FEBS Letters 585 (2011) 193–198

journal homepage: www.FEBSLetters .org

Identification and functional characterization of a poly(A)-binding proteinfrom Leishmania infantum (LiPABP)

Natalia Guerra a, María Vega-Sendino b, M. Isabel Pérez-Morgado a, Edurne Ramos a, Manuel Soto b,Víctor M. Gonzalez a, M. Elena Martín a,⇑a Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, IRyCIS, 28034 Madrid, Spainb Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular, Universidad Autónoma de Madrid, CSIC-UAM, 28049 Madrid, Spain

a r t i c l e i n f o

Article history:Received 10 September 2010Revised 22 November 2010Accepted 23 November 2010Available online 26 November 2010

Edited by Michael Ibba

Keywords:Poly(A)-binding proteinTranslation regulationLeishmania infantum

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.11.042

Abbreviations: Li, Leishmania infantum; MAPK, mitoPABP, poly(A)-binding protein; RRM, RNA-recognitiododecyl sulphate–polyacrylamide gel electrophoresis;visceral leishmaniasis⇑ Corresponding author. Address: Servicio de Bioqu

Ramón y Cajal, Ctra. Colmenar km 9100, E-28034 Ma9016.

E-mail address: m.elena.martin@hrc.es (M.E. Mart

a b s t r a c t

Gene expression regulation in Leishmania has been related to post-transcriptional events involvingmainly sequences present in the 50 and 30 untranslated regions. PABPs are high-affinity poly(A)-binding proteins that are implicated in the regulation of translation initiation, RNA stability andother important biological processes. We describe a PABP from Leishmania infantum (LiPABP) thatshows a very high homology with PABPs from other eukaryotic organisms, including mammals andother parasites. LiPABP conserves the main domains present in other PABPs, maintains poly(A)-binding properties and is phosphorylated by p38 mitogen-activated protein kinase. Using the serafrom dogs infected with L. infantum, we demonstrate that LiPABP is expressed in L. infantumpromastigotes.� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction for RNA recognition, but is necessary for interaction with factors

The poly(A)-binding protein (PABP) of eukaryotes binds the 30

end poly(A) tail of the mRNAs and participates in different cellularprocesses such as translational initiation and termination and inmRNA turnover [1–3]. When bound to the poly(A) tail, PABP circu-larizes mRNA molecules via its interaction with translational initi-ation factors at the cap, enhancing translational initiation andstabilizing mRNA [1–3]. In addition, interaction with the releasefactor eRF3 and dissociation of PABP from the poly(A) tail is neces-sary for progressive deadenylation and consequent decay for manytranscripts [4–7]. Furthermore, this protein seems to be involved inmRNA transport from the nucleus to the cytoplasm [1,8].

PABPs interact with poly(A) via RNA-recognition motifs (RRMs)that appear to be present in proteins in all types of organisms, sug-gesting that this is an ancestral motif with important functions inRNA biology. On the other hand, the C-terminal region of PABPcontains an highly conserved carboxy-terminal helical domainnamed MLLE, previously known as PABC [9], that is not required

chemical Societies. Published by E

gen-activated protein kinase;n motif; SDS–PAGE, sodiumUTR, untranslated region; VL,

ímica-Investigación, Hospitaldrid, Spain. Fax: +34 91 336

ín).

such as the release factor eRF3 [4] and the PABP-interacting pro-teins Paip1 and Paip2 [10]. Finally, it exits a proline and gluta-mine-rich region that connect the RRMs to the MLLE domain thatis implicated in the interaction between different PABP moleculesbinding the poly(A) tail [11]. This region, the least conserved seg-ment of the protein, also contains arginine residues that are targetfor the arginine methyl transferase PRMT4/CARM1.

Leishmania is a parasitic protozoan of the trypanosomatids fam-ily that possesses a digenetic life cycle with two discrete morpho-logical phases: the promastigote, which develops extracelullarlywithin the gut of the insect vector, and the amastigote that is spe-cialized to survive within the macrophage’s phagolysosome of thevertebrate host. It is assumed that trypanosomatids parasites needa regulated expression of stage-specific genes to survive extremeenvironmental changes in which PABP regulation could play somerole.

Different PABPs have been described in trypanosomatids. Thefirst PABP homologue reported in this family was in Trypanosomacruzi (TcPABP1) [12]. Afterwards, a Trypanosoma brucei orthologue86.4% identical to the T. cruzi protein was characterized [13]. Next,a Leishmania major PABP1 (LmPABP1), only 35% identical to eitherof the Trypanosoma PABP homologues, has been found [11]. Thislow level of identity could indicate that this protein is not thehomologue of the Trypanosoma PABP. Finally, an in silico analysisof a putative PABP from Leishmania amazonensis (LaPABP) has beenreported [14]. In this paper we described the first PABP homologue

lsevier B.V. All rights reserved.

194 N. Guerra et al. / FEBS Letters 585 (2011) 193–198

from Leishmania infantum (Li), called LiPABP. LiPABP conserves themain domains present in other PABPs and maintains poly(A) bind-ing properties. Moreover, we demonstrate the presence of LiPABPin L. infantum promastigotes.

2. Materials and methods

2.1. Cell culture and extract preparation

L. infantum (MCAN/ES/96/BCN150) promastigotes were grownat 26 �C in RPMI 1640 medium (PAA Laboratories), supplementedwith 10% (v/v) foetal calf serum (PAA Laboratories), 10 U/ml peni-cillin and 100 lg/ml streptomycin (Gibco) and lysated in ice-coldbuffer A (20 mM Tris–HCl, pH 7.6, 1 mM dithiothreitol (DTT),1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride (PMSF),1 mM benzamidine, 2 mM sodium molybdate, 2 mM sodium b-glycerophosphate, 0.2 mM sodium orthovanadate, 120 mM KCl,1 lg/ml leupeptin and pepstatin A, and 10 lg/ml antipain) contain-ing 1% TritonX-100 and then centrifuged at 17 000�g for 20 min. Protein deter-mination was performed by the Bradford method [15].

2.2. Sequence alignment, domain assignment and homology modellingof LiPABP

Homologous sequence to higher eukaryotic of LiPABP(XP_001469222/LinJ35.4250) was deduced from the Leishmaniagenome database (LeishDB; www.sanger.ac.uk), using as querythe sequence of the human protein. Determination of LiPABP se-quence, molecular weight and isoelectric point were done withthe ExPasy program (www.espasy.ch), and conserved domainsdetermining was performed with SMART program (http://smar-t.embl-heidelberg.de/). The PABPs sequence alignment analysiswas produced with CLUSTALW2 program. The structural character-ization was obtained by CPH models and the homology modellingwas performed using the SWISS-MODEL tool that enables anautomated comparative protein modelling. Finally, the potentialfunctional sites were identified with ELM server (http://elm.eu.org/news.html).

2.3. Cloning and plasmid construction

The L. infantum PABP coding sequence was PCR amplified fromgenomic DNA of L. infantum parasites. The oligonucleotides For-ward, 50-CGGGATCCATGGCCTTCACTGGTCCGAATC-30 and Reverse,50-CGGGATCCTCA AACGCTCATG-30, designed from LinJ35.4250 se-quence, were used. BamHI restriction sites included in the primersto facilitate cloning are underlined. The resultant PCR product wasinserted into pBluescript II SK (Stratagene). LiPABP was introducedinto the XbaI/EcoRI site of the eukaryotic expression plasmidpcDNA3-myc to produce the fusion protein myc-LiPABP. LiPABPwas cloned into the BamHI site of pQE30 (Qiagen) to obtain6�HIS-LiPABP recombinant protein (rLiPABP). The constructs wereanalyzed by sequencing.

2.4. Expression and purification of recombinant LiPABP

Recombinant LiPABP was expressed in M15[pREP4] Escherichiacoli strain cells (Qiagen) and purified from inclusion bodies asindicated in Ramos et al. [16]. Fractions were collected and thepresence of LiPABP was analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and silver stain.The fractions containing LiPABP were pooled and concentratedand aliquots were stored at �80 �C. Protein determination was per-formed as above.

2.5. Cell culture and transient transfections

Transient transfections with pcDNA3-myc-LiPABP were essen-tially performed as described previously [17]. After the time oftransfection the medium was removed and the cells washedtwice with ice-cold buffer A as above and lysed in the same buffercontaining 1% Triton X-100 (50 ll/106 cells). Cell lysates werecentrifuged at 12 000�g for 15 min and the supernatants werekept at �80 �C until used. Protein determination was performedas above.

2.6. Western blot analysis

Samples were subjected to SDS–PAGE, transferred onto PVDFmembranes (GE Healthcare), incubated with the primary antibod-ies indicated in Figure legends and later with the secondary perox-idase-conjugated antibodies (GE Healthcare) and developed withECL reagent (GE Healthcare). Stained bands were scanned andquantified with an analyzer equipped with the Image QuantTL soft-ware package (GE Healthcare).

2.7. ELISA assays and anti-LiPABP antibody purification

Serum samples, generously provided by Dr. Carlos Gómez Nie-to, were collected from 25 dogs suffering symptomatic canine vis-ceral leishmaniasis (VL) and control sera were obtained fromthree healthy animals. All sera were positive when were testedby indirect immunofluorescence as described [18]. In the ELISAassays, 96-well Maxisorp plates (Nunc) were coated with 100 llof the LiPABP recombinant protein (2 lg/ml) in phosphate salinebuffer (PBS) for 18 h at 4 �C and processed as previously described[19].

Specific antibodies against L. infantum PABP were affinity-purified from a pool of four positive anti-PABP sera. For thatpurpose 0.7 mg of the rLiPABP was covalently bound to cyanogenbromide (CNBr)-activated Sepharose 4B (GE Healthcare) andpacked into a column. Coupling and blocking were carried outaccording to the manufacturer’s instructions. One ml of the mixedsera was passed through the antigen column. After washing, thespecific antibodies were eluted from the column with 0.1 Mglycine, pH 2.8 and equilibrated to pH 7.5 with 1 M Tris–HCl. Thesolution of antibodies was restored to the original volume of thepooled sera.

2.8. Cell-free protein expression

TNT Quick Coupled Transcription/Translation System based onrabbit reticulocyte lysates (Promega) was used as recommendedin the manufacturer’s instructions. Briefly, 500 ng of pcDNA3-myc-LiPABP plasmid was added to the mix reaction and incubatedfor 30 min at 30 �C. Five micro liters of the mix were loaded in a10% SDS–PAGE gel.

2.9. Poly(A) activity assay

First, poly(A)-Sepharose was formed by the covalent coupling ofpoly(A) (Invitrogen) to CNBR-Sepharose 4B following the manufac-turer instructions (GE Healthcare). To determine the capacity/abil-ity of LiPABP for binding poly(A), 100 lg of lysates from L. infantumpromastigotes or 25 lg of lysates from HEK293T cells expressingmyc-LiPABP, in 200 ll of binding buffer (20 mM Tris–HCl, pH 7.4,1 mM DTT, 1 mM MgCl2, 5 mM EDTA and 1 mM PMSF), were incu-bated with 50 ll of poly(A)-Sepharose (50%) for 1 h at 4 �C in theabsence or the presence of 25 lg of poly(A) or poly(U). The beadswere washed three times with binding buffer and proteins wereeluted with Loading/Sample buffer.

N. Guerra et al. / FEBS Letters 585 (2011) 193–198 195

2.10. Phosphorylation of LiPABP in vitro

Recombinant LiPABP protein (2 lg) was incubated in the ab-sence or the presence of 6.25 ng of eukaryotic recombinant p38

Fig. 1. Clustering of representative PABPs sequences from different species. Thetree was obtained from the alignment of the sequences in ClustalW2 and displayedwith the program MEGA4. The scale bar indicates 0.1 substitutions per site. Tc, T.cruzi; Tb, T. brucei; Lm, L. major; Li, L. infantum; h, human. References are inparenthesis.

Fig. 2. Comparison between human PABP and LiPABP. (A) ClustalW2 alignment compmotives RNP1 and RNP2 in the RRMs domains are highlighted in blue and violet, respectivfor RNA binding. The conserved octomeric sequence located in the C-terminal domain isTheoretical model of the RRM I and RRM II domains showing RNP1 and RNP2 coloured asRRM domain. (C) Structure of the C-terminal domain of LiPABP showing the conservedSwiss-PDB Viewer.

mitogen-activated protein kinase (MAPK) (Upstate) in kinase buf-fer containing 30 lM ATP and 3 lCi of [c-32P] ATP (Hartmann Ana-lytic) at 30 �C for 20 min. Kinase reactions were stopped by mixingwith Loading/Sample buffer, subjected to 10% SDS–PAGE and auto-radiography or visualized using a Typhoon 9400 Phosphorimager(GE Healthcare).

3. Results and discussion

The poly(A)-binding protein with homology to human PABPfrom L. infantum, named LiPABP, was obtained from a L. infantumgenomic library. A single 1770 pb product was cloned and se-quenced. The reading frame results in a protein containing 585amino acids, an expected molecular weight of 65 kDa and a basictheoretical isoelectric point of 9.14. The analysis of the deducedamino acid sequence and the alignment of this sequence withthose of PABPs from human and other trypanosomatids showedhigh identity with some of them. Thus LiPABP is approximately70% identical to the PABPs from T. cruzi [12] and T. brucei [13]but only has a 33% of identity to LmPABP1 [11]. During the reviewof this paper, two other PABPs from L. major have been character-ized [20]. One of them, named LmPABP2, could be the orthologue ofLiPABP (95% identical). Phylogenetic and molecular evolutionaryanalyses of all PABPs human and trypanosomatids were conducted

aring the PABPs sequences from H. sapiens and L. infantum. The highly conservedely, and the MLLE domain in green. Asterisk indicated conserved residues important

highlighted in yellow. The residues implicated in methylation are shown in grey. (B)in A. The secondary structure shown at the top of the figure corresponds to a singleoctomeric sequence in yellow. Figures were prepared using the software package

196 N. Guerra et al. / FEBS Letters 585 (2011) 193–198

using MEGA version 4 [21]. As can be seen in Fig. 1, LiPABP is in thesame branch that the recently published LmPABP2 and both comefrom a common branch shared with TbPABP1 and TcPABP1.

LiPABP sequence conserves the four characteristic RRMs, lo-cated in the positions 9–81 (RRM1); 96–169 (RRM2); 184–263(RRM3); 287–364 (RRM4), the C-terminal domain, located in thepositions 509–579, and the sequence between the N-terminalRRMs and the C-terminal domain that contains only a 10% of pro-line, but is rich in glycine residues (19%) (Fig. 2A). It has been pre-viously described that the first two RRMs are sufficient for specificpoly(A) binding [23] and RRMs 3 and 4, lacking poly(A)-bindingspecificity, may mediate recognition of AU-rich elements (ARE)motifs implicated in mRNA stability present in the 30 untranslatedregion of many mRNAs [24]. The structure of the domains of thehuman PABPC2 shows that the RRMs are globular domains com-posed of a four-stranded anti-parallel b sheet backed by two a heli-ces [22]. The central two b strands of each RRM include two highlyconserved sequence motifs, RNP1 and RNP2. We have generated atheoretical three-dimensional model of LiPABP that show the fouranti-parallel strands forming the b-sheet responsible for the RNAbinding and two hydrophobic a-helices in the opposite face(Fig. 2B). Finally, it has been also described that the globular C-ter-minal domain of PABPs from Homo sapiens [10] and T. cruzi [25]contains five a helices. However, LiPABP shows only four a helicesin this region, as described for the MLLE domain from Saccharo-myces cerevisiae [26], probably due to the fusion of helices 1 and

Fig. 3. Endogenous LiPABP is expressed in L. infantum promastigotes. (A) ELISA reactivityagainst recombinant LiPABP. Solid lines, mean values (b, P < 0.01). The U-Mann–Whitney(B) 25 lg of lysate from HEK293T cells and 1 lg of recombinant LiPABP were blotted withECL system. After washing, the membrane was reprobed with a commercial human anti-cytoplasmic levels of LiPABP. Different amounts of recombinant LiPABP (12.5–100 ng)antibodies and developed as above. The bands were quantified and the values obtained

2 (Fig. 2C). Moreover, the motif KITGMLLE located in helix 3 de-scribed in human PABP [9,10] and that is involved in the interac-tion with regulatory proteins and translation factors containing apeptide named PAM2, is also conserved in LiPABP.

Different Leishmania intracellular proteins are antigenic in hu-man and dogs suffering VL, including the eIF4A translation initia-tion factor [27]. Thus, we next performed ELISA experiments toanalyze the immunoreactivity of sera from dogs with symptomaticcanine VL against LiPABP. Results indicated that LiPABP is recog-nized by the immune system of the vertebrate host, since 100%of the dogs with symptomatic canine VL showed absorbance valuesabove the negative sera (Fig. 3A). We used the four most antigenicsera to obtain a purified anti-LiPABP serum that was able to recog-nize the recombinant LiPABP but not the endogenous PABP pre-sents in HEK293T cells (Fig. 3B), indicating that the immuneresponse is specifically elicited against the LiPABP as also occurswith other conserved parasite intracellular antigens. In addition,the serum detected a band of approximately 65 kDa in lysates fromL. infantum promastigotes corresponding to endogenous LiPABP(Fig. 3C). We have also used this serum to quantify the cytoplasmiclevels of LiPABP. The results indicated the presence of 2.5 ng ofLiPABP per microgram of protein in lysates from stationary cul-tures containing 107 promastigotes/ml (Fig 3C).

To confirm the function of LiPABP as a poly(A) binding pro-tein we performed in vitro binding assay in which lysates fromL. infantum promastigotes were incubated with poly(A)-Sephar-

of sera from dogs diagnosed of canine visceral leishmaniosis (VL) and control seratest was used to analyze the data of the sera from symptomatic and control animals.the anti-LiPABP antibodies purified from canine VL sera (1/100) and developed withPABP antibody (Cell signaling; 1/1000) and developed as above. (C) Quantitation ofand two amounts of total proteins (25 and 50 lg) were blotted with anti-LiPABPfor cytoplasmic protein extrapolated.

N. Guerra et al. / FEBS Letters 585 (2011) 193–198 197

ose in the absence or the presence of poly(A) or poly(U) as com-petitors. Endogenous LiPABP bound to the resin was analyzed byWestern blot with the purified dog anti-LiPABP serum. As shownin Fig. 4A, the amount of LiPABP bound to poly(A)-Sepharose de-creased significantly (around 20%) in the presence of freepoly(A). However, the addition of poly(U) did not appreciablydecrease the amount of LiPABP bound to poly(A)-Sepharose. Toreinforce this result we have also performed the same experi-ments with lysates from HEK293T cells expressing myc-LiPABP.As shown in Fig. 4B, the band (70 kDa) corresponding to LiPABPbound to poly(A)-Sepharose decreased 75% in the presence ofpoly(A) meanwhile poly(U) did not produce any effect. Unex-pectedly, the anti-myc antibody also detected a second band(50 kDa) corresponding to other form of the protein that alsobound poly(A) with the same affinity. Both two bands were alsoobtained using other human cell lines such as MCF7 or HepG2(data not shown). However, only the upper band was produced

Fig. 4. LiPABP maintains poly(A)-binding activity and is phosphorylated by p38 MAPK. Rexpressed in HEK293T cells (B) bound to poly(A)-Sepharose in the presence or not ocorresponding to 50 lg of L. infantum lysate or 10 lg of lysate from cells expressing mycthe values normalized relative to the LiPABP bound in the absence of competitor. Bars**P < 0.001. (C) Five micro liters of the reaction mixture containing myc-LiPABP transcribdiluted 1/1000. A reaction without plasmid was used as a control. 10 lg of lysate fromsame gel. (D) Recombinant p38 MAPK was used to phosphorylate LiPABP in an in vitroPhosphorylation of the myelin basic protein (MBP) by p38 MAPK was used as a positivereported by ELM database software, is shown at the bottom of the figure. An asterisk m

when the plasmid containing LiPABP was transcribed and trans-lated in an in vitro system suggesting that the lower bandshould be a product of posttranslational processing (Fig. 4C).Similar results have been previously reported for RBP63 fromCrithidia fasciculata [28] where two polypeptides of 56 and48 kDa, degradation products of the RBP63 protein processednear the C-terminal end, are observed. Breakdown products arealso observed during the purification of PABPs from other kine-toplastides [12,13,29].

Finally, for further characterization of LiPABP, we have checkedthe ability of p38 MAPK to phosphorylate LiPABP, as occurs for hu-man PABP [30]. As shown in Fig. 4D, LiPABP is substrate of p38MAPK. The analysis of the LiPABP sequence identifies a potentialp38 MAPK phosphorylation site in the C-terminal domain of theprotein that is also present in human PABP. It has been describedthat p38 MAPK phosphorylation of human PABP plays some rolein RNA deadenylation [30].

epresentative blots showing the amount of endogenous LiPABP (A) or myc-LiPABPf free poly(A) or poly(U) competitors are shown at the top of the figures. Input-LiPABP was loaded in parallel. Bands were quantified as described in Section 2 andrepresent the mean ± S.E.M. of three different experiments. Statistical significance:ed and translated in vitro were blotted with anti-myc (Santa Cruz Tech.) antibody

HEK293T cells previously transfected with pcDNA3-myc-LiPABP was loaded in thekinase assay. Autoradiography (top) and Coomassie staining (bottom) are shown.

control. A putative p38 MAPK phosphorylation site located in the LiPABP sequence,arks a putative degradation product in panels B and C.

198 N. Guerra et al. / FEBS Letters 585 (2011) 193–198

In conclusion, we described a PABP homologue from L. infantum,named LiPABP, that contains the main domains present in PABPsfrom other organisms, maintains poly(A) binding capability andis phosphorylated by p38 MAPK. Furthermore, the expression ofendogenous LiPABP is confirmed by sera from dogs with canine VL.

Acknowledgements

We thank M. Gómez-Calcerrada for her technical assistance.This work was funded by Grant PI05/0453 and PI08/0101 fromthe Fondo de Investigaciones Sanitarias (FIS) of the Ministerio deCiencia e Innovación (Spain). N.G. and E.R. are supported bycontracts from the FIS and V.M.G. and M.E.M. are researchers fromFIBio-HRC supported by Consejeria de Sanidad (CAM). An institu-tional grant from Fundación Ramón Areces for the CBMSO is alsoacknowledged.

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