synthase 2 mice and Biol. 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The fatty acid 2000; PubMed Scopus Google and acid K. The acid 2007; PubMed Scopus Google the fatty the lipids and the enzymes have as for the of or S. and new PubMed Scopus Google Scholar, C. D. J.C. C. and metabolism of 2008; PubMed Scopus Google Scholar). We were to the of the fatty acid and in a with enzymatic assays are to this R.A. S. The fatty acid 2000; PubMed Scopus Google for a and H. K. H.J. of sensitive fluorescent assays for fatty acid 2007; PubMed Scopus Google We studies the kinetic of these alkyne-labeled substrates, which would the by a convenient and direct assay The various substrates the of the assay can be to more acyltransferases and and other hydrolases general applicability for the kinetic of many enzymes in lipid metabolism. alkyne lipids that we tested in this pilot study were as substrates by enzymes. in affinity of the enzymes with the natural substrates was in kinetic We enzymatic assays with alkyne lipids as a and convenient alternative to fluorescent or radioactive assays. As with the the separation in assay not of the lipids. can be by a (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google on the of the alkyne label in the not all can be followed with alkyne of fatty acids to the of the label (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google Scholar), as the of to by D. studies that the alkyne label not with the affinity of enzymes to the substrates, especially the alkyne label is to the terminus of the fatty acids of and are synthetically available (13Thiele C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (86) Google Scholar, S.B. R. M.D. and of using Chem. Biol. PubMed Scopus Google and can as a of a of lipid substrates with the of at and using chemical and enzymatic to this can be synthesized from we alkyne lipids to more and more available in the as substrates for enzymatic assays as as for identification The of lipids to the dye is in the separation is with the as for radiolabeled lipids or and the of the are in the The of radiolabeled assays to the use of alkyne lipid substrates be neither nor overly lipids are substrates for use in in vitro enzymatic as are convenient as we for the first display the kinetic characteristics of the natural substrates. The would like to and K. for the mice in the with A boron dipyrromethene ceramide synthase fatty acid lysophosphatidic acid lysophosphatidic acid acyltransferase lysophosphatidylcholine lysophosphatidylcholine acyltransferase acyltransferase nitrobenzoxadiazole acid phosphatidic acid acid