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Acta Tropica 101 (2007) 80–89

Short communication

The Calcineurin A homologue from Trypanosoma cruzi lackstwo important regulatory domains�

Valeria Ruiz Moreno, Fernan Aguero, Valeria Tekiel, Daniel O. Sanchez ∗Instituto de Investigaciones Biotecnologicas, Universidad Nacional de General San Martın, CONICET, Buenos Aires, Argentina

Received 27 April 2006; received in revised form 29 September 2006; accepted 29 November 2006Available online 17 January 2007

Abstract

A novel protein from the parasite Trypanosoma cruzi homologous to calcineurin (serine–threonine phosphatase 2B) was identifiedand characterized. The Calcineurin A gene is present as a single copy gene per haploid genome and encodes a protein of 43 kDathat is expressed in all major developmental stages of T. cruzi. Surprisingly, it is mainly localized in the cell nucleus, in sharpcontrast with its mammalian counterpart. The T. cruzi calcineurin A protein presents the three invariants motifs characteristic of thePPP serine–threonine phosphatase superfamily. However, out of the four domains typically present in all calcineurin described todate, the T. cruzi calcineurin A possess only two domains: the catalytic and the calcineurin B binding domain. Sequence similaritysearches in the T. cruzi, Trypanosoma brucei and Leishmania major genomes revealed that only L. major presents a gene encodinga putative protein containing the four domains. On the other hand, the T. cruzi Calcineurin B subunit showed a conserved structure,and a reasonable level of similarity over the entire length with calcineurin B proteins from other organisms. Interaction betweenCalcineurin A and Calcineurin B was analyzed by yeast Two-Hybrid and GST pull-down assays.© 2006 Elsevier B.V. All rights reserved.

Keywords: Calcineurin; Trypanosoma cruzi; Protein phosphatase PP2B; Calcineurin A; Calcineurin B

1. Introduction

Trypanosoma cruzi is the flagellate parasite causativeof Chagas’ disease that affects about 20 million of peo-

Abbreviations: Cn, calcineurin; CnA, Calcineurin A subunit;CnB, Calcineurin B subunit; TcCnA-like, T. cruzi calcineurin A-like;TcCnB, T. cruzi calcineurin B; CaM, calmodulin; AI, auto inhibitory;CyP, cyclophilin; CsA, cyclosporin A; OA, okadaic acid

� Nucleotide sequence data reported in this paper have been sub-mitted to the EMBL/GenBank/DDBJ databases with the accessionnumbers AJ878872 (TcCnA-like) and AJ878873 (TcCnB).

∗ Corresponding author at: Parque Tecnologico Miguelete, Institutode Investigaciones Biotecnologicas, IIB-UNSAM, Av. Gral Paz EntreAlbarellos y Constituyentes, Edificio 24, B1650KNA San Martın,Buenos Aires, Argentina. Tel.: +54 11 4580 7255;fax: +54 11 4752 9639.

E-mail address: dsanchez@iib.unsam.edu.ar (D.O. Sanchez).

ple in Latin America (Moncayo, 1997). The parasitelife cycle involves four stages: the intracellular amastig-ote and extracellular trypomastigote forms specific tothe mammalian host and the epimastigote and meta-cyclic trypomastigote forms in the invertebrate (vector)host. As a digenetic parasite, T. cruzi must respondto extracellular and intracellular signals as it adapts tonew environments within different hosts (Parsons andRuben, 2000). In eukaryotes, this coordination is gener-ally achieved by regulatory circuits that involve proteinkinases, protein phosphatases, G proteins and secondmessengers, being phosphorylation and dephosphoryla-tion one of the major mechanisms of signal integration(Cohen, 1992; Parsons and Ruben, 2000).

Several kinases and phosphatases that may beinvolved in signal transduction have been described inT. cruzi, among them: a cAMP-dependent protein kinase

0001-706X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2006.11.008

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(Ochatt et al., 1993; Ulloa et al., 1988), a protein kinaseC (Gomez et al., 1989), a Ca2+/Calmodulin-dependentprotein kinase (Ogueta et al., 1996) and, more recently,two type 1 (Orr et al., 2000) and one type 2A (Gonzalezet al., 2003) serine and threonine phosphatases.

Calcineurin (Cn) is a highly conserved eukaryotic ser-ine and threonine phosphatase that belongs to the PP2Bgroup of the phosphoprotein phosphatase (PPP) fam-ily. The active protein is a heterodimer formed by acatalytic subunit (Calcineurin A, ∼60 kDa) and a reg-ulatory subunit (Calcineurin B, ∼19 kDa) (Rusnak andMertz, 2000). The Calcineurin A (CnA) subunit com-prises the catalytic domain and three regulatory domains:the Calcineurin B (CnB) binding domain, the calmod-ulin (CaM) binding domain and an auto inhibitory (AI)domain (Rusnak and Mertz, 2000). Among serine andthreonine phosphatases, Cn is the only known to be undercontrol of Ca2+/CaM. Besides, Cn is the main targetof the immuno-suppressant drug cyclosporin A (CsA),which upon formation of a complex with cyclophilin(CyP) binds to Cn inhibiting its phosphatase activity (Liuet al., 1991).

In T. cruzi, a role for Ca2+ has been proposed in theinvasion process where changes in Ca2+ concentrationwithin the parasite and the host cell have been reported(Moreno et al., 1994). Moreover, developmental changesand cell growth rate might also have Ca2+-dependentcomponents. Previously, it has been shown that CsAhas an inhibitory effect on the in vitro replication of T.cruzi epimastigotes (Bua et al., 2004; McCabe et al.,1985). Moreover, a Cyp gene has been identified andcharacterized (Bua et al., 2001). These findings suggestthat a phosphatase similar to Cn might be present inthis parasite. We report here the characterization of theT. cruzi homologue of the highly conserved eukaryoticCalcineurin phosphatase.

2. Materials and methods

2.1. T. cruzi cultures and cloning of TcCnA-like andTcCnB

The CL clone of T. cruzi was used. Epimastigoteswere grown at 28 ◦C in liver infusion tryptose broth sup-plemented with 10% fetal bovine serum. Trypomastigoteand amastigotes were obtained in vitro by infection ofHeLa cells grown in modified Eagle’s medium (MEM)supplemented with 3% fetal bovine serum at 37 ◦C. Par-asites were harvested and stored at −80 ◦C until used.

Isolation of RNA was performed using Trizol(GIBCO) according to the manufacturer’s protocol. Anhemi-nested RT-PCR amplification strategy was used

to obtain the full-length TcCnA-like and TcCnB genesequences. Reverse transcription was done by using CnAor CnB specific anti sense primers TcCnA1 (5′-GGCG-GCAGCGTTCTTTTG-3′) and TcCnB1 (5′-TGAT-GGCGACGGCAAGAT-3′), respectively. The first PCRround was done with a primer corresponding to partof the sequence of the T. cruzi miniexon (ME: 5′-AACGCTATTATTGATACAGTTTCTGT-3′) togetherwith the TcCnA1 or TcCnB1 primers. The hemi-nestedPCR was done with internal anti sense primers TcCnA2(5′-TGCCTTTAGCCGCTCTCGT-3′) or TcCnB2 (5′-TACGATGTTGATGGCGAC-3′), for CnA and CnBrespectively, and the ME primer. PCR products werecloned into pGEM-T Easy vector (Promega Corp.,Madison, USA) and sequenced.

2.2. In silico analysis

Sequence similarity searches were done using theBLAST (Basic Local Algorithm Search Tool) suite ofprograms on NCBI (National Center for Biotechnol-ogy Information) and TIGR (The Institute GenomicResearch) databases. Alignment of different CnA andCnB amino acid deduced sequences was done usingthe CLUSTAL W alignment method (Thompson et al.,1994). In the case of T. cruzi we also performed oursearches against a database of predicted proteins anno-tated by the sequencing consortium (accessible throughTcruziDB, http://tcruzidb.org). In the case of Try-panosoma brucei and Leishmania major we performedBLAST searches against finished sequences availablefrom the GeneDB website (http://www.genedb.org)using our TcCnA-like protein as query. Protein motifsearches were performed using PROSITE and Pfam pat-terns.

2.3. Southern and Northern blot analysis

For Southern analysis, T. cruzi genomic DNA (5 �g)was digested with various restriction enzymes. Restric-tion fragments were separated by electrophoresis on0.8% agarose gels and transferred to a nylon membrane.The blot was probed with TcCnA-like cDNA sequencecorresponding to TENS 1318 (nucleotides 574–1373of GenBank accession AJ878872) labeled with 32P byPCR. After hybridization at 65 ◦C overnight, the mem-brane was washed in 40 mM Na2HPO4, 0.1% SDS for1 h at the same temperature. For Northern blot, totalRNA (20 �g/lane) isolated from trypomastigotes, epi-mastigotes and amastigotes was electrophoresed on a1.5% formaldehyde gel, blotted on a nylon membraneand developed with a 32P-labeled probe specific for the

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complete TcCnA-like ORF sequence. After hybridiza-tion at 63 ◦C overnight, the membrane was washed for10 min at room temperature in 2× SSC/0.5% SDS, thenfor 30 min at 63 ◦C in the same washing buffer and finallyfor 30 min at 63 ◦C in 0.2× SSC/0.5% SDS. The mem-brane was also probed with a ribosomal RNA probe.Ribosomal RNA bands were used as molecular weightmarkers and mRNA levels were normalized using therRNA probe.

2.4. Expression of recombinant protein in bacteriaand production of polyclonal antiserum

The complete TcCnA-like ORF sequence, wasobtained by PCR using Pfu DNA polymerase and theprimers 5′-CCGGAATTCATGTATTCTGAACGAGG-GCT-3′ (sense) and 5′-CCGGAATTCTTAACTCA-TTTCCTCCTCTT-3′ (anti sense). The products werecloned into the pGEM-T Easy vector and sequenced.The cloned TcCnA-like ORF was sub-cloned into theEcoRI site of the pGEX 1�-T vector (Pharmacia) bytaking advantage of the EcoRI sites included in theprimers (underlined on primer sequences). pGEX 1�-T constructs were transformed into E. coli BL21 andpositive clones were induced with 0.25 mM IPTG at28 ◦C overnight. Bacterial pellets were lysed and thesoluble GST–TcCnA-like fusion protein was purifiedusing glutathione agarose. Purity of the fusion proteinwas checked by SDS-PAGE before producing the anti-serum. Purified fusion protein was injected into rabbits(50–100 �g) using Freund’s complete (first injection) orincomplete (subsequent injections) adjuvant. The poly-clonal antiserum obtained was tested by Western blotanalysis using the purified fusion protein.

2.5. Immunoblot analysis and fluorescencemicroscopy

For Western blot, total extracts from different forms ofthe parasite were prepared. Parasites were harvested andresuspended in SDS-PAGE loading buffer containing1% SDS. Blots were developed with rabbit TcCnA-likeantiserum previously depleted with an E. coli extractexpressing heterologous GST. Peroxidase-conjugatedanti-rabbit IgG was used as the secondary antibody. Anti-body complexes were detected using Supersignal WestPico Chemiluminescent substrate (PIERCE) accordingto the manual instructions.

For immunofluorescence, epimastigotes (1–5 × 106)were harvested and washed in PBS and HeLa cells con-taining T. cruzi amastigotes were washed once withPBS and incubated with trypsin at 37 ◦C for 3 min.

Cells were then resuspended in MEM supplementedwith 3% fetal bovine serum and incubated at 37 ◦C on aglass coverslip in a 24-well plate (1 × 104 cells per well)overnight. Infected cells and epimastigotes were fixedin 4% paraformaldehyde; epimastigotes were spread ona slide pre-treated with poly-l-lysine. Incubations weredone in PBS, 3% swine normal serum, and 2% BSA.Permeabilization was carried out with 0.1% saponine for30 min. Samples were incubated at room temperature for1 h with the primary antiserum (1:500) and, after wash-ing, the secondary antibody conjugated either with Alexa594 or FITC was added (1:1000). Hoechst 33423 wasused to visualize DNA in the samples. Coverslips weremounted in PBS containing 50% glycerol and visualizedusing a Nikon E600 epifluorescence microscope.

2.6. Yeast Two-Hybrid analysis

The TcCnA-like and TcCnB ORFs were cloned intoyeast plasmids pGBKT7 and pGADT7-Rec, respec-tively. pGBKT7 expresses the protein fused to the GAL4DNA binding domain (DNA-BD) and carries the TRP1selectable marker. pGADT7-Rec expresses the proteinfused to the GAL4 activation domain (AD) and car-ries the LEU2 selectable marker. Two-Hybrid interactionanalysis was carried out by co-transformation of plas-mids containing GAL4 DNA-BD/TcCnA-like and theGAL4-AD/TcCnB into yeast strain AH109 according tothe Yeast Protocols Handbook (CLONTECH). TcCnA-like–TcCnB interaction was selected in yeast mediumlacking Ade, His, Leu and Trp (selection medium).To check transformation efficiency and markers, eachplasmid was transformed into AH109 and plated onyeast medium without Trp or Leu. As negative controlseach plasmid carrying the fusion protein was trans-formed into AH109 and plated on selection medium.Results were obtained after plating the yeast strainAH109 transformed with each plasmid alone (pGBKT7-BD/TcCnA-like and pGADT7-AD/TcCnB) or withboth of them (pGBKT7-BD/TcCnA-like + pGADT7-AD/TcCnB). Plates were incubated at 30 ◦C for 4 days.Colonies obtained in the co-transformation plate werere-streaked in selection medium containing the chro-mogenic substrate X-�-Gal.

2.7. GST pull-down assay

GST pull-down assays were carried out as describedin Kaelin et al. (1991). Briefly, 5 �g purified GSTor GST–TcCnA-like fusion protein immobilized onglutathione-sepharose beads (Pharmacia) and 5 �g ofpurified recombinant TcCnB, without GST tag, were

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incubated at 4 ◦C for 1 h. After three washes with abuffer containing 20 mM Tris, pH 7.4, 0.1 mM EDTAand 50, 100, 150 or 300 mM NaCl, pulled-down proteincomplexes were subjected to Western blot analysis.

3. Results and discussion

In order to identify and characterize potential drugtargets in T. cruzi, we have searched public as well aslocal databases for orthologues of known drug targets.As a result, we have identified a homologue of Cn, whichwas further analyzed. The T. cruzi CnA (TcCnA) sub-unit was initially identified during a gene discovery workperformed in our lab (Verdun et al., 1998). As part ofthis work we identified an EST (TENS 1318) similar tothe rat CnA subunit (Swissprot P20651). The T. cruziCnB (TcCnB) was detected by searching the GenBankdatabase, where another T. cruzi EST (TENG 0261), withhigh similarity to the CnB subunit from N. gruberi andD. melanogaster was identified. Complete sequencingof both cDNA clones, obtained from an epimastigotecDNA library from T. cruzi CL Brener, showed that bothcDNAs contained the poly A tail but lacked the 5′ end.Therefore, we used a hemi-nested RT-PCR amplifica-tion strategy to obtain the full-length gene sequences.We obtained one PCR product that corresponded to the5′ end of TcCnB and two PCR products correspondingto different 5′ ends of TcCnA. Complete sequencing ofboth TcCnA cDNAs revealed the same ORF of 1149 bp,encoding a protein of 43 kDa (Fig. 1A). The completesequence of the TcCnB gene revealed an ORF of 531 bpthat encodes a protein of 19 kDa (Fig. 1B).

Alignment of the TcCnA deduced protein with knownCnA sequences showed an overall sequence identity of52–53% with Neurospora crassa, D. melanogaster andH. sapiens CnA sequences (Fig. 1A). The three invari-ant motifs (-GDxHG-, -GDxVxRG-, -GNH-) present inall PPP serine–threonine phosphatases (Cohen, 1997)are present as exact matches in our TcCnA (Fig. 1A).TcCnA contains two distinct functional regions: a cat-alytic domain (residues 1–333) and a putative TcCnBbinding domain (334–382). However, unlike other CnAproteins it lacks the regulatory domains present down-stream of this putative CnB binding domain. The missingdomains include the CaM binding domain and the AIdomain that are present in all CnA subunits published todate (Fig. 1A). We have also observed that the TcCnAdeduced protein sequence contains a Cys residue in theconserved motif SA(P/S)NYC (Fig. 1A). A Leu residueis generally observed at this position in other CnA pro-teins and the presence of a Cys residue in this positionhas been correlated with an increased susceptibility to

okadaic acid (OA), which is characteristic of PP1 andPP2A proteins (Bialojan and Takai, 1988). According tothese findings we decided to call our TcCnA protein asTcCnA-like.

Since the T. cruzi genome (El-Sayed et al., 2005) andthose of two related trypanosomatid parasites – T. bru-cei and L. major – have just been published (Berrimanet al., 2005; Ivens et al., 2005) we searched the avail-able genomic data for homologues of our TcCnA-likegene to investigate whether the CaM binding and AIdomains were also missing in these genes. In the case ofT. cruzi, a BLAST search using the TcCnA-like sequenceas query revealed two genes (Tc00.1047053508413.40,Tc00.1047053510755.138), that, except for having alonger N-terminus (probably due to the annotator’schoice of an upstream translational start codon) were98–99% identical to our TcCnA gene. A third gene(Tc00.1047053510187.500), reported as homologue toPP2B catalytic subunit A2, was also detected, but in thiscase the identity was quite low (51%). In all three casesthe encoded protein did not contain the CaM bindingnor AI domains. In the case of T. brucei and L. major weperformed BLAST searches using our TcCnA-like pro-tein as query. This comparative analysis also showed thepresence of short CnA genes lacking some of the regula-tory domains. The T. brucei genome shows the presenceof two different CnA-like genes, one of them (T.bru1 inFig. 1A) is similar to the TcCnA-like described hereinand lacks both the CaM binding and the AI domain; theother (T.bru2 in Fig. 1A) contains a CaM binding domainbut lacks the AI domain. The L. major genome showsone CnA-like gene (L.maj2 in Fig. 1A) lacking the AIdomain (similar to T.bru2) and one apparently completeCnA gene (L.maj1 in Fig. 1A). Thus, it appears that intrypanosomatids the presence of CnA-like genes with avariable complement of regulatory domains is common.Moreover, the observation that the three investigatedgenomes each contain a different set of CnA/CnA-likegenes might represent an indication of recent evolution-ary changes.

Finally, because the T. cruzi genome has beensequenced by the whole genome shotgun strategy, andalso because of its highly repetitive nature, we wantedto discard the possibility of missing a CnA gene withadditional CaM and/or AI domains due to assembly arti-facts. To investigate this, we set out to perform similaritysearches with TBLASTN using the L. major completeCnA protein as query against the T. cruzi CL-Brenerunassembled reads (GSS sequences). This search did notreveal matches spanning the CaM binding and/or autoin-hibitory domains, thus suggesting that the two PP2Bproteins lacking the mentioned domains as annotated by

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Fig. 1. Multiple sequence alignment of Cn proteins. (A) Alignment of TcCnA-like (T. cru) and PP2B proteins from N. crassa (N. cra, Swissprot ID:Q05681), D. melanogaster (D. mel, Swissprot ID: P48456) and H. sapiens (H. sap, Swissprot ID: Q08209) and with conceptually translated genomicsequences homologous to PP2B from T. brucei (T. bru1, Genedb: Tb10.70.0350, GenBank: EAN78060; T. bru2, Genedb: Tb09.160.0480, GenBank:EAN76278) and L. major (L. maj1, Genedb: LmjF26.2530, EMBL: CAJ05636; L. maj2, Genedb: LmjF36.1980, EMBL: CAJ09154). The threeinvariant amino acid motifs (-GDxHG-, -GDxVxRG-, -GNH-) are underlined in black. The hydrophobic domain that binds CnB is highlighted abovethe alignment. The motif that is involved in the sensitivity to okadaic acid (SA(P/S)NYC) is underlined in gray. (B) Alignment of TcCnB and CnBproteins from other species: LmCnB (L. major, Genedb: LmjF21.1630, EMBL: CAJ050636); TbCnB (T. brucei, Genedb: Tb10.70.7970, GenBank:EAN77469); CANB YEAST (S. cerevisiae); CANB SCHPO (S. pombe); CANB2 HUMAN (H. sapiens); CANB2 DROME (D. melanogaster).EF HAND motifs detected with the ScanProsite tool are shown in boxes. The 12-residue loop within the EF HAND that is involved in Ca2+ bindingis marked with a line within the box. The position of the invariant Glu or Asp residue involved in Ca2+ coordination is also indicated. The arrowheaddenotes the conserved Gly residue involved in myristoylation. Identical residues are black shaded. Conserved residues are shown in different shadesof gray according to BLOSUM62.

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the sequencing consortium are not assembly artifacts.Moreover, we obtained a similar result by followinga similar, though more sensitive search strategy usingthe GeneWise program together with a profile HiddenMarkov Model (HMM) of the metallo-phosphoesterasedomain present in all PPPs (Pfam accession PF00149).Using this HMM as query against T. cruzi unassembledshotgun reads we were able to uncover sequences corre-sponding to different members of the PPP superfamily.We then assembled these sequences and inspected theresulting contigs. After including new shotgun readsobtained from sequence similarity searches using theends of the contigs as queries (to extend the contigs inboth directions), we obtained only two contigs (out of45) with high similarity to our TcCnA cDNA or to otherPP2B proteins. None of the contigs showed the presenceof additional domains.

In light of this findings and because Cn is an het-erodimer, we also performed a comparative sequenceanalysis using the TcCnB protein as the starting point.A comparison against CnB proteins from other organ-isms showed a reasonable level of similarity over theentire length of the protein (Fig. 1B) with the T. bruceiCnB protein being the closest, with 63% identity. In CnBproteins most of the length of the protein is composedof highly specialized Ca2+-binding domains (EF-hands).These domains consist of a 12-residue loop flanked onboth sides by a 12-residue �-helical domain. The Ca2+

ion is coordinated by the 12-residue loop of which theimportant residues for Ca2+ coordination are the 1st, 3rd,5th, 7th, 9th and an invariant acidic residue (usuallyGlu) in the 12th residue (Rigden and Galperin, 2004).In TcCnB, out of the four EF hand domains known to bepresent in all CnB proteins, only the last two are com-pletely conserved (Fig. 1B). The second EF hand can bedetected by motif searches (matches PROSITE patternPS00018), but it lacks the invariant residue (Glu or Aspat position 12 in the loop) needed for Ca2+ coordina-tion. The first EF hand is not detected by motif searches(although it is detected by a more sensitive search usingthe profile HMM with Pfam accession PF00036) andshows considerable variation in the putative Ca2+ bind-ing loop including the absence of the essential acidicresidue needed for Ca2+ coordination. Interestingly, theother trypanosomatid CnB proteins also show less con-servation in their first and second EF hand domains(Fig. 1B). For both LmCnB and TbCnB all four EFhands are detected by motif searches, although the first2 EF hands show a lower score or are predicted as par-tial matches (TbCnB). However, and unlike the TcCnBprotein both the T. brucei and L. major CnB proteinsshow the required acidic residue conserved in the 12th

residue. Finally, it is known that CnB proteins fromother organisms contain a conserved myristoylation sitenear the N-terminus. Trypanosomatid CnB proteins alsoshow differences in the conservation of this site: none ofthe trypanosomatid CnB proteins match the PROSITEmyristoylation motif (PS00008). For both L. major andT. brucei this might indicate that the myristoylation siteis absent, as the required glycine residue (the one thatgets myristoylated) is not conserved (see the alignmentin Fig. 1B). On the other hand, this Gly residue is con-served in the TcCnB protein. This leaves the possibilitythat this site might be functional, though in this case, dif-ferent than the canonical site described by the PROSITEpattern.

To study TcCnA-like gene organization and genecopy number we next carried out Southern blot anal-ysis of T. cruzi genomic DNA (Fig. 2A). GenomicDNA digestion with non-probe-cutter enzymes showsone hybridization band in all cases except for the restric-tion enzymes AccI and AvaII (Fig. 2A, lanes 1 and 2).This observation could be explained by the presenceof polymorphic sites outside of the probe sequence.The single-probe-cutter enzyme AluI shows two smallhybridization bands, as expected (Fig. 2A, lane 5). Theseresults suggest the existence of one gene of TcCnA-likeper T. cruzi haploid genome, and further support the anal-ysis based on sequence similarity searches performedagainst the draft T. cruzi genome, where two TcCnA-likecopies (most probably corresponding to the two allelesof the gene) were found.

In order to identify the TcCnA-like transcript, North-ern blot analysis with total RNA from different formsof T. cruzi was performed (Fig. 2B). The probe encom-passing the TcCnA-like ORF hybridizes with two mRNAtranscripts of about 2.8 and 3 kb that are larger than theone deduced from cDNA sequencing (1373 pb). Similardiscrepancies have been reported previously for a fam-ily of RNA binding proteins in T. cruzi (De Gaudenziet al., 2003). The same hybridization pattern is seenin all developmental stages of the parasite but highermRNA levels are apparent in trypomastigotes (Fig. 2B).In epimastigotes, the steady-state level of TcCnA-likemRNAs is actually low and it is only detected after over-exposing the film (not shown). In contrast to TcCnA-like,the TcCnB probe hybridizes with a unique mRNA tran-script that matches the size of the sequenced cDNA. Thetranscript is present in all the parasite forms assayed,although at different levels, being more abundant in thetrypomastigote forms (not shown).

The complete ORF of TcCnA-like was cloned fusedto GST and the purified recombinant GST–TcCnA-likeprotein was used to generate a specific antiserum that

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Fig. 2. Analysis of TcCnA-like gene copy number, mRNA and protein expression and interaction with TcCnB. (A) Southern blot analysis of T. cruzigenomic DNA. Total DNA was digested with restriction enzymes either having no internal site: AccI (1), AvaII (2), HaeIII (3) and AvaI (4) or havingone internal site: AluI (5) within the probe. The blot was probed with TcCnA-like cDNA sequence corresponding to TENS 1318. The molecularsize marker used was Lambda DNA digested with BstII. (B) Northern blot analysis of different forms of T. cruzi. Total RNA from trypomastigotes(T), epimastigotes (E) and amastigotes (A) was electrophoresed, blotted on a nylon membrane and developed with a 32P-labeled probe specificfor the complete TcCnA-like ORF sequence. The membrane was also probed with a ribosomal RNA cDNA probe. (C) Western blot analysis ofprotein expression. Lysates from 30 × 106 parasites were run in SDS-polyacrylamide gels, transferred to nitrocellulose membranes and developedusing an anti-TcCnA-like-GST serum and chemiluminescence. E: epimastigotes, A: amastigotes, T: trypomastigotes. (D) Sub-cellular localizationof the TcCnA-like protein in amastigotes and epimastigotes. Cells were first incubated with anti-TcCnA-like antibody followed by Alexa 594(amastigotes) or FITC (epimastigotes) conjugated secondary antibodies. Below the amastigote figures are amplified sections showing TcCnA-likenuclear (arrowhead) and cytoplasmatic (arrow) localization. Nucleus and kinetoplast were stained with Hoechst 33423. (E) Interaction analysisbetween TcCnA-like and TcCnB (Two-Hybrid system). The TcCnA-like and TcCnB ORFs were cloned into yeast plasmids pGBKT7 and pGADT7,respectively. Table: Results obtained after plating the yeast strain AH109 transformed with each plasmid alone (pGBKT7-BD/TcCnA and pGADT7-AD/TcCnB) or with both of them (pGBKT7-BD/TcCnA + pGADT7-AD/TcCnB). X-�-Gal assay: Colonies obtained in the co-transformation platewere re-streaked in selection medium containing the chromogenic substrate X-�-Gal.

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was employed to study the expression of the TcCnA-like protein in the different stages of the parasite. Aunique protein band that matched the predicted molec-ular weight of the TcCnA-like protein was detected(43 kDa, Fig. 2C). We next carried out immunofluores-cence analysis to study the sub-cellular localization ofthe TcCnA-like protein in the different forms of the par-asite. The TcCnA-like protein showed a predominantlynuclear localization in both T. cruzi amastigotes and epi-mastigotes (Fig. 2D). The protein was also present in thecytoplasm showing a diffuse and punctuate distribution,although at lower levels based on the intensity of fluo-rescence detected. The same localization was observedin trypomastigotes and metacyclic trypomastigotes (notshown). Preimmune rabbit serum showed only back-ground levels of fluorescence (not shown). These resultsare opposite to those observed in mammalian cells,where Cn is mainly found in the cytoplasm and istranslocated to the nucleus in low and variable amountsupon interaction with its receptor (Bollen and Beullens,2002).

As mentioned before, the TcCnA-like protein con-tains a carboxy terminal region presumably involved inthe interaction with TcCnB. Binding between CnA andCnB subunits occurs through hydrophobic interactions(Watanabe et al., 1995). The putative CnB-bindingdomain of the TcCnA-like protein, although lessconserved at the primary sequence level, shows ahydrophobicity profile that is strikingly similar to theone observed in other CnA proteins (data not shown).In order to see if the TcCnA-like and TcCnB proteinscan form an heterodimer, we used a Two-Hybrid assay.Yeast growth in selective medium was only observedwhen the vectors pGADT7 carrying the coding sequenceof TcCnB and pGBKT7 carrying the coding sequenceof TcCnA-like were co-transformed into the yeast strainAH109. No colonies were observed when yeast weretransformed with each fusion vector alone and platedin the same selective medium (table in Fig. 2E). Inother words, selectable marker expression under GAL4 regulation was due to the interaction between TcCnA-like and TcCnB. This result was further confirmed byre-streaking co-transformed yeast colonies in the sameselective medium carrying the chromogenic substrateX-�-gal where all colonies assayed developed blue color(X-�-Gal assay in Fig. 2E). Interaction between bothproteins was also tested using a GST pull-down assay.The results obtained under different wash stringencyconditions (from 50 to 300 mM NaCl) were alwaysnegative (not shown). Even though results obtainedby these two different approaches were conflicting,we favor the hypothesis of a weak but functional CnB

binding domain in the TcCnA-like protein. This sug-gestion is based on: (i) the conservation of the carboxyterminal of TcCnA, including the conservation of thehydrophobic nature of this region, which is importantfor the interaction and (ii) the fact that the interactiondetected with the Two-Hybrid system occurred in anintracellular environment. If this were the case, thenegative result obtained in the pull-down assay mightbe explained by our inability to find the optimal in vitroconditions to detect this weak interaction.

In Leishmania, Ca2+ and CaM-dependent phos-phatase activity have been detected (Banerjee et al.,1999; Rascher et al., 1998) but not in T. cruzi whereall attempts to detect this phosphatase activity wereunsuccessful (our results, data not shown). The compar-ative analysis described in this work showed that of thethree parasite’s genome only L. major has a completeCnA gene, that is, containing all the domains present inclassical Cn proteins. This result is in agreement witha previous report showing that L. major has a Ca2+-and CaM-dependent phosphatase activity (Rascher etal., 1998). The same cannot be said for T. cruzi and T.brucei. Despite that Cn has been highly conserved fromyeast to mammals, our results suggest that in trypanoso-matids this phosphatase has diverged from the originalmodel through loss of a variable number of regulatorydomains, thus generating shorter versions of the classicalCn. In T. cruzi this novel version of Cn lacks its Calmod-ulin and Autoinhibitory domains. Although the classicalmechanism of Cn regulation is by Ca2+/CaM (Klee etal., 1988), it is possible to speculate that in T. cruzi (andperhaps also in other trypanosomatids) this phosphatasemay have lost its regulation by Ca2+. Alternatively, try-panosomatid CnA-like proteins lacking the CaM bindingdomains could be regulated by Ca2+ through its reg-ulatory subunit (CnB), which itself is a Ca2+-bindingprotein, or perhaps through other interacting proteins.Further characterization of these TcCnA-like proteinswould be necessary to elucidate their biological role inthese trypanosomatids.

Acknowledgements

We thank Dr. Antonio Gonzalez for kindly providingthe EST TENG 0261. Preliminary genomic data obtainedfrom http://www.tigr.org/, was provided by the TIGR-SBRI-KI Sequencing Consortium supported by NIHgrants AI45038, AI45061 and AI45039. This study wassupported in part by grants from the World Bank/UnitedNations Development Project/World Health Organiza-tion Special Program for Research and Training inTropical Disease (TDR Project 970629), the Agencia

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Nacional de Promocion Cientıfica y Tecnologica, theCarrillo-Onativia Fellowship, the Ministerio de Salud,and the Fundacion Antorchas, Argentina.

VR was supported from a fellowship from the Con-sejo de Investigaciones Cientıficas (CIC), Buenos Aires,Argentina. VT is a postdoctoral fellow and FA andDOS are members of the research career of the Con-sejo Nacional de Investigaciones Cientıficas y Tecnicas(CONICET), Argentina.

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