Cell-free Synthesis and Functional Characterization ofSphingolipid Synthases from Parasitic TrypanosomatidProtozoa*□S

Received for publication, March 26, 2010, and in revised form, May 3, 2010 Published, JBC Papers in Press, May 10, 2010, DOI 10.1074/jbc.M110.127662

Elitza S. Sevova‡1,2, Michael A. Goren§1,3, Kevin J. Schwartz‡, Fong-Fu Hsu¶, John Turk¶, Brian G. Fox§�,and James D. Bangs‡4

From the ‡Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health,Madison, Wisconsin 53706, the §Department of Biochemistry and the �Center for Eukaryotic Structural Genomics, College ofAgriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706, and the ¶Department of Medicine, WashingtonUniversity School of Medicine, St. Louis, Missouri 63110

The Trypanosoma brucei genome has four highly similargenes encoding sphingolipid synthases (TbSLS1–4). TbSLSs arepolytopic membrane proteins that are essential for viability ofthe pathogenic bloodstream stage of this human protozoan par-asite and, consequently, can be considered as potential drugtargets. TbSLS4 was shown previously to be a bifunctionalsphingomyelin/ethanolamine phosphorylceramide synthase,whereas functions of the others were not characterized. Using arecently described liposome-supplemented cell-free synthesissystem,which eliminates complications frombackground cellu-lar activities, we nowunambiguously define the enzymatic spec-ificity of the entire gene family. TbSLS1 produces inositolphosphorylceramide, TbSLS2 produces ethanolamine phos-phorylceramide, and TbSLS3 is bifunctional, like TbSLS4.These findings indicate that TbSLS1 is uniquely responsible forsynthesis of inositol phosphorylceramide in insect stage para-sites, in agreement with published expression array data (17).This approach also revealed that the Trypanosoma cruziortholog (TcSLS1) is a dedicated inositol phosphorylceramidesynthase. The cell-free synthesis systemallowed rapid optimiza-tion of the reaction conditions for these enzymes and site-spe-cific mutagenesis to alter end product specificity. A single resi-due at position 252 (TbSLS1, Ser252; TbSLS3, Phe252) stronglyinfluences enzymatic specificity. We also have used this systemto demonstrate that aureobasidin A, a potent inhibitor of fungalinositol phosphorylceramide synthases, does not significantlyaffect any of the TbSLS activities, consistent with the phyloge-

netic distance of these two clades of sphingolipid synthases.These results represent the first application of cell-free synthe-sis for the rapid preparation and functional annotation of inte-gralmembrane proteins and thus illustrate its utility in studyingotherwise intractable enzyme systems.

Sphingolipids (SLs)5 are ubiquitous in eukaryotic mem-branes, where they perform multiple functions. Besides actingas structural components of biologicalmembranes, they partic-ipate in protein sorting and cell signaling viamembrane rafts (1)and serve as critical apoptotic and anti-apoptotic second mes-sengers. As such, sphingolipids have important influence inhuman disease (2). SLs also act as precursors of other essentiallipid biosynthetic pathways, e.g. generation of ethanolaminephosphate for use in the Kennedy pathway (3) and subsequentsynthesis of phospholipids and glycosylphosphatidyl inositols.The common SL synthesis pathway (Fig. 1) begins with thecondensation of serine and palmitate by serine palmitoyl trans-ferase to make the sphingoid base 3-ketodihydrosphinganine,which is then reduced to make sphinganine. Sphinganine is theacceptor for N-fatty acylation by ceramide synthase to makedihydroceramide, which is then desaturated to make ceramide.Ceramide synthase also utilizes sphingosine generated by cera-mide turnover. Specific sphingolipid synthases (SLS) thentransfer the polar head groups from phosphoglycerolipiddonors to generate the phosphosphingolipids: sphingomyelin(SM), ethanolamine phosphorylceramide (EPC), and inositolphosphorylceramide (IPC).The major phosphosphingolipid in mammals is SM,

although EPC is present, whereas in fungi, themajor phosphos-phingolipid is IPC. Among the parasitic trypanosomatid proto-zoa, Leishmania synthesizes IPC exclusively (4, 5). The SouthAmerican trypanosome Trypanosoma cruzi synthesizes IPC(6–8) but has also been reported to contain SM (9, 10). The

* This work was supported by United States Public Health Service,NIAID, National Institutes of Health Grant R01 AI35739 (to J. D. B.), NIGMS,National Institutes of Health Grant RO1 GM50853 (to B. G. F.), and NIGMS,National Institutes of Health Protein Structure Initiative Grant 1 U54GM074901 (to J. L. Markley, G. N. Phillips, Jr., and B. G. F.). The WashingtonUniversity Department of Medicine Mass Spectroscopy Facility is sup-ported by United States Public Health Service Grants P41-RR00954, P60-DK20579, and P30-DK56341.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S3.

1 Both authors contributed equally to this article.2 Supported by National Institutes of Health “Microbial Pathogenesis and

Host Responses Training Grant” (T32 AI055397) to the University ofWashington-Madison.

3 Supported by the National Science Foundation East Asia and Pacific Sum-mer Institutes for U.S. Graduate Students.

4 To whom correspondence should be addressed. Tel.: 608-262-3110; Fax:608-262-8418; E-mail: jdbangs@wisc.edu.

5 The abbreviations used are: SL, sphingolipid; BSF, bloodstream form; PCF,procyclic form; SLS, sphingolipid synthase; SM, sphingomyelin; EPC, etha-nolamine phosphorylceramide; ORF, open reading frame; IPC, inositol phos-phorylceramide; nt, nucleotide(s); HA, hemagglutinin; PC, phosphatidyl-choline; PE, phosphatidylethanolamine; PI, phosphatidylinositol; AbA,aureobasidin A; NBD, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino);MES, 4-morpholineethanesulfonic acid; CHAPS, 3-[(3-cholamidopro-pyl)dimethylammonio]-1-propanesulfonic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 27, pp. 20580 –20587, July 2, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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African trypanosome Trypanosoma brucei synthesizes SM,EPC, and IPC (11). Interestingly, SL synthesis is developmen-tally regulated in that IPC is only synthesized in the procyclicinsect stage (PCF), and EPC synthesis is restricted to the mam-malian bloodstream form (BSF), whereas SM synthesis is con-stitutive throughout the life cycle (11).SL synthesis in mammals is carried out by a family of related

sphingomyelin synthases (SMSs) with distinct specificity andfunction (12–15). SMS1 is responsible for de novo synthesis ofSM in the Golgi, SMS2 regenerates SM from ceramide derivedfrom turnover in the plasma membrane, and SMSr synthesizesEPC in the ER. The trypanosomatids have an orthologousfamily of SLSs. In Leishmania major, the single SLS presenthas been shown to be an IPC synthase (LmjIPCS) (11, 16).T. brucei has a family of four paralogs (TbSLS1–4), one ofwhich (TbSLS4) has been shown by transgene expression inL. major to be a bifunctional SM/EPC synthase (11). Theactivities of TbSLS1–3 are unknown, although TbSLS1mRNA levels are specifically up-regulated in PCF trypano-somes, suggesting that this enzyme could be the agent ofstage-specific IPC synthesis (17). T. cruzi has a single SLSlocus with minor polymorphisms in the two allelic genes. Allof these SLS enzymes are modeled to have a six-transmem-brane topology with putative catalytic residues located onthe lumenal side of the organellar membrane (11, 12). Fur-thermore, simple reciprocal homology searches (BlastP) areconsistently positive between the mammalian and trypano-somatid orthologs but fail to detect any of the fungal IPCsynthases, suggesting a more distant relationship for thisclade of SLS enzymes.

In this work, we use a cell-free synthesis system designed forin vitro expression of active polytopic membrane proteins (18)to define the enzymatic specificity of the trypanosomatid SLSenzymes. Our results reveal unexpected diversity in the biosyn-thetic specificities of these enzymes and explain the basis forstage-specific content of sphingolipids in African trypano-somes. Additional data are presented raising doubt about theutility of aureobasidin A, a potent inhibitor of fungal IPC syn-thases, as a chemotherapeutic agent targeting trypanosomatidSLSs. Finally, thiswork illustrates and validates the utility of thisnovel expression system for structure-function investigation ofcomplex polytopic membrane proteins.

EXPERIMENTAL PROCEDURES

In Vitro Expression Constructs—Complete SLS open readingframes (ORFs) were amplified by PCR using specific primerscontaining 5� SgfI and 3� PmeI sites for cloning into the 5� SgfIand 3� EcoICRI sites of the pEU-C-His FlexiVector plasmid (19,20). Pfu high fidelity DNA polymerase (Agilent Technologies,Stratagene, San Diego, CA) was used for all cloning reactions.The plasmid contains (in order, 5�–3�: Sp6 RNA polymerasepromoter, 5� internal ribosome entry site, restriction sites forinsertion of cloned ORFs, in-frame C-terminal His6 tag, 3�homology region for cloning efficiency, and 3�-UTR for trans-lational efficiency). Specific trypanosomatid SLSORFs (TryTrpdatabase accession numbers) were: TbSLS1, nt 1–1011,Tb09.211.1030; TbSLS2, nt 1–969, Tb09.211.1020; TbSLS3, nt1–987, Tb09.211.1010; TbSLS4, 1–1041, Tb09.211.1000;LmjIPCS, nt 1–1014, LmjF35.4990; and TcSLS1.1, nt 1–1005,Tc00.1047053506885.124. The start codons for the T. bruceiparalogs were as defined by reverse transcription PCR in Ref.11. The numbering of TcSLS alleles is according to (16).T. bru-cei SLS genes were amplified from Lister 427 strain genomicDNA, T. cruzi SLS (TcSLS1.1) was amplified from CL-Brennerstrain genomic DNA, and L. major IPCS was amplified from aprevious PCR clone of the FV39-clone 5 gene (11). T. cruzi SLShas a second allelic copy (TcSLS1.2, Tc00.1047053510729.290)with minor polymorphisms relative to TcSLS1.1; this geneproduct was not characterized. All PCR products were verifiedby sequencing using primers flanking the FlexiVector insertionsite. The deduced amino acid sequences are compared with thehomologous ORFs from their respective annotated genomestrains in supplemental Fig. S1. EachTbSLS productwas clonedand sequenced from at least two independent PCR reactions,and the deduced amino acid sequences reported here forTbSLS1, TbSLS3, and TbSLS4 were identical for each indepen-dent clone. Two distinct but highly related PCR productswere consistently obtained from TbSLS2 reactions (data notshown). These are likely to be polymorphic alleles. TbSLS2.1was expressed in cell-free translation but was inactive in cat-alytic assays (data not shown) and is not considered furtherin this work. TbSLS2.2 was active; its sequence is reported insupplemental Fig. S1 and is characterized herein. TbSLS2.2is referred to as TbSLS2 throughout the remainder of text.Overall, these analyses reveal that the Lister 427 strainTbSLS sequences display minor polymorphisms relative totheir homologs from the TREU 927 genome strain.

FIGURE 1. Sphingolipid synthesis pathway. 3-KDS, 3-keto-dihydrosphin-gosine; PL, phospholipid; DAG, diacylglycerol. Homologs of all indicatedenzymes (italics) are found in the T. brucei and L. major genomes: serinepalmitoyl transferase, Tb927.4.1020 (subunit 1) and Tb10.70.3220 (subunit 2);3-ketosphinganine reductase, Tb927.10.4040; ceramide synthase,Tb927.8.7730; dihydroceramide desaturase, Tb927.6.3000; sphingolipid syn-thases, Tb09.211.1000-1030. The proposed site of action of aureobasidin A(AbA?) is indicated.

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Site-specific Mutagenesis—Mutagenesis was performedusing the PCR-based QuikChange� Multi Site-directedMutagenesis kit (Stratagene). The cloned TbSLS1 and TbSLS3genes in the FlexiVector and were used as templates. TbSLS1was altered by an S252F mutation, and TbSLS3 was altered bythe equivalent F252S mutation. Mutagenic primer sequencesare presented in supplemental Table S1. Mutated DNA washeat-shocked into XL10-Ultracompetent cells (Stratagene),and mutant TbSLS sequences were confirmed by plasmidsequencing.Preparation of Liposomes—Liposomes were prepared as

described previously (18, 21) using an Avanti mini-extruderand soybean lecithin (Soy PC, 20%, category no. 541601G,Avanti Polar Lipids, Alabaster, AL). These liposomes containsoy phosphatidylcholine (PC), phosphatidylethanolamine (PE),and phosphatidylinositol (PI) in an approximate molar ratio of2:2:1. Tomake liposomes of defined phospholipid composition,PC (16:0/18:2, Avanti product no. 850458P), PE (16:0/18:2,Avanti product no. 850756P), and PI (16:0/18:2, Avanti productno. 840044P) were mixed in equimolar ratios and processed asabove.In Vitro Transcription and Cell-free Synthesis—In vitro tran-

scription of cloned SLS ORFs with bacteriophage Sp6 RNApolymerase (Promega, Madison, WI) was carried out asdescribed previously (18, 21). Standard cell-free translation (18,21) was carried out in 50-�l reactions in 12 MWCO dialysiscups (Cosmo Bio, Tokyo, Japan) with wheat germ extract (CellFree Sciences, Yokohama, Japan) and 60�g of liposomes. Reac-tions were initiated with mRNA from a transcription reactionprogrammedwith 4�g of plasmidDNAand allowed to proceedovernight at 26 °C. Proteoliposomes (70 �l) were recoveredfrom two pooled translation reactions by floatation, and a sam-ple (5 �l) was reserved for estimation of translation product bySDS-PAGE and Coomassie Blue staining relative to knownamounts of creatine kinase. Typical recovery in floated proteo-liposomes was 18–28 �g of specific translation product. Totalphosphate analysis of Folch extracted lipids by the phosphorusash method (22) indicates a typical total phospholipid contentof 2.1 mM in the final recovered proteoliposomes. Proteolipo-somes were stored at �80 °C until use. Pilot experimentsindicated that SLS activities in proteoliposomes werestable through four cycles of freeze-thaw (�80 °C, roomtemperature).Liposome Sphingolipid Synthesis Assay—Standard reaction

buffer was MES-buffered saline (MBS: 50 mM MES, pH 6.5, 50mM NaCl, 5 mM KCl, 1 mM EDTA). For pH titrations, HEPESwas substituted for MES in some cases (HBS). Reactions con-tained proteoliposomes (typically 25–40 �l containing �10 �gof translation product) in a 100-�l volume andwere initiated byaddition of 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosine (NBDC6-ceramide, Invitrogen,Molecu-lar Probes) to final concentrations ranging from 0.1–5 �M asindicated in the text. Reactions were terminated by addition of200 �l distilled H2O, followed by 2.0 ml of chloroform:metha-nol (1:1 v/v). Samples were mixed, insoluble material wasremoved by centrifugation, and the organic extract was driedunder N2. Dried lipids were subjected to n-butanol/water par-titioning, and lipids recovered from the butanol phase were

fractionated by thin layer chromatography in chloroform:methanol:acetic acid:water (25:15:4:2, v/v/v) on TLC Silica Gel60 plates (Merck, Gibbsburg, NJ). Fluorescent lipids were visu-alized on a Storm 860 imaging system with ImageQuant soft-ware. NBD-C6-SM (Avanti Polar Lipids) and NBD-C6-IPC(preparation described in Ref. 11) were run as mobility stand-ards. The identity of NBD-C6-EPC products were previouslyinferred by relative mobility (11).Maintenance and Generation of Transgenic Trypanosomes—

Standard propagation of BSF Lister 427 strain Trypanosomabrucei brucei is described elsewhere (11). TheTbSLS1ORFwasPCR-amplified from genomic DNA with primers designed tointroduce an in-frame C-terminal HA epitope tag (YPYDVP-DYA), and flanking 5� HindIII and 3� XmaI sites for insertioninto the multicloning region of our constitutive pXS5 expres-sion vector (23). The construct was linearized with XhoI,electroporated into BSF trypanosomes, and clonal neor celllines derived as described recently (24). Likewise, metabolicradiolabeling and immunoprecipitation have been describedpreviously.Preparation of Trypanosome Membranes—Semi-intact try-

panosome membranes were prepared by a modification ofMasterson et al. (25). Cultured BSF trypanosomes constitu-tively expressing TbSLS1HA were harvested at late log phase(1–2 � 106/ml) and washed in HBS, pH 7.4, containing 70 mM

D-glucose. Cells were pelleted and resuspended (1.1 � 108/ml)on ice in distilled H2O containing 0.1 mM tosyl-lysyl-choro-methylketone and 2 �g/ml each leupeptin, antipain, pepstatin,and chymostatin. After 5 min, 0.1 volume of 10� MBS wasadded, and the membranes were washed twice in MBS. Thesemi-intact membranes were resuspended in MBS at 108 cellequivalents/ml and stored at �80 °C until use (not longer than3 days). NBD-SL synthesis was assayed exactly as describedabove for liposomes using 107 cell equivalents of membranesper reaction.Preparation of Perforated Yeast Spheroplasts—A modified

protocol of Rexach et al. (26) was followed. TheGPY60 Saccha-romyces cerevisiae strain (MAT�leu2-3, 112 ura3-52 his4-579trp1-289 prb1 gal2pep4::URA3) was grown to early logphase (2–4 A600/ml) at 30 °C in YP medium. An equivalent of1,500 A600 cells (1 A unit is defined as 107 cells) were pelleted(3,000 rpm for 5min) and resuspended to 50A600/ml in 0.1 mM

Tris, pH 9.4, 10 mM dithiothreitol (5 min, room temperature).Cells were centrifuged (3,000 rpm for 2 min at room tempera-ture) and resuspended to 50A600/ml in spheroplastingmedium(0.75� YP, 10 mM Tris, pH 7.5, 0.7 M sorbitol, 0.5% glucose)with yatalase (2 mg/ml, 10 lytic units/mg, Takara Bio, Inc.,Otsu, Japan) and lyticase (3 mg/ml, 2000 units/mg, SigmaAldrich). Cells were incubated at 30 °C, and samples were peri-odically diluted 1:100 in H2O for A600 determination. WhenmeasuredA600 was�30% of the initial value, spheroplasts wereharvested (3,000 rpm, 2 min, room temperature) and resus-pended (50 A600/ml) in regeneration medium (0.75� YP, 0.7 M

sorbitol, 1% glucose). After 30 min at 30 °C, spheroplasts wereagain harvested (3,000 rpm, 5 min, 4 °C) and resuspended inlysis buffer (20 mM HEPES, pH 6.8, 400 mM sorbitol, 150 mM

KOAc, 2 mM MgOAc, 0.5 mM EGTA) and pelleted (5,000 rpm,5 min, 4 °C). This step was repeated. Perforated spheroplasts

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were washed and resuspended to 300 A600 in MBS, aliquoted,and frozen over liquid nitrogen vapors for 45 min. Perforatedspheroplasts were then stored at �80 °C.Data Analyses—Statistical analyses and curve fitting were

performed in Prizm4 (GraphPad Software, La Jolla, CA).

RESULTS

Production of Active TbSLS4 in a Wheat Germ Cell-freeSystem—Wepreviously characterized TbSLS4 as a bifunctionalSM/EPC synthase by the laborious process of generating atransgenic L. major promastigote cell line constitutivelyexpressing the TbSLS4 gene (11). Analyses by ESI mass spec-trometry and by in vivo incorporation of NBD-ceramide sub-strate unequivocally demonstrated the presence and synthesis,respectively, of both SM and EPC, which are absent in wild typeL. major (27). To continue our characterization of the TbSLSgene family and the orthologous trypanosomatid SLSs in amore expeditious manner, we have exploited a recently devel-oped system for in vitro synthesis of active polytopicmembraneproteins (18, 21). In this system, mRNA for the gene of interestis generated by in vitro transcription and is in turn used toprogram cell-free translation in a wheat germ extract. Thetranslation reaction is supplemented with unilamellar lipo-somes prepared from soy phospholipids composed predomi-nantly of PC, PE, and PI (�2:2:1 ratio). The liposomes presum-ably capture and stabilize nascent transmembrane domains asthey emerge from the ribosome thereby facilitating proteinfolding within the membrane bilayers. Although the precisebiophysics and biochemistry of this process are not fully under-stood, proper folding can be monitored by enzymatic assay(18, 21).To validate this system for assaying TbSLS function, we pro-

grammed cell-free synthesis with TbSLS4 mRNA and recov-ered proteoliposomes by floatation (Fig. 2A). A band ofexpected size (Fig. 2A, star) was recovered. This band is notobserved in cell-free reactions programmedwith othermRNAs

such as bacteriorhodopsin. Variable amounts of wheat germproteins were present in the proteoliposomes. These contami-nants are typical and have been partially characterized (18). Therecovered proteoliposomes were assayed for SL synthesis usingexogenous fluorescent NBD-ceramide substrate and endoge-nous liposome phospholipids as head group donors (Fig. 2B).Consistent with our previous characterization of this enzyme,NBD-SM, and to a lesser extent NBD-EPC, were produced in atime- and transcript-dependent manner. The wheat germextract and proteoliposomes containing bacteriorhodopsin(Fig. 2B) had no SL synthesis activity. This system was used tofurther investigate and optimize enzymatic activity (Fig. 3).Synthesis of both NBD-SM and NBD-EPC were found to belinear for �3 h with maximal production of each at 2.5–5 �M

NBD-ceramide substrate. The kinetic profiles of product for-mation were essentially identical at 0.25 (Fig. 3A) and 2.5 �M

(data not shown) NBD-ceramide, indicating that the extent ofthe reaction was not controlled by substrate depletion. The pHoptimum was fairly sharp at 6.5–7.0, and reactions were mod-estly superior at 30 °C. Divalent cations had no effect on activ-ity. Exogenous detergents had differential effects on TbSLS4activity (Fig. 3F). Both Triton X-100 and dodecyl-maltosidedramatically reduced (�90%) both SM and EPC synthesis, butCHAPS had minimal effect on either activity. Based on theseresults, final reaction conditions for subsequent assays were setas 2.5 h at 30 °C in MBS buffer, pH 6.5, with 2.5 �M

NBD-ceramide.Enzymatic Specificity of Trypanosomatid Sphingolipid

Synthases—The trypanosomatid SL synthases were producedindividually in the cell-free system. For these experiments, lipo-somes were prepared with equimolar pure preparations of PC,PE, and PI to further normalize reactions for substrate concen-tration. Consequently, these assays represent a fair comparisonof the relative activity and specificity of each enzyme. SDS-PAGE of recovered proteoliposomes indicated that each SLSwas synthesized to similar levels (Fig. 4A). The enzymatic activ-ities of each were determined in parallel in standard NBD-cer-amide incorporation assays programmed with normalizedamounts of TbSLS protein (Fig. 4B). As demonstrated above,TbSLS4 synthesized both NBD-SM and NBD-EPC (Fig. 4B,lane 4). Although not apparent in this image, TbSLS4 alsomadea trace amount of NBD-IPC (see also Fig. 6, lanes 5 and 6).TbSLS3 has identical activity to TbSLS4 (Fig. 4B, lane 3), alongwith the production of a minor amount of IPC. Interestingly,TbSLS2 made predominantly NBD-EPC (Fig. 4B, lane 2), withminor amounts of NBD-SM andNBD-IPC detected.More sur-prisingly, TbSLS1 predominantly synthesized NBD-IPC withonly minor amounts of NBD-SM and NBD-EPC detected (Fig.4B, lane 1). As expected (11, 16), LmjIPCS synthesized NBD-IPC exclusively (Fig. 4B, lane 6). Finally, TcSLS1.1 also madeNBD-IPC exclusively (Fig. 4B, lane 5). These results indicatethat the TbSLSs have overlapping enzymatic profiles, but eachhas a distinct dominant specificity.Modulation of TbSLS Specificity by Site-specific Mutagenesis—

Thedifferences inTbSLS specificities are striking given that theprimary sequences of the four TbSLSs are �90% identical(excluding the C-terminal cytoplasmic domain), and, whenconservative substitutions in transmembrane helices are dis-

FIGURE 2. Cell-free synthesis of active TbSLS4. TbSLS4 was synthesized inthe cell-free system, and proteoliposomes were recovered by floatation andassayed for conversion of NBD-ceramide substrate. A, samples from the gra-dient were fractionated by SDS-PAGE and stained with Coomassie Blue. Gra-dient fractions are indicated from top (T) to bottom (B), and the direction offloatation is indicated (arrow). A star indicates a TbSLS4-specific band recov-ered in the proteoliposomes. This band is absent in floated fraction 2 fromreactions programmed for synthesis of bacteriorhodopsin (BO, indicated byarrowhead). B, recovered TbSLS4 or bacteriorhodopsin proteoliposomeswere used for NBD-SL synthesis over a 4-h time course in MBS (pH 6.5, 0.25 �M

NBD-ceramide, 30 °C); products were visualized by TLC and phosphorimag-ing. Mobilities of NBD standards, solvent front (F), NBD-ceramide (C), andorigin (O) are indicated.

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counted, e.g. Leu for Ile, identity exceeds 95% (see supple-mental Fig. S1 in Ref. 11 for a comprehensive alignment). Thisleaves few residues to account for the differences in substratespecificity. The chemistry of SL synthase reactions is thought toproceed via formation of a covalent phosphoenzyme interme-diate (28). Thus, specificity is likely to be defined by residues inthe lumenal catalytic domain that interact with the polar headgroups of the phospholipid donors. The aligned trypanosoma-tid SLS sequences (Fig. 5A) reveal a single residue immediatelyadjacent to a catalytic histidine in lumenal LoopV that is invari-ably associated with either IPC (Ser252) or SM/EPC (Phe252)synthesis by the trypanosomatid SLSs. Conversion of this resi-due inTbSLS1 (S252F) dramatically reduced IPC synthesis (Fig.5B, compare lanes 1 and 2). In contrast, conversion of the sameresidue in TbSLS3 (F252S) enhanced IPC synthesis withoutaffecting innate SM or EPC synthetic activity, essentially creat-ing a trifunctional enzyme (Fig. 5B, compare lanes 3 and 4).These results provide the first functional mapping of substratebinding determinants in the sphingolipid synthase family andsuggest that differences in amino acid sequence are the primarydeterminants of substrate/product specificity, as opposed tosubstrate availability, in the native organisms.Aureobasidin A Is Not a TbSLS Inhibitor—Fungal IPC syn-

thases are potently inhibited (subnanomolar) by aureobasidinA (AbA) (29), and fungal growth is blocked in the low nM range

(30). It has recently been reported(31) thatAbA is a potent inhibitor ofTbSLS4 (IC50 of 0.42 nM) whenassayed in membranes preparedfrom transgenic yeast and likewisethat AbA is an inhibitor of growth ofcultured BSF trypanosomes (EC50 �250 nM). Given the reported lack ofinhibition by AbA with T. cruzi IPCsynthase in native membranes(tested at 7.3 �M) (8) or LmjIPCS inmembranes from transgenic yeast(IC50 � 100 �M) (31), we decided tore-examine the inhibitory activity ofAbA against the T. brucei ortho-logues using three distinct assays.First, 500 nMAbAhad little effect onthe formation of any of the NBD-SLproducts of TbSLS1 (Fig. 6A, lanes 1and 2), TbSLS2 (Fig. 6A, lanes 3 and4), orTbSLS4 (Fig. 6A, lanes 5 and 6)produced using the cell-free system.These data indicate that AbAis not an inhibitor of the TbSLSswhen assayed in isolation. Second,to rule out that insensitivity ofrecombinant TbSLSs to AbAwas anartifact of the cell-free system, weassayed native TbSLS activities insemi-intact membranes preparedfrom a transgenic BSF cell line con-stitutively expressing HA-taggedTbSLS1. This system allows simul-

taneousmonitoring of synthesis of SM, EPC, and IPC in a singleassay. Immunoprecipitation of metabolically radiolabeled con-trol versusTbSLS1HA cells confirmed expression of a polypep-tide of the expected size (Fig. 7A, star), and SL synthesis assaysrevealed specific production of NBD-IPC in addition to thenormal production of NBD-SM and NBD-EPC (Fig. 7B).ESI-MS analyses of total lipid extracts also confirmed elevatedsteady state levels of d18:1/16:0-IPC in this cell line(supplemental Fig. S2). By using membranes from this cell line,the effect of 500 nMAbA on synthesis of all three SLs was deter-mined (Fig. 7C). AbA gave no inhibition of NBD-SM synthesisand onlyminor inhibition ofNBD-EPC andNBD-IPC synthesis(20–25%). This is in contrast to NBD-IPC synthesis by yeastmembranes containing yeast IPC synthase, whichwas inhibitedby �90% (a typical reaction with yeast membranes is presentedin supplemental Fig. S3). Collectively, these data indicate thatthe TbSLS enzymes, in both native and recombinant forms, arerelatively refractory (IC50 � 500 �M) to the inhibitory action ofAbA. Finally, we determined the effect of AbA on growth ofcultured BSF trypanosomes (Fig. 8). Proliferation was increas-ingly impaired at concentrations of �250 nM and was com-pletely ablated at 2.5 �M. Similar results were obtained withmultiple lots of commercially available AbA. The EC50 forgrowth inhibition was determined to be 0.8�M, consistent with

FIGURE 3. Optimization of cell-free enzymatic parameters. Formation of NBD-SM (black symbols and bars)and NBD-EPC (open symbols and bars) were used to optimize enzyme assay conditions for TbSLS4 synthesizedin the cell-free system. Unless specifically varied, reaction conditions in all cases were 4 h at 30 °C in MBS, pH 6.5,with 0.25 �M NBD-ceramide. Fluorescent products were extracted and analyzed by TLC as in Fig. 2. SM and EPCproducts were individually quantified by phosphorimaging and normalized to maximum production (A–C) orto standard control conditions (D–F). All assays were performed in triplicate, and results are presented asmean � S.E. A, kinetics of product formation. B, NBD-ceramide substrate dependence. C, pH dependence usingMES (MBS, pKa of 6.15) or HEPES (HBS, pKa of 7.55) buffering agent. D, temperature dependence. E, effect of 10mM CaCl2, MgCl2, or MnCl2. Note that under standard buffer conditions all reactions contain 1 mM EDTA.F, effect of detergents: Triton X-100, 0.1% (v/v); CHAPS, 0.1% (w/v); n-dodecyl-maltoside, 0.1% (w/v). Thedetergent:phospholipid molar ratios for these assays were Triton X-100 (TX100), 2.8; CHAPS, 2.7; and n-dodecyl-maltoside, 3.3.

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the relative lack of effect on SL synthesis by AbA in both thecell-free and semi-intact membrane systems.

DISCUSSION

T. brucei has a unique linear array of four tandemly linkedgenes encoding sphingolipid synthases. Previously, we demon-strated that TbSLS4 is a bifunctional SM/EPC synthase, but theactivities of the remaining paralogswere uncertain (11). Using arecently developed cell-free system for synthesis of active poly-topic membrane proteins, we now have determined unambig-uously the dominant activities of the entire enzyme family.TbSLS1 is an IPC synthase, TbSLS2 is an EPC synthase, andTbSLS3 and TbSLS4 are bifunctional SM/EPC synthases. Theassignment of primary IPC synthesis activity to TbSLS1 con-firms a recent prediction to this effect made on the basis ofstage-specific patterns ofTbSLS gene expression (17). Our con-clusion is supported by the specific synthesis of IPC in BSF cellsectopically expressing TbSLS1. Thus, TbSLS1 is responsible forthe stage-specific production of IPC in PCF trypanosomes. Wehave also used the cell-free system to confirm that LmjIPCS is anexclusive IPC synthase and to establish for the first time that theT. cruzi ortholog (TcSLS1.1) is an IPC synthase.T. cruzi has a sec-ond allelic copy (TcSLS1.2, Tc00.1047053510729.290) that hasminor polymorphisms, and it is possible that this also is an IPCsynthase. However, T. cruzi has also been reported to contain SM(9, 10, 32), so it may be that TcSLS1.2 has SM synthase activity.Our determinations of TbSLS activities were made in lipo-

somes containing equimolar concentrations of all three poten-

tial head group donor phospholipids (PC, PE, PI), and thus pro-vide new information on the specificity of each enzyme.Sequence alignments and site-specific mutagenesis indicatethat these specificities are exquisitely controlled by subtle dif-ferences in active site residues. For instance, Ser252 in Loop V ispermissive for synthesis of all three SL products, but Phe252 isrestrictive for synthesis of the cationic SLs, i.e. SM and EPC.

Recently, the enzymatic activity of TbSLS4 was investigatedby Mina et al. (31) using transgene expression in a conditionalaur1 (IPC synthase) mutant of S. cerevisiae. Their conclusionsdiffer in several key aspects from our current and previously

FIGURE 4. Enzymatic activities of trypanosomatid sphingolipid syn-thases. The trypanosomatid SLSs were individually synthesized in the cell-free system using equimolar PC/PE/PI liposomes, and proteoliposomes wererecovered by floatation. A, samples of proteoliposomes containing the indi-vidual trypanosomatid SLS translation products were analyzed by SDS-PAGEand Coomassie Blue staining. The positions of molecular mass standards(kDa) and TbSLS translation products are indicated. This gel was used to nor-malize the amount of TbSLS protein used in subsequent synthesis reactions.B, proteoliposomes containing equivalent amounts of TbSLS translationproducts (�10 �g) were assayed for activity in a standard 2.5-h reaction at30 °C with 2.5 �M NBD-ceramide. NBD-SL product analysis was performed inparallel on a single TLC plate. A phosphorimage is presented with mobilitystandards indicated.

FIGURE 5. Site-specific mutagenesis of TbSLSs. A, alignment of trypanoso-matid SLS Loop V residues. Top, diagram of the modeled topology of trypano-somatid SLSs (adapted from Refs. 28 and 11). Transmembrane domains andsolvent exposed loops are numbered in Arabic and Roman numerals, respec-tively. Key catalytic residues are silhouetted. Residue 252 targeted formutagenesis is indicated by an open circle. Bottom, alignment of the Loop Vregion from trypanosomatid SLSs as indicated. Key catalytic residues areunderlined, and residue 252 is silhouetted. Proven enzymatic activities are inparentheses. Numbering is according to the T. brucei orthologs. B, TbSLS1 andTbSLS3 mutants were produced in the cell-free system, and normalizedamounts of translation product (�10 �g) were assayed for enzymatic activityas in Fig. 4. Top, SDS-PAGE and Coomassie Blue staining of purified proteoli-posomes. Equivalent synthesis of matched wild type and mutant TbSLSs isevident. The vertical white stripe indicates intervening lanes that were digitallyexcised from the gel. Equivalent amounts of each cell-free translation productwere used in the SLS assays. Bottom, NBD-SL product analysis was performedin parallel on a single plate. A phosphorimage is presented with mobilitystandards indicated. WT, wild type.

FIGURE 6. Effect of AbA on SLS activities in the cell-free system. TbSLSswere produced in the cell-free system using standard equimolar donor lipo-somes. A, proteoliposomes containing TbSLS1 (lanes 1 and 2), TbSLS2 (lanes 3and 4), or TbSLS4 (lanes 5 and 6) were assayed in standard reactions for syn-thesis of NBD-SLs as in Fig. 4. Reactions were performed in the absence (�) orpresence (�) of 500 nM AbA. B, products on TLC plates were quantified andare presented as percent of the AbA minus control (mean � S.D., n 3) foreach TbSLS.

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published findings. Mina et al. (31) found that TbSLS4 is abifunctional IPC/SM synthase. Other than leaky expression ofendogenous IPC synthase in the yeast cell line, we can offer no

certain explanation for this result. However, multiple lines ofevidence support our conclusion that TbSLS4 is a bifunctionalSM/EPC synthase with minimal IPC synthase activity. First,transgene expression ofTbSLS4 inL. major, which only synthe-sizes IPC, resulted in synthesis and steady state accumulation ofboth SM and EPC (11). Second, unique production of TbSLS4in the cell-free system, which is devoid of other SLS activities,results in robust synthesis of SM and EPC but little IPC. Finally,TbSLS4 is constitutively expressed in both BSF and PCFtrypanosomes (17), yet significant IPC is only found in PCFstage parasites (11). The later two results in particular would bequite unlikely if native TbSLS4 had significant IPC synthaseactivity. The fact that TbSLS1, a confirmed IPC synthase, isactive when expressed ectopically in BSF trypanosomes alsorules out any inherent problemswith IPC synthesis in this stageof the life cycle.Another discrepancy with the findings of Mina et al. (31) is

the sensitivity of TbSLS4 activity to inhibition byAbA. By againusing the transgenic yeast system, they found an IC50 of 0.42 nMfor putative synthesis of IPC, a value that is remarkably similarto the published sensitivity of yeast IPC synthase (�0.2 nM)(29). This result is quite surprising because the trypanosomatidSLSs are phylogenetically distant from fungal IPC synthases(16), and neither of the closely related T. cruzi (8) nor L. major(31) orthologs are sensitive to AbA. Furthermore, the bifunc-tional TbSLS4-mediated SMsynthesis assigned in the yeast sys-tem by Mina et al. (31) should have also been sensitive to AbA,but these data were not reported (31).We independently investigated the AbA sensitivity of the

TbSLSs in three complementary ways. First, we find thatrecombinant TbSLS1, TbSLS2, and TbSLS4 in the cell-free sys-tem are not significantly inhibited by AbA. Likewise, we findthat native SLS activities in semi-intact trypanosome mem-branes containing all four TbSLSs are relatively insensitive toAbA. It is worth noting that in both these systems, TbSLS activ-ities were assayed at 500 nM AbA, a concentration �1000-foldhigher than the IC50 reported by Mina et al. (31). Again, onepossible explanation of this discrepancy is leaky expression ofendogenous IPC synthase in the yeast system. Alternatively, acritical protein interaction partner for yeast IPC synthase hasrecently been identified (Kei1p) (33). Kei1p is essential for bothyeast IPC synthase activity and for its sensitivity to AbA. Per-haps this protein, assuming it is present in aur1 cells, affectedthe activity of TbSLS4 when expressed in yeast. Finally, we dofind that AbA inhibits growth of cultured BSF trypanosomes,but at a higher concentration (EC50 of 0.8 �M) than reported byMina et al. (31) (EC50 � 0.25 �M). In summary, the presentresults indicate that AbA is not a potent inhibitor of TbSLSactivities. Instead, inhibition of parasite growth is likely due tooff-target effects. Alternatively, even weak inhibition of TbSLSactivities may subtly alter ratios of ceramide substrate and di-acylglycerol product, both of which are potent intracellular sig-naling molecules and can have apototic effects (2, 34).In closing, it is worth commenting on the advantages of the

cell-free system used in this work. The speed and simplicitywith which we were able to generate and assay the individualTbSLS enzymes (days to weeks) greatly exceeds the time com-mitment and complexity of our prior strategy of transgene

FIGURE 7. Effect of AbA on SLS activities in semi-intact trypanosomemembranes. Permeabilized whole cell membranes were prepared fromtransgenic BSF cells ectopically expressing TbSLS1:HA (indicated as TbSLS1).A, immunoprecipitation of metabolically radiolabeled control and transgeniccells extracts with anti-HA antibody. A band of the expected size for TbSLS:HAis specifically detected in the TbSLS1:HA membranes (star). B, NBD-SL synthe-sis assays were performed under the standard conditions defined in the cell-free system. Reactions were programmed with 2 � 107 cell equivalents ofsemi-intact membranes. Reaction products were analyzed by TLC and phos-phorimaging. In addition to NBD-SM and NBD-EPC synthesized in controlmembranes, TbSLS1:HA-containing membranes also synthesize NBD-IPC.C, the effect of 500 nM AbA on NBD-SL synthesis in TbSLS:HA semi-intactmembranes was determined. As a positive control for inhibition, assays werealso programmed with 5 �g of perforated membranes prepared from S. cer-evisiae. Products on TLC plates were quantified by phosphorimaging, anddata are presented as percent of the AbA minus control (mean � S.D., n 5).

FIGURE 8. Effect of AbA on growth of BSF trypanosomes. A, BSF trypanosomecultures were initiated in the presence of increasing concentrations of AbA, andcell density was monitored for 24 h by microscopic cell counting. B, cell densitiesat 24 h were normalized as a percent of control AbA minus cultures. All data arepresented as mean � S.D. for triplicate independent cultures.

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expression in Leishmania (months) (11). This method was par-ticularly advantageous in pursuing mutagenesis studies of thedeterminants of enzymatic specificity. In addition, the ability tosynthesize and assay a single enzyme in isolation eliminates anyconfounding issues of background activities associated withendogenous expression in living systems. For instance, it wouldnot have been possible to unambiguously determine the IPCsynthase activity of TbSLS1 in transgenic Leishmania becauseof the background activity of endogenous LmjIPCS. The wheatgerm system can also be used for large scale protein production(in mg quantities) for subsequent structure/function studies(21), which provides another advantage relative to the presentlyknown methods to produce SLS enzymes. All of these qualitiesare potentially applicable to the study of other polytopic mem-brane proteins. Consequently, this system should become animportant tool for investigation of a wide variety of previouslyinaccessible biological processes.

Acknowledgments—We thank Dr. Stephen Beverley (WashingtonUniversity, St. Louis, MO) for thoughtful discussions. We are gratefultoDr. Rick Tarleton (University of Georgia) for T. cruzi genomicDNA.

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G. Fox and James D. BangsElitza S. Sevova, Michael A. Goren, Kevin J. Schwartz, Fong-Fu Hsu, John Turk, Brian

from Parasitic Trypanosomatid ProtozoaCell-free Synthesis and Functional Characterization of Sphingolipid Synthases

doi: 10.1074/jbc.M110.127662 originally published online May 10, 20102010, 285:20580-20587.J. Biol. Chem. 

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