A Novel Phosphatidylinositol-Phospholipase C of Trypanosoma cruzi That Is Lipid Modified and Activated during Trypomastigote to Amastigote Differentiation

A Novel Phosphatidylinositol-Phospholipase C of Trypanosoma cruzi That Is Lipid Modified and Activated during Trypomastigote to Amastigote Differentiation
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   A Novel Phosphatidylinositol-Phospholipase C of   Trypanosoma cruzi That Is Lipid Modified and Activated during Trypomastigote to Amastigote Differentiation* (Received for publication, September 30, 1999) Tetsuya Furuya‡, Corinna Kashuba, Roberto Docampo, and Silvia N. J. Moreno§  From the Laboratory of Molecular Parasitology, Department of Pathobiology, College of Veterinary Medicine, University of  Illinois at Urbana-Champaign, Urbana, Illinois 61802 The phosphoinositide (PI)-specific phospholipase Cgene( TcPI-PLC )oftheprotozoanparasite Trypanosomacruzi  was cloned, sequenced, expressed in  Escherichiacoli , and the protein product (TcPI-PLC) was shown tohave enzymatic characteristics similar to those of mam-malian    -type PI-PLCs. The  TcPI-PLC  gene is expressedat high levels in the epimastigote and amastigote stagesof the parasite, and its expression is induced during thedifferentiation of trypomastigotes into amastigotes,where TcPI-PLC associates with the plasma membraneand increases its catalytic activity. In contrast to otherPI-PLCs described so far, the deduced amino acid se-quenceofTcPI-PLCrevealedsomeuniquefeaturessuchasan  N  -myristoylationconsensussequenceatitsamino-terminal end, lack of an apparent pleckstrin homologydomain and a highly charged linker region between thecatalytic X and Y domains. TcPI-PLC is lipid modified invivo , as demonstrated by metabolic labeling with[ 3 H]myristate and [ 3 H]palmitate and fatty acid analysisof the immunoprecipitated protein, and may constitutethe first example of a new group of PI-PLCs. Many pathogenic parasites have developed the ability to livein two distinct hosts, one vertebrate and the other invertebrate.Such parasites include  Trypanosoma cruzi , the etiologic agentof Chagas’ disease or American trypanosomiasis. During its lifecycle,  T. cruzi  has to adapt to environments of different tem-perature, osmolarity, ionic composition, and pH, and some of these adaptation processes are paralleled by morphological andfunctional changes. Very litttle is known about the signaling mechanisms involved in these processes.Phosphoinositide-specific phospholipases C (PI-PLCs) 1 cata-lyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate(PIP 2 ) to  D - myo -inositol-1,4,5-trisphosphate (IP 3 ) and  sn -1,2-diacylglycerol (DAG) (1, 2). Both products of this reaction func-tion as second messengers in eukaryotic signal transductioncascades. The soluble IP 3  triggers release of calcium from in-tracellular stores (1). The membrane-resident DAG controlscellular protein phosphorylation states by activating variousprotein kinase C isozymes (2). Three classes of mammalianPI-PLCs with 10 different isozymes have been characterized(  1-  4,    1-   2, and   1-  4) (3). The activity of    - and    - isozymes(145–150 kDa) is regulated by G protein-coupled and tyrosinekinase-linked receptors, respectively. These isozymes are re-lated to the much smaller   -isozymes (  85 kDa) (3). It seems very likely that PI-PLC-   evolved first, because every PI-PLCcloned so far from a non-mammalian species (for example,  Dictyostelium , yeast, higher plants, and  Chlamydomonas ) isclearly a   -isoform (3). It is currently not known how  -isozymes are regulated  in vivo  (4). It is possible that they areregulated only by calcium ions (3) although the idea of PI-PLC-   being regulated by GTP-binding proteins is one whichhas increasing support (3, 5). Results from several laboratories(6–8) have suggested that, at least in yeasts, PI-PLC-   isrequired for a number of nutritional and stress-related re-sponses. It has also been postulated that PI-PLC-   could havea role in differentiation of   Dictyostelium discoideum  (9). Tran-scription of this PI-PLC-   appears to be enhanced during cellaggregation, it decreases during slug formation, and increasesin the culminating fruit body (9).  D. discoideum  PI-PLC-   is Gprotein-coupled (10).The understanding of factors controlling phospholipid me-tabolism in parasitic protozoa is very poor, although theselipids could be involved in several important events in theseeukaryotic cells. The presence and operation of the inositolphosphate/diacylglycerol signaling pathway was demonstratedin epimastigotes of   T. cruzi  (11). IP 3  and DAG formation wasstimulated by Ca 2  in digitonin-permeabilized cells, thus sug-gesting the presence of a PI-PLC (11). The presence of differentinositol phosphates in amastigotes (12) and trypomastigotes(13) was reported later. A shift in the levels of phosphoinositidemetabolites after incubation of epimastigotes with carbamoyl-choline (14) and the stimulation of IP 3  and DAG production andepimastigote proliferation by fetal calf serum (15) were alsoreported. A PI-PLC activity was also detected in epimastigotelysates using PI as substrate (15). A synthetic peptide corre-sponding to a chicken   D -globin fragment was able to increaseinositol monophosphate (IP) and IP 3  levels in epimastigotes(16) and stimulate transformation of epimastigotes into trypo-mastigotes (metacyclogenesis) (17). A protein kinase C was alsocharacterized in  T. cruzi  epimastigotes (18). This enzyme re-quires phosphatidylserine and Ca 2  for activity and is stimu-lated by DAG (18). * This work was supported in part by a grant-in-aid from the Amer-ican Heart Association, Illinois Affiliate (to S. N. J. M.). The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “ advertisement ”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ Supported by a student fellowship of the American Heart Associa-tion, Illinois Affiliate.§ To whom correspondence should be addressed: Laboratory of Mo-lecular Parasitology, Dept. of Pathobiology, College of Veterinary Med-icine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave., Urbana, IL 61802. Tel.: 217-333-2746; Fax: 217-244-7421; 1 The abbreviations used are: PI-PLC, phosphatidylinositol-phospho-lipase C; PIP 2 , phosphatidylinositol 4,5-bisphosphate; IP 3 ,  D -myo-inosi-tol 1,4,5-trisphosphate; DAG, diacylglycerol; IP, inositol monophos-phate; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serumalbumin; MES, 4-morpholineethanesulfonic acid; Mops, 4-morpho-linepropanesulfonic acid; PCR, polymerase chain reaction; kb, kilobasepair(s); ORF, open reading frame; bp, base pair(s); PAGE, polyacryl-amidegelelectrophoresis;PBS,phosphate-bufferedsaline;FAME,fattyacid methyl ester; GPI, glycosylphosphatidylinositol; FCaBP, flagellarcalcium-binding protein; PH, pleckstrin homology. T HE  J OURNAL OF  B IOLOGICAL  C HEMISTRY   Vol. 275, No. 9, Issue of March 3, pp. 6428–6438, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.  Printed in U.S.A. This paper is available on line at 6428   b  y g u e  s  t   onM a  y2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  In this work we report the cloning, sequencing, and expres-sion of a gene  (TcPI-PLC ) encoding a PI-PLC from  T. cruzi . Thegene is expressed at high levels in the epimastigote andamastigote stages, and its expression is induced during thedifferentiation of trypomastigotes into amastigotes, where itsproduct (TcPI-PLC) is associated with the plasma membraneand increases its activity. In contrast to other PI-PLCs,  T. cruzi PI-PLC is lipid modified  in vivo , and has a highly chargedlinker region between the catalytic X and Y domains. EXPERIMENTAL PROCEDURES Culture Methods—T. cruzi  amastigotes and trypomastigotes (Y strain) were obtained from the culture medium of L 6 E 9  myoblasts by amodification of the method of Schmatz and Murray (19) as we havedescribed before (12, 20). The contamination of trypomastigotes withamastigotes and intermediate forms or of amastigotes with trypomas-tigotes or intermediate forms was always less than 5% unless otherwisestated.  T. cruzi  epimastigotes (Y strain) were grown at 28 °C in liverinfusion tryptose medium (21) supplemented with 10% newborn calf serum. Protein concentration was determined using the Bio-Rad pro-tein assay. Trypomastigotes were induced to transform into amastig-otes axenically as described previously (22). Briefly, trypomastigoteswere washed once with DMEM containing 0.4% bovine serum albumin(DMEM-BSA) at pH 7.5. The cells were resuspended at a concentrationof 5    10 7  /ml in DMEM-BSA at either pH 5 or 7.5. The DMEM wasbuffered with 20 m M  MES (pH 5.0) or 20 m M  Hepes (pH 7.5). Parasiteswere incubated at 35 °C and harvested at different time points. Forovernight incubations, parasites previously incubated at acidic pH for4 h were centrifuged and resuspended in DMEM-BSA at pH 7.5. Chemicals— Newborn calf serum, MES, Hepes, CNBr-activatedSepharose 4B, and EGTA were purchased from Sigma. Trizol Reagent,and  Taq  polymerase were from Life Technologies, Inc., Gaithersburg,MD. PolyATract TM mRNA isolation system, pGEM-T Easy vector, Ri-boprobe  in vitro  Transcription System, and Prime-a-Gene Labeling System were from Promega, Madison, WI. The   ZAP-Express phageand pBluescript KS(  ) vectors were from Stratagene, La Jolla, CA.Sequenase sequencing kit was from U. S. Biochemical Corp.[  - 32 P]dCTP (3000 Ci/mmol), [  - 32 P]UTP (3000 Ci/mmol), and the Bio-trak [ 3 H]IP 3  assay system were from Amersham Pharmacia Biotech.[9,10- 3 H]Myristic acid (10–60 Ci/mmol), [9,10- 3 H]palmitic acid (30–60Ci/mmol), and EN 3 HANCE were from NEN Life Science Products Inc.,Boston, MA. pCR 2.1-TOPO cloning kit was from Invitrogen, Carlsbad,CA. Zeta Probe GT nylon membranes and the protein assay were fromBio-Rad. Digoxigenin (DIG System) was from Roche Molecular Bio-chemicals. The primers were purchased from Genosys BiotechnologiesInc.,Woodlands,TX.The  Escherichiacoli expressionvectorpET28c,theQuick 900 cartridge, and the His-bind buffer kit were from Novagen,Madison, WI. The Protease Inhibitor Mixture Set III was from Calbio-chem, La Jolla, CA. Protein A/G PLUS-agarose was from Santa CruzBiotechnology, Santa Cruz, CA. BF 3 -methanol was from Superco, Belle-fonte, PA. All other reagents were analytical grade.  Nucleic Acid Analysis—  All basic recombinant DNA techniques fol-lowed standard procedures described previously (23) unless otherwisenoted. For Southern blotting DNA was electrophoresed in 1.0% agarosewith TAE (40 m M  Tris, 20 m M  acetic acid, 1 m M  EDTA, pH 8.0) bufferand transferred to Zeta Probe GT nylon membrane. DNA was isolatedby standard procedures (23). Total RNA was isolated with Trizol rea-gent following the manufacturer’s recommendations. The polyadeny-lated RNA was obtained using a PolyATract mRNA isolation system.RNA was electrophoresed in 1.0% agarose gels with 2.2  M  formalde-hyde, 20 m M  Mops (pH 7.0), 8 m M  sodium acetate, 1 m M  EDTA andtransferred to Zeta Probe GT nylon membranes. DNA probes wereprepared using random hexanucleotide primers and Klenow fragmentof DNA polymerase I (Prime-a-Gene Labeling System) and[  - 32 P]dCTP. RNA probes were prepared from linearized double-stranded DNA templates with either T3 or T7 promoter sequencesupstream of the probe sequence using T3 or T7 RNA polymerase (Ri-boprobe  in vitro  Transcription System) and digoxigenin (DIG System).To amplify the PI-PLC gene of   T. cruzi , the polymerase chain reaction(PCR) was performed with 30 cycles of 94 °C for 1 min, 40 °C for 1 min,and 72 °C for 1 min, using 1.25 units of   Taq  DNA polymerase with 50ng of   T. cruzi  genomic DNA, 1   M  of each oligonucleotide primer, 20 m M Tris-HCl (pH 8.4), 50 m M  KCl, 2 m M  MgCl 2 , and 0.2 m M  of eachdeoxynucleoside trisphosphate in a PTC-100 Programmable ThermalController (MJ Research, Inc., Watertown, MA). The PCR productswere separated in an agarose gel, purified, and cloned into the pGEM-Teasy or pCR 2.1-TOPO. The sequences of the primers were: 5B, 5  -CA(C/ T)AA(C/T)AC(A/T/C)TA(C/T)(C/T)T-3   and 5R, 5  -GG(C/T)TT(A/C/G/ T)A(A/G)(A/C/G/T)AC(A/G)TA(A/C/G/T)CC-3  . To make a  T. cruzi  sub-genomic library, the genomic DNA was completely digested by  Bam HIand DNA fragments of 2–6 kb were purified from the gel and ligatedinto   ZAP-Express vector. The resulting library was screened by plaquehybridization with the PCR clone as a probe in a manner describedpreviously (23). DNA sequencing (24) was performed either manuallywith Sequenase sequencing kit or automatically with Dye TerminatorCycle sequencing kit and a 373A DNA Automatic Sequencer (Perkin-Elmer Applied Biosystem, Foster City, CA) at the Biotechnology Cen-ter, University of Illinois at Urbana-Champaign. DNA and deducedamino acid sequence were analyzed with the Wisconsin Sequence Anal-ysis Package (version 8.0, GCG, Madison, WI). Hydropathy plot analy-sis was performed by using the Kyte and Doolittle method (25). The TcP0  (26) DNA used as a control in the Northern blots was obtained byamplifying   T. cruzi  genomic DNA by PCR with the specific primers(5  -GTACGAGGAGCGTTTCAATG-3   and 5  -ATCATCCTCCTCTTCG-GGTT-3  ) and the product was purified and cloned into pCR 2.1-TOPO.cDNA was synthesized by using reverse transcriptase, total RNA, andoligo(dT) as a primer. The cDNA was amplified by PCR using primerscorresponding to the  T. cruzi  splice leader sequence (5  -ATAGAACAG-TTTCTGTAC-3  ) and a  TcPI-PLC  sequence (5  -CTTGATCACTTTGAT-GCAGG-3  ). PCR conditions were the same as described above exceptthat the annealing temperature was 60 °C. The sequence of   TcPI-PLC was deposited in GenBank under the accession number AF093565.  Expression and Purification of TcPI-PLC from E. coli— To obtain acontiguous  TcPI-PLC  open reading frame (ORF), two sequences whichcovered the 5  - and 3  -portions of   TcPI-PLC  were combined and clonedinto pBluescript KS(  ) (pTcPI-PLCorf). An  Nhe I site was created im-mediately upstream of the initiation codon to insert the  TcPI-PLC  ORFinto the  E. coli  expression vector pET28c. A 162-bp sequence at the5  -end of   TcPI-PLC  ORF was amplified by PCR using primers, one withthe  Nhe I site followed by  TcPI-PLC  5  -end sequence including theinitiation codon (5  -TGCTAGCATGGGTCTTTGTACGAGTAA-3  ), andthe other with the sequence 143 bp downstream of the initiation codon(5  -TAGGATATCACGCGAAGCTC-3  ). The PCR product was insertedinto the pTcPI-PLCorf to produce pTcPI-PLC-  Nhe I. The whole ORF and300 bp of 3  -end untranslated sequence of TcPI-PLC was excised fromthe pTcPI-PLC-  Nhe I and inserted into the multiple cloning site of pET28c (pET-TcPI-PLC).In order to overexpress  TcPI-PLC -encoding protein (TcPI-PLC),  E.coli  strain BL21(DE3) was transformed by pET-TcPI-PLC. The trans-formed cells were grown in LB medium and the gene expression wasinduced by adding isopropyl-1-  - D -galactopyranoside at a final concen-tration of 1 m M  when the cell density reached an  A 600  of 0.6. The cellswere harvested after 3 h incubation at 18 °C and used directly forSDS-PAGE analysis, or sonicated (4  30 s with 30 s intervals at 4 °C)in 5 m M  imidazole, 500 m M  NaCl, and 20 m M  Tris-HCl (pH 7.9) andcentrifuged at 21,000    g  at 4 °C for 20 min for separation into pelletand supernatant fractions. The supernatant was used for purification of active TcPI-PLC, and the pellet was used to extract TcPI-PLC forantibody production.For purification of the expressed TcPI-PLC, a nickel resin column(Quick 900 cartridge) and buffer system (His-bind buffer kit) were used.The supernatant fraction was applied to the column. After washing once with 60 m M  imidazole, 500 m M  NaCl, and 20 m M  Tris-HCl (pH 7.9),TcPI-PLC was eluted with 1  M  imidazole, 500 m M  NaCl, and 20 m M Tris-HCl (pH 7.9). The purified TcPI-PLC was transferred into dialysistubes (  M  r  cut off 3,500) and concentrated by incubating the tube inpolyethylene glycol (  M  r  15,000–20,000) powder at 4 °C until the volumewas one-tenth of the srcinal. The proteins were analyzed by SDS-polyacrylamide gel electrophoresis and silver staining (Bio-Rad).  PI-PLC Assay— PI-PLC activity was measured as the release of wa-ter-soluble radioactivity from [2- 3 H]inositol-labeled PI or PIP 2  by aprocedure described before (6) with minor modifications. Briefly, stocksolutions containing either PI or PIP 2  in organic solvent were dried justprior to use under a stream of nitrogen and suspended in reaction bufferby sonication for 10 s in a Branson digital sonifier. Reaction mixturescontained cold PI or PIP 2  at various concentrations, 15,000 to 20,000cpm of [ 3 H]PI (or [ 3 H]PIP 2 ), 1 mg of sodium deoxycholate/ml, 100 m M NaCl, 50 m M  Hepes-HCl (pH 7.0), 2.5 m M  EGTA, 0 to 10 m M  CaCl 2 , andan appropriate amount of enzyme in a total volume of 100   l. The finalpH in the reaction mixture was pH 7.15 unless otherwise noted. Precisefree Ca 2  concentrations were achieved by using a CaCl 2 -EGTA bufferand calculated using the free distributed software MaxChelator using the Bers stability constants (27, 28). Reactions were initiated by theaddition of enzyme, carried out for 10 min at 30 °C and terminated by  A Novel Phospholipase C in T. cruzi  6429   b  y g u e  s  t   onM a  y2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  the addition of 0.5 ml of chloroform-methanol-HCl (100:100:0.6) fol-lowed by 0.15 ml of 5 m M  EGTA in 1  N  HCl. Samples were subjected to vigorous vortex mixing for 30 s and centrifuged at 21,000   g  for 2 minto separate the organic and aqueous phases. The aqueous phase (0.25ml) was removed, dissolved in 5 ml of a liquid scintillation fluid, andcounted in a scintillation counter. Enzyme amount was adjusted in eachassay so that a linear time course could be obtained during the 10-minreaction.  Production of Polyclonal Antibody against TcPI-PLC—  A polyclonalantibody against TcPI-PLC was generated using the  E. coli -expressedrecombinant protein. For this purpose, the inclusion bodies in the pellet(insoluble) fraction of TcPI-PLC-expressing   E. coli  were used because of their large quantity. This fraction was suspended in 8  M  urea, 100 m M Tris-HCl (pH 8.0) and centrifuged at 21,000    g  for 15 min at roomtemperature to remove endogenous  E. coli  proteins. The pellet was thenwashed once with PBS, solubilized in 62.5 m M  Tris-HCl (pH 6.6), 3%SDS, 10% glycerol, 5% (v/v)   -mercaptoethanol, 0.2% bromphenol blue,and separated by SDS-PAGE (7.5%). TcPI-PLC was visualized by stain-ing with 2.5% Coomassie Brilliant Blue in water for 2 h and destaining in water for 2 h. The protein band corresponding to TcPI-PLC wasexcised from the gel, ground, mixed with water, incubated for 3 h atroom temperature with constant mixing and the supernatant collectedafter centrifugation at 1000   g  for 5 min. After repeating this processthree times, the supernatants were pooled and concentrated to 500  g/ml by constant air flow. A rabbit was injected subcutaneously with50   g of TcPI-PLC emulsified in Freund’s complete adjuvant, followedby five booster injections of 50   g of TcPI-PLC in Freund’s incompleteadjuvant at 2-week intervals. Sera were collected before the initialinjection (preimmune), prior to each booster injection, and 2 weeks afterthe final booster. The antiserum after the final booster had a titer of more than 1:20,000 as determined by Western blotting. Affinity purifi-cation of anti-PI-PLC antibodies was carried out by elution from acolumn to which the hexahistidine-TcPI-PLC fusion protein had beencoupled. The affinity column was prepared by conjugating purifiedhexahistidine-tagged TcPI-PLC (derived from a 1-liter culture of thetransformed  E. coli ) to CNBr-activated Sepharose 4B (0.5 ml of beads)as described by the manufacturer. The affinity matrix was incubatedwith 10 ml of anti-TcPI-PLC antiserum (1:2 dilution in PBS) for 16 h at4 °C, washed 4 times with 20 volumes of phosphate-buffered saline, andthe antibodies eluted in 0.2  M  glycine (pH 2.8), 1 m M  EDTA. Theantibodies were immediately neutralized with 0.1 volume of 1  M  Tris(pH 9.5), supplemented with sodium azide to a final concentration of 0.05% and stored at 4 °C.  SDS Electrophoresis and Western Blotting— The electrophoretic sys-tem used was essentially as described by Laemmli (29). Aliquots of different stages of   T. cruzi  (at the protein concentration indicated under“Results”) were mixed with an equal amount of non-reducing 2  SDSbuffer (125 m M  Tris-HCl, pH 6.6, 20% glycerol (v/v), 6.0% SDS (w/v),and 0.4% (w/v) bromphenol blue) and boiled for 5 min prior to applica-tion to SDS-polyacrylamide gels at the concentration indicated under“Results.” Electrophoresed proteins were transferred to a polyvinyli-dene difluoride membrane (Bio-Rad, Hercules, CA) by the method of Towbin  et al.  (30), using a Bio-Rad transblot apparatus. Following transfer, the membrane was blocked in 5% nonfat dry milk in TPBS(0.1% Tween 20, 80 m M  Na 2 HPO 4 , 20 m M  NaH 2 PO 4 , 100 m M  NaCl, pH7.5) for 30 min at room temperature. Affinity purified anti-TcPI-PLC inTPBS was then applied at room temperature for 60 min. The membranewas washed three times for 15 min each with TPBS. After incubating with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:10,000) and washing three times with TPBS, immunoblots were visu-alized on blue-sensitive x-ray film (Midwest Scientific, St. Louis, MO)using the ECL chemiluminiscence detection kit and following the in-structions of the manufacturer (Amersham Life Science).  Immunofluorescence Microscopy—  After washing three times withPBS, parasites were fixed with 4% formaldehyde in PBS for 1 h at roomtemperature, and allowed to adhere to poly- L -lysine-coated glass slides(Sigma). After permeabilization with 0.3% Triton X-100 in PBS for 3min and blocking with 3% bovine serum albumin in PBS for 20 min, theparasites were incubated with 1:150 dilution of the antibody againstTcPI-PLC, followed by 1:300 of fluorescein-5-isothiocyanate-conjugatedgoat anti-rabbit IgG antibody, both at room temperature. The imageswere obtained with an Olympus BX-60 fluorescence microscope. Theimages were recorded with a CCD camera (model CH250; PhotometricsLtd., Tucson, AZ) and IPLab software (Signal Analytics, Vienna, VA) asdescribed previously (31).  Metabolic Labeling—  After a 4-h incubation of trypomastigotes in theacidic medium (pH 5.0) (DMEM containing 0.4% fatty acid-free BSA and 20 m M  MES), metabolic labeling was carried out by adding [9,10- 3 H]myristate (25   Ci/ml), or [9,10- 3 H]palmitate (50   Ci/ml) to the me-dium at pH 7.5 (DMEM containing 0.4% fatty acid-free BSA and 20 m M Hepes). The cells were labeled for 16 h at 35 °C and harvested forimmunoprecipitation. Epimastigotes were metabolically labeled by in-cubating the cells in MEM with the same radiochemicals as above with2% dialyzed fetal calf serum for 16 h at 28 °C.  Immunoprecipitation—  All procedures were carried out at 4 °C. Thecells were lysed in radioimmunoprecipitation analysis (RIPA) buffer (50m M  Tris-HCl, pH 7.4, 150 m M  NaCl, 0.5% Nonidet P-40, 0.5% sodiumdeoxycholate, 0.1% SDS, 1 m M  EGTA, and 1 m M  MgCl 2 ) containing 1m M  4-(2-aminoethyl)-benzenesulfonylfluoride-HCl, 0.8   M  aprotinin, 50  M  bestatin, 15   M  E-64, 20   M  leupeptin, 10   M  pepstatin A (ProteaseInhibitor Mixture Set III), 100 m M  N   -  p -tosyl- L -lysine chloromethylketone (TLCK), and 2 m M  EDTA. The lysate was centrifuged at21,000   g  for 20 min, and the supernatant was collected and incubatedwith protein A/protein G-conjugated agarose beads (Protein A/G PLUS-agarose) to adsorb nonspecifically bound proteins. After centrifugation,the supernatant was collected, and mixed with anti-TcPI-PLC anti-serum at a final dilution of 1:100. After overnight incubation, theprotein-antibody complex was selectively adsorbed by incubation withProtein A/G Plus-agarose for 1 h. The beads were collected by centrif-ugation at 14,000   g  for 2 min and washed four times with RIPA bufferand once with 50 m M  Tris-HCl (pH 7.5). The collected beads were mixedwith an equal volume of 2    SDS electrophoresis buffer (125 m M Tris-HCl, pH 6.6, 10% (v/v)   -mercaptoethanol, 20% glycerol, 6.0% SDS(w/v), and 0.4% (w/v) bromphenol blue) and heated at 96 °C for 2 min.For [ 3 H]myristate or [ 3 H]palmitate-labeled protein, sample buffer with-out Tris was used to avoid deacylation of the protein at high tempera-ture (32). The proteins were separated by SDS-PAGE, and fluorographywas performed by treating the gel with EN 3 HANCE according to themanufacturer’s instructions, dried, and exposed to blue-sensitive x-rayfilm (Midwest Scientific, St. Louis, MO) at   80 °C. For deacylation of TcPI-PLC in the gel, the gel was washed twice for 30 min with 1  M hydroxylamine (pH 7.5), or 1  M  Tris-HCl (pH 7.5) (control) and rinsedthree times for 5 min with water prior to the fluorography. Quantitation of IP  3  in T. cruzi— The cellular content of IP 3  wasdetermined with a competitive binding protein assay. This assay wasperformed on acid-transformed (pH 5.0) and control (pH 7.5) trypomas-tigotes at different incubation times. Aliquots of the parasite suspen-sions were removed, centrifuged (2,000    g  for 10 min), and washedonce in DMEM-BSA (pH 7.5). The cells were resuspended at a concen-tration of 8.3  10 8 cells/ml in the same buffer. An aliquot was removedfor protein determination followed immediately by addition of ice-cold20% perchloric acid to a final concentration of 4% HClO 4 . The lysatewas incubated on ice for 20 min and centrifuged at 2000   g , for 10 min.The supernatant was adjusted to pH 7.5 by the addition of 10  M  KOHand the suspension was centrifuged again at 2000   g  for 10 min. IP 3 was quantified in the supernatant by measuring the ability of theparasite extract to displace [ 3 H]IP 3  from its binding protein as de-scribed in the manufacturer’s instructions.  IdentificationofFattyAcids— DeacylationoftheimmunoprecipitatedTcPI-PLC was carried out basically as described previously (32). A complex of [ 3 H]myristate-labeled TcPI-PLC (from 1    10 9 cells), wasadsorbed to polyclonal antibody anti-TcPI-PLC, which was in turnbound to protein A/G-conjugated agarose beads as described above. Thiscomplex was resuspended in 100   l of 2  SDS-PAGE buffer and heatedat 90 °C for 2 min. The resulting supernatant was adjusted to a volumeof 750   l by adding water. A similar complex of [ 3 H]palmitate-labeledTcPI-PLC, anti-TcPI-PLC, and agarose beads obtained as describedabove was resuspended in 250   l of 1  M  NH 2 OH (pH 7.0), and incubatedfor 20 min at room temperature, followed by centrifugation at 14,000   g  for 1 min and withdrawal of the supernatants. Two more hydroxyla-mine treatments were performed and the eluates were pooled. Fifty-four   l of concentrated HCl was added to both  3 H-acylated proteinpreparations to acidify the solutions. Fatty acids released from both 3 H-acylated preparations were recovered into hexane by three extrac-tions, each with 500   l of the solvent. The hexane phases for eachsamplewerepooled,back-extractedtwicewith700  lof10m M HCl,anddried under nitrogen gas. Methyl ester derivatives of the fatty acidswere generated by resuspending the dried fatty acids in 200   l of BF 3 -methanol and heating for 2 min at 100 °C. Two-hundred   l of 5  M NaCl, 0.5  M  acetic acid were added and the fatty acid methyl esters(FAMES) extracted three times, each time with 200   l of toluene.Toluene extracts were combined, dried under nitrogen gas, and resus-pended in 15   l of the solvent. The samples were analyzed by reversephase-high performance thin layer chromatography using AdsorbosilRP HPTLC plates (10  10 cm) (Alltech, Deerfield, IL) with chloroform/ methanol/water (15:45:3, v/v/v) as the mobile phase. The developed  A Novel Phospholipase C in T. cruzi 6430   b  y g u e  s  t   onM a  y2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  plate was air-dried and FAMES detected with an automatic TLC-linearanalyzer (Berthold Analytical Instruments, Inc., Nashua, NH). Stand-ard FAMES of [ 3 H]myristate and [ 3 H]palmitate were prepared in thesame manner described above. RESULTS Cloning and Sequencing of a PI-PLC Gene of T. cruzi (TcPI- PLC)— To clone the PI-PLC gene of   T. cruzi , a region contain-ing a conserved PI-PLC sequence was amplified from  T. cruzi genomic DNA. In order to design degenerate oligonucleotideprimers, amino acid sequences of three unicellular eukaryoticspecies (  Saccharomyces cerevisiae, Schizosaccharomyces pombe,  and  Dictyostelium discoideum ) were retrieved fromGenBank and regions with the highest similarity were locatedin the catalytic domains (X and Y domains) of the proteins (Fig.1  A ). Six degenerate oligonucleotide primers (three primerseach for the forward and reverse direction) were selected ac-cording to these domains and the PCR was carried out with  T.cruzi  genomic DNA as a template. Among those used, fourcombinations with four primers (two for the forward and twofor the reverse direction) gave distinctive bands of the expectedsizes using PCR conditions of relatively low stringency (anneal-ing temperature of 40 °C). One of the PCR products with theprimer pair 5B and 5R (the corresponding regions are shown inFig. 1  A ) and with a size of 1.0 kb was purified, ligated into thecloning vector pGEM-T Easy, and sequenced. Its deducedamino acid sequence showed marked similarity to those of PI-PLCs of other species (more than 40% identity and 60%similarity) and showed motifs characteristic of the X and Y domains of PI-PLCs. This PCR clone was named PLC-PCR(Fig. 1  B ).Southern blotting was performed with PLC-PCR as a probeto confirm the presence of this gene in the  T. cruzi  genome (Fig.1 C ). All restriction enzymes except  Eco RI gave single, strong bands, which were distinct from one another, indicating thepresence of the PI-PLC gene in the  T. cruzi  genome. Afterobtaining the entire ORF of this PI-PLC gene ( TcPI-PLC ) byscreening a  T. cruzi  subgenomic library, Southern blotting wasalso performed with the 5  -proximal (from nucleotide 1 to 915)or 3  -proximal (from nucleotide 1716 to 2272) region of   TcPI- PLC  to confirm that the bands in the Southern blotting repre-sented only one gene. Observation of more than one band with  Eco RI was not reproducible (data not shown) and could be dueto the star activity of the enzyme at the high concentrationused.In order to clone the entire  TcPI-PLC , a subgenomic libraryof   T. cruzi  was constructed. Since  Bam HI digestion gave astrong band at 4.4 kb in the Southern blotting (Fig. 1C),  T.cruzi  genomic DNA was digested with  Bam HI and DNA in the4.4 kb size range was purified from the gel, and ligated into a  ZAP Express phage vector. The resulting   T. cruzi  subgenomiclibrary was screened by plaque hybridization with PLC-PCR asa probe. After screening 5.0    10 4 plaques, 18 positive phageclones were selected. After  in vivo  excision to obtain the insertstogether with the phagemid vector, the DNA was digested with  Bam HI and  Eco RV and three distinct digestion patterns (four,five, and nine clones with each pattern) were observed (Fig.1  B ). Nucleotide sequencing results revealed that all clonescontained sequences overlapping with the sequence of PLC-PCR (Fig. 1  B ). Two groups (Fig. 1  B, groups I   and  II  ) sharedidentical 5  -regions of the ORF, one of which contained anadditional  Eco RV site in its intergenic sequence upstream of the ORF, and the third group contained the rest of the 3  -regions of the ORF (Fig. 1  B, group III  ), together forming acontiguous ORF of 2175 base pairs encoding a protein (TcPI-PLC) of 725 amino acid with a calculated molecular mass of 82kDa (Fig. 2  A ). The portions containing the initiation and stopcodon were sequenced twice in both directions to confirm thebeginning and end of the ORF. The variation between groups Iand II in the 5  -end flanking region could be due to allelic variation at the same locus because no evidence of multiplecopies of the gene or tandem repeats were detected in Southernblots with completely or partially digested genomic DNA. Thenumber of clones obtained also agrees with the molar ratio thatwould result after  Bam HI digestion of a diploid DNA with anallelic variation at the same locus (Fig. 1  B ). This type of allelic variation at the same locus was reported for the polyubiquitingene of   Trypanosoma brucei  (33).The nucleotide sequence and deduced amino acid sequenceobtained according to universal codon usage were analyzed toidentify homologies with other genes. This analysis revealedseveral domains characteristic of PI-PLCs (Figs. 2A and 3). F IG . 1.  Amino acid sequences used to design degenerate oligo-nucleotideprimerstoamplifythePI-PLCgeneof  T.cruzi bythePCR technique (  A ), scheme of the  PI-PLC  gene ( TcPI-PLC ) andthe PCR clone (PLC-PCR) (  B ), and Southern blotting analysis of the  TcPI-PLC  gene in genomic DNA ( C ).  A,  a comparison of thePI-PLC amino acid sequences of   S. cerevisiae  (ScPLC1, accession num-ber L13036),  S. pombe  (SpPLC1, accession number D38309), and  D.discoideum  (DdPLC, accession number Q02158) in their two catalyticdomains is shown.  Numbers  show the positions of amino acid residuesin the ScPLC1.  Arrows  indicate the amino acid sequences correspond-ing to the degenerate oligonucleotide primers synthesized.  B,  the posi-tion of the primers used (5B and 5R), the different groups of clonesobtained (groups I-III), and the  Bam HI and  Eco RV sites in  T. cruzi genomic DNA are also shown.  C,  total genomic DNA (10   g/lane) wasdigested with various restriction enzymes and analyzed as describedunder “Experimental Procedures.” Size markers are indicated on the left . The names of the restriction enzymes used are shown  above  eachlane.  A Novel Phospholipase C in T. cruzi  6431   b  y g u e  s  t   onM a  y2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om  TcPI-PLC and other PI-PLCs share a high level of sequenceidentity in the X and Y domains (44.5–55.5 and 33.3–46%identity, respectively) and moderate identity in the C2 domain(20.6–31.9% identity). TcPI-PLC contained an apparent EF-hand motif (Figs. 2  A  and 3) but did not contain either an extraCOOH-terminal domain like the   -isotype PI-PLCs or extradomains between X and Y domains as in the    -isotype PI-PLCs(Fig. 3). An hydropathy plot did not indicate any significanthydrophobic regions in TcPI-PLC (data not shown). The do-main organization of TcPI-PLC was similar to that of the mam-malian   -type PI-PLC subfamily as well as to that of otherunicellular eukaryotic PI-PLCs.In addition to the similarities to other PI-PLCs mentionedabove, there are some unique features found in the deducedamino acid sequence of TcPI-PLC. First, it was surprising tofind an  N  -myristoylation consensus sequence at the amino-terminal end of the amino acid sequence, since this has notbeen reported for any other member of the PI-PLC family todate (Figs. 2  B  and 3). Second, there are unusual clusters of negatively charged (amino acids 402–426; 17 out of 25 residueswere negatively charged) and mixed-charged (amino acids 437–457; 13 and 5 out of 21 residues were positively and negativelycharged, respectively) amino acid residues in the linker regionbetween the X and Y catalytic domains (Fig. 2  A ). Third, a PHdomain at the amino-terminal region, which is present in mostPI-PLCs, is not found in the TcPI-PLC amino acid sequence.These unique features may indicate some important differ-ences in regulation of   T. cruzi  PI-PLC from other PI-PLCs.  Expression, Purification, and Catalytic Activity of Recombi-nant TcPI-PLC— TcPI-PLC was expressed in  E. coli  to study itsenzymatic activity. The two pieces of the  TcPI-PLC  ORF se-quences (5  and 3  regions) from the genomic clones were com-bined in the cloning vector pBluescript KS(  ) to form a contig-uous and complete  TcPI-PLC  ORF. In order to express TcPI-PLC with a hexahistidine tag, an  Nhe I site was introduced atthe 5  -terminal end of the ORF by using the PCR. The resulting  TcPI-PLC  ORF was inserted into the prokaryotic expression vector pET28c in a way that allowed the hexahistidine tag andthe  TcPI-PLC  to be translated in-frame (pET-TcPI-PLC). An  E.coli  strain, BL21(DE3), was transformed by this plasmid andused for the induction of TcPI-PLC expression.The TcPI-PLC expression was induced by adding isopropyl-1-  - D -galactopyranoside to the culture of   E. coli  transformed bypET-TcPI-PLC. After 3 h of induction, the cells were disruptedand separated into pellet and supernatant fractions, and pro-teins in these fractions were separated using SDS-polyacryl-amide gels. In both pellet and supernatant fractions, the ex-pressed TcPI-PLC appeared as a protein with an approximatesize of 80 kDa, which is very close to the size predicted by itsamino acid sequence (82 kDa) (Fig. 4,  lanes 3  and  4 ).Taking advantage of the specific binding between the hexa-histidine tag and nickel ions, the recombinant TcPI-PLC waspurified by a one-step affinity chromatography procedure. Thesupernatant fraction of the TcPI-PLC-expressing   E. coli  ob-tained above was applied to the nickel affinity column and theTcPI-PLC was eluted from the column by a buffer containing ahigh concentration of imidazole (1.0  M ). As shown in the SDS-PAGE (Fig. 4,  lane 5 ), near homogenous TcPI-PLC was ob-tained by this method. A PI-PLC assay was carried out withPIP 2  as a substrate to measure the specific enzymatic activityin each fraction. PI-PLC activity was found in the fraction of the transformed  E. coli  obtained after nickel affinity chroma-tography. This purified recombinant TcPI-PLC was subse-quently used for further characterization of the enzyme.The rate of enzymatic reaction at different PIP 2  substrateconcentrations was determined. The enzyme reaction was car- F IG . 2.  Nucleotide and predicted protein sequences of   TcPI- PLC  (  A ) and  N  -myristoylation consensus sequence as comparedwith the amino-terminal ends of TcPI-PLC and FCaBP (  B ).  A, amino acid residues are numbered in the  left  margin; nucleotides arenumbered in the  right  margin. Catalytic domains (X and Y) are  boxed. Hatched lines  show a stretch of unusual, negatively charged and mixedcharged amino acid clusters between the X and Y domains. A potentialamino-terminal  N  -myristoylation site is  double-underlined . An EF-hand motif is in  bold italic . The amino acid sequences corresponding tothe highly conserved catalytic domains employed in the design of de-generate oligonucleotides are  underlined . The conserved amino acidresidues that were described to be in contact with IP 3  and Ca 2  in thecatalytic domains, and those of the calcium-binding sites in the C2domain of PI-PLC-  1 (38) are in  bold. B,  the glycine at position 1 andserine at position 5 are in  bold . At position 2 (*2), uncharged residuescould be present. Many of the reported myristoylated proteins have alysine at position 6 (*6).F IG . 3.  Structural comparison of the domain organization of PLCs from  T. cruzi  (TcPI-PLC),  D. discoideum  (DdPLC),  S. cer-evisiae  (ScPLC1),  Arabidopsis thaliana  (AtPLC1, accessionnumber U13203), and mouse PI-PLCs (PLC   1, PLC  1, andPLC   1, accession numbers AAD32616, AAD01749, and Q62077,respectively).  A Novel Phospholipase C in T. cruzi 6432   b  y g u e  s  t   onM a  y2  0  ,2  0 1  6 h  t   t   p :  /   /   w w w . j   b  c  . or  g /  D o wnl   o a  d  e  d f  r  om
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