Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus

Biochimica et Biophysica Acta 1631 (2003) 26 – 34 www.bba-direct.com

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus Adriana Esteves *, Virginia Portillo, Ricardo Ehrlich Seccio´n Bioquı´mica, Facultad de Ciencias, Igua´ 4225, 11400 Montevideo, Uruguay Received 7 November 2001; received in revised form 3 October 2002; accepted 17 October 2002

Abstract This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host – parasite adaptation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Fatty acid binding protein; Echinococcus granulosus; Genomic structure; EgFABP1; EgFABP2

1. Introduction Fatty acid binding proteins (FABPs) belong to a family of small (14 – 15 kDa) cytosolic proteins, which bind noncovalently hydrophobic ligands [1]. They are believed to be implicated in fatty acid intracellular uptake and transport, regulation of lipid metabolism and protection from the deleterious action exerted by free long-fatty acids. Specific functions have not yet been established for any FABP. They have been recently called fatty acid chaperones, emphasising their ability not only to protect and shuttle fatty acids within the cell but actively participate in the acquisition or removal of fatty acids from intracellular sites [2]. Several groups have been identified, each named after the tissue in which they predominantly occur. According to their cellspecific expression, abundance and high affinity for fatty acids, individual FABPs might perform distinct roles within various tissues. All the members of the family share a superimposable h-barrel tertiary structure, despite their

* Corresponding author. Tel.: +598-2-525-2095; fax: +598-2-5258617. E-mail address: [email protected] (A. Esteves).

variable primary structure identity (20 – 70%). They are widely distributed from lower invertebrates to mammals (reviewed in Refs. [2 –4]). The study of FABPs has a particular interest in parasitic platyhelminths. First, these parasites are unable to synthesise de novo most of their own lipids, in particular long chain fatty acids and cholesterol [5 and references therein], suggesting that these molecules should be obtained from the host, a process in which FABPs could play an important role. Secondly, parasitic platyhelminths are dependent on carbohydrates for their energy metabolism [6]. In addition, a functional h-oxidation pathway has not yet been demonstrated in cestodes [7] and there is no evidence for storage of triglycerides in platyhelminths. Finally, immunisation with Sm14, a Schistosoma mansoni FABP [8], and Fh15, a fatty acid from Fasciola hepatica [9], as well as with FgFABP, a Fasciola gigantica FABP [10], has conferred significant levels of protection against challenge infections with S. mansoni and F. hepatica, in animal models [11 – 13]. We have previously reported the identification, cloning, sequencing, immunohistochemical localisation, 3D structure analysis and evolutionary relationships of an Echinococcus granulosus FABP, named EgDf1 (presently referred as EgFABP1) [14 – 16]. According to phylogenetic algorithms,

1388-1981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1388-1981(02)00321-9

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sequence homology and 3D structure, this protein belongs to the group of heart-FABPs (H-FABPs) [15,17]. This group of proteins might be implicated in oxidation processes, a role that cannot be attributable to EgFABP1 according to the metabolic pathways described for platyhelminths parasites. Although in vitro putative ligands were determined [17], we have no information about its function. EgFABP1 appeared differentially expressed in the larval stage of the parasite, being present in the proliferative domain of the germinal layer of the hydatid cyst and in the protoscoleces (PS). In this work we report the genomic structure and expression studies of a second Echinococcus FABP gene, named EgFABP2. The description of the genomic structure of EgFABP1-coding domain and comparative analysis of both genes and proteins are also presented.

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lysis buffer (100 mM NaCl, 10 mM Tris –HCl pH 8.0, 25 mM EDTA pH 8.0, 0.05% w/v sodium dodecyl sulfate) and 0.2 mg/ml proteinase K, at 50 jC for 4 h. 2.3. cDNA synthesis Total RNA was extracted from E. granulosus PS using the acid guanidinium isothiocyanate – phenol – chloroform extraction method [21]. Samples were treated with DNAase I RNAse free and RNAase inhibitor (RNAsin, Promega) for 1 h at 37 jC following phenol and chloroform extraction and ethanol precipitation. Treated RNA, 10 Ag, was incubated for 1 h at 37 jC with 20 Al of 1  reverse transcriptase MmLV buffer (Promega), 10 pmol of poly-dT oligonucleotide, 250 AM each of deoxyribunucleotides and 200 U of reverse transcriptase MmLV (Promega). The reaction product was kept at 20 jC until use.

2. Material and methods 2.4. Sequence analysis 2.1. Cloning strategies 2.1.1. EgFABP2 A genomic library of E. granulosus constructed in EEMBL3 was screened with the cDNA clone EgFABP1 as a probe, at high stringency conditions. DNA from a positive phage containing an insert of 15 kb was purified [18] and mapped with BamHI, SalI, EcoRI and KpnI restriction enzymes. Restriction fragments were analysed by electrophoresis and Southern blot [19] using the same probe. EcoRI– KpnI fragments of 2.5 and 4.5 kb were subcloned and sequenced. An EcoRI –EcoRI fragment of 7 kb, containing the above-mentioned fragments, was also subcloned. Clones were named according to the plasmid name, the cloning sites and length of the insert (pUC18 – E.E7.0, pSK – E.K2.5 and pSK –E.K4.5). The 4.5-kb fragment was also analysed with restriction endonucleases BclI and PvuI. 2.1.2. EgFABP1 Polymerase chain reaction (PCR) using EgFABP1 coding region specific primers (5V-AATGGATCCATGGAGGCATTCCTTGGTACC-3V and 5V-TTAACTAGTCGCCACCTTTGAGTAGGTTCG-3V) containing 5V and 3V coding regions ends, respectively, and E. granulosus DNA as template was performed using an annealing temperature of 55 jC during 32 cycles of 1 min each. Cloning site sequences were added to both primer 5Vends (underlined). PCR amplification product was separated on a 1.5% agarose gel, excised from the gel, purified, cloned in pKS-T and sequenced. 2.2. DNA extraction DNA was purified from the larval stage of E. granulosus as described in Ref. [20] with the following modifications: 100 Al of protoscoleces (PS) was incubated with 900 Al of

DNA sequencing was performed according to Sanger and Coulson [22] and using automatic methods. In all the cases both strands were sequenced. When necessary, specific primers were synthesised. Sequence analysis was performed using SIGNAL SCAN program for promoter analysis [23]; sequence alignment was performed using Clustal-W program. Spatial (3D) structures of EgFABP1 and EgFABP2 obtained through Swiss Model facilities were compared using Swiss PdbViewer program. Isoelectric point (pI) was calculated by standard computer programs. 2.5. Primer extension EgFABP2 transcription initiation site was determined by the primer extension method [24] using 30-Ag PS mRNA. Total RNA was extracted from E. granulosus as described above. Synthetic oligonucleotide (5V-GCTGATTATCAAGTTGGGCTTC-3V) (10 pmol) complementary to the coding region, 107 base pairs (bp) downstream the ATG codon and 164 bp from TATA box, was end-labelled using [g 32P]ATP (0.05 Ci) and T4 polynucleotide kinase [25]. Transcription was carried out using 200U of reverse transcriptase (MTV, Gibco). Radiolabelled DNA was analysed by electrophoresis through a polyacrylamide 7 M urea gel. Adjacent lanes were loaded with sequence reactions corresponding to the clone pUC – E.E7.0 sequenced with the same oligonucleotide [22]. 2.6. Search for intraspecific variants Single cyst DNAs from nine individual cysts were analysed by PCR using specific primers: for EgFABP1 5V-ATGGAGGCATTCCTTGGTACC-3Vand 5V-CGCCACCTTTGAGTAGGTTCG-3V; for EgFABP2 5V-GAATCGAATAGTCGTCAGC-3V and 5V-GATGAGTAGCA-

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Fig. 1. Genomic organisation of EgFABP2 gene. (A) Restriction map of the clone EEg15 containing EgFABP2 gene. Promoter and coding regions are indicated by blank and filled boxes, respectively. E: EcoRI; K: KpnI; S: SalI. Southern-blot-positive fragment is indicated by a dark line. (B) Primer extension analysis of the promoter region. The sequence surrounding the start point of transcription is indicated; the arrow indicates the start point base. The lane presenting the primer extension reaction is indicated. (C) Putative transcription factor response element consensus sequences and TATA box obtained using the SIGNAL SCAN program are indicated: AP2 (TGGGA), GRE half-site (TGTTCT), GATA (GATAGA), CAT box (CCAAT), PPRE (TTTCTCTn42TGGAAATAGGTCA); SP1 (GGGCAG, TGCAC), TATA box (TATAAA). CTTCTC sequence and motifs shared with other E. granulosus promoters are underlined. Initial base of transcription is indicated with (*). Numbering is given from the transcription start site.

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GCCTCCC-3V. Primers were designed near start and stop codons, in regions presenting low identities between both genes. Primer positions are indicated in Fig. 2. Annealing temperature was 58 jC during 32 cycles of 1 min each. Amplification products were digested with restriction enzymes (BclI and PvuI) with single and specific sites for each gene (see Fig. 2 for restriction sites location). Restriction products were analysed by electrophoresis on 6% polyacrylamide gels stained with AgNO3.

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template and the same EgFABP1 and EgFABP2 specific primers used in the analysis of variants. PCR amplification conditions are the same as above. The specificity of the products was analysed by restriction digestions.

3. Results 3.1. Genomic organisation of EgFABP1 and EgFABP2 genes

2.7. Analysis of EgFABP1 and EgFABP2 expression Reverse transcription-polymerase chain reaction (RTPCR) was performed using cDNA from larval stage as

A genomic clone, containing a 15-kb insert and named EEg15, was isolated from an E. granulosus library constructed in EEMBL3. Restriction analysis of the isolated

Fig. 2. Alignment of EgFABP1 and EgFABP2 genomic coding regions. 3Vdomain of the EgFABP2 promoter, including putative TATA box (bold) and transcription starting point (*) are showed in lower case letters. EgFABP1 (first line) and EgFABP2 (second line) genomic coding sequences are showed in capital letters. Intron sequences are in bold; start and stop codons are in bold and underlined. Arrows indicate primers used in PCR reactions: filled line for EgFABP1 and doted line for EgFABP2; cloning sites added are also indicated. BclI, KpnI and PvuI sites are underlined. Sequence numbering is indicated with a dot each 10 nucleotides and a number at the end of the lines, beginning from ATG codon. (*) identities; ( – ) gaps.

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stream of the KpnI site of the pSK –E.K4.5 clone (Fig. 2). We discarded the presence of the EgFABP1 gene in the 4.5kb sequence by the absence of specific restriction sites (KpnI and BclI). By primer extension analysis we identified an adenine 13 bp upstream of the translation initiation ATG codon as the unique start site of transcription (Figs. 1B and 2). The EgFABP2 proximal regulatory domain contains a canonical TATA box 44 bp upstream of the identified start point of transcription, two Sp1 binding site sequences at positions 67 and 264 and a CAAT box at 93 (Fig. 1C). Imperfect (10/13) consensus DR1 response element to peroxisome proliferator-activated receptors (PPRE), including 5V-flank region (6/7) and half-site of glucocorticoids response element (GRE) at positions 111, 166 and 84, respectively, were also identified (Fig. 1C). Putative enhancer elements AP2, GRE and GATA were located between 1600 and 1000 bp upstream of the start site (Fig. 1C). Interestingly, a CTTTCT motif was found twice, at positions 205 and 650 in the direct strand and once in the complementary strand at position 2271. Similar motifs, included in a longer conserved sequence, were found in other E. granulosus promoters [26,27]. The ATG codon is included in the sequence GTCAGCAATGG and presents three mismatches with the consensus optimal context GCCA/GCCATGG [28].

Fig. 3. Analysis of intraspecific variants. (A) Electrophoresis of PCR products from protoscoleces DNA isolated from nine individual cysts (1 – 9) amplified with specific primers surrounding ATG and STOP codons for EgFABP1 (A) and EgFABP2 (B) (primer location are indicated in Fig. 2). (C) Restriction analysis of the amplified products using BclI and PvuI: 1 to 3 DNA amplified with EgFABP1 primers and 4 to 6 DNA amplified with EgFABP2 primers. N/d: nondigested. Electrophoresis analysis was done in 6% polyacrylamide gels stained with AgNO3.

clone was performed and submitted to Southern blot hybridisation using the coding region of EgFABP1 as a probe. A positive EcoRI – EcoRI clone of 7 kb (pUC18 – E.E7.0) containing a KpnI restriction site was selected and subcloned, as well as both EcoRI –KpnI restriction fragments obtained through the digestion of the 7-kb fragment (pSK – E.K2.5 and pSK – E.K4.5) (Fig. 1A). The pKS – E.K4.5 clone was also positive for the EgFABP1 probe. Sequence analysis of the complete pSK – E.K2.5 clone (2727 bp) indicates the presence of several consensus sites of eukaryotic promoters (Fig. 1C). The 3V domain of the clone contains an open reading frame for the N-terminal domain of a second FABP from E. granulosus (EgFABP2). The remaining coding domain of this gene is located down-

Fig. 4. RT-PCR analysis of EgFABP1 and EgFABP2 expression. Electrophoresis of amplified protoscolex cDNA using specific primers for EgFABP1 (A) and EgFABP2 (B). (A) 1: molecular weight marker, 2: amplified protoscolex cDNA. (B) 1: molecular weight marker, 2: amplified protoscolex cDNA. (C) Control experiments with specific restriction enzymes of RT-PCR products obtained with EgFABP1 and EgFAB2 specific primers. 1: protoscolex cDNA amplified with EgFABP1 primers and digested with BclI; 2: protoscolex cDNA amplified with EgFABP2 primers and digested with PvuI. Electrophoresis analysis was done in 6% polyacrylamide gels stained with AgNO3.

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Fig. 5. Comparison of EgFABP1 and EgFABP2 primary structure. (A) Amino acid sequence alignment of EgFABP1 (first line) and EgFABP2 (second line). Structural elements are indicated; h strands: hA – hJ and a helices: a1 and a2. Amino acid numbering is indicated with a dot for each 10 amino acids and a number at the end of the lines. (B) Hydropathicity profiles of EgFABP1 (top) and EgFABP2 (bottom) calculated using Protscale facilities. Arrows indicate divergent regions. Axes: Kyte and Doolite scores, and amino acid positions.

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The EgFABP1 genomic clone, which was obtained through E. granulosus DNA amplification using specific primers for the coding ends, was also sequenced. EgFABP2 shares 88% of identity with EgFABP1 at the nucleotide level. Both genes have a unique intron of 79 and 80 bp, respectively, 348 bp downstream the ATG codon (Fig. 2). These introns have an identity of 69%. Exon– intron boundaries are GT –AG, for both genes, as described [29]. These introns are located at the same position of the third intron described for other FABP genes [30 – 35]. 3.2. Search for intraspecific variants In order to determinate if both genes correspond to variants present in different parasite isolates, we analysed DNA extracted from PS of single cysts. It is assumed that PS from a single cyst constitutes a clonal population. We carried out PCR analysis amplifying DNA extracted from nine different cysts from a bovine host and using specific primers of each gene. The specificity of the products was analysed by using BclI and PvuI restriction enzymes. The analysis revealed the presence of both genes in all samples (Fig. 3). 3.3. Expression studies We analysed the expression of EgFABP1 and EgFABP2 in the PS by RT-PCR. As shown in Fig. 4, EgFABP1 and EgFABP2 are co-expressed in this larval stage. 3.4. Protein sequence analysis EgFABP2 is clearly included in the FABP family presenting the highest sequence identity (76%) and similarity (96%) scores with EgFABP1, using Clustal W algorithms (Fig. 5A). Both proteins differ in 32 amino acids, out of which 13 are nonconservative changes. Four of this amino acids (97D/V, 98D/G, 100T/D, 102V/T) are located in the protruding hG-hH loop. Conservative substitutions are equally distributed through all the protein structure. Analysing phylogenetic relationships, EgFABP2 appears, as EgFABP1, related to the H-FABPs group. The theoretical isoelectric point (pI) was 5.4. This pI differs from EgFABP1 theoretical and experimental value of 7.7 already reported [17]. Comparison of hydropathicity profiles of both E. granulosus FABPs is shown in Fig. 5B. Major differences are observed in the region 97D/V – 102V/T mentioned above. This sequence has no counterparts in any member of the family and could be highly immunogenic considering its hydrophilicity. Modelled structure of EgFABP1 and EgFABP2 obtained through Swiss Model facilities (not shown), using myelin P2 protein as template, were compared looking for structural elements that could be related to different physiological properties. Both a-C backbones are almost superimposable.

4. Discussion Hydatid disease is a major zoonosis spread all over the world and caused by the parasitic platyhelminth E. granulosus. The molecular and cellular bases of E. granulosus development are still largely unknown. Several molecular markers, which can identify cells or particular stages during the parasite life cycle, have been characterised by our group [14,36 – 41]. In particular, we have previously characterised a gene, named EgFABP1, coding for a FABP, which is developmentally regulated. Immunochemical approaches revealed that EgFABP1 expression is increased during germinal layer proliferation to PS [14]. In this work we report the genomic structure, primary biochemical characterisation and expression of a second FABP from E. granulosus, named EgFABP2. The complete genomic sequence of the gene, including the promoter region, was determined. We also include the description of the genomic structure of EgFABP1 coding domain and a comparative analysis of both genes and their corresponding proteins. Southern blot analysis in low stringency condition (not shown) does not suggest the presence of more genes of the same type in the E. granulosus genome. The promoter region of EgFABP2 presents several consensus putative cis-acting regions found in eukaryotic promoters including consensus proximal modules (TATA box, Sp1 binding site, CAAT box), as well as specific response elements in proximal (PPRE, GRE half-site) and remote positions (GATA, AP2, GRE half-site among others). At 44 bp upstream from the transcription start point a typical positioned TATA box is present. The unique start point was identified as the adenine 13 bp upstream the initiator ATG. The context surrounding ATG start codon, with a Pu3 and G4 +, indicates an efficient translation initiation [42]. The leader sequence length is of 12 bp consistent with the consensus of 10– 20 bp and the GC content (50%) consistent with translation initiation standard contexts. Higher GC contents were reported to difficult translation [43]. It is worth mentioning that the presence of putative glucocorticoid half-site (GRE) and peroxisome proliferator (PPRE) response elements in EgAFBP2 promoter region correlate with the presence of similar functional elements in reported FABPs promoters [44 –50], according to computational analysis [23]. The PPRE site identified in EgFABP2 presents 10/13 identities with respect to the 3V consensus direct repeat element AGGTCA A/T AGGTCA; furthermore, a 5V flank consensus sequence, as reviewed in Ref. [51], is also present. The EgFABP2 putative PPRE presents the highest identity score with the response element described in adipocyte FABP named ARE6 [52]. With respect to GRE, putative canonical 3Vhalf sites are present. The corresponding neighbouring sequences in EgFABP2 promoter presents only limited identity with the described 5Vvariable GRE half-site. It is worth mentioning that the lipid binding protein gene of adipocyte, which is

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activated by glucocorticoids, lacks 5Vconsensus GRE halfsite. Genomic structures were established for a number of FABP genes: rat liver (L-FABP) [30], human intestinal (IFABP) [31], mouse adipocyte (A-LBP) [32], swine adipocyte [49], rabbit myelin P2 [34], human CRBPI [53] and rat intestinal CRBPII [35]. The organisation of these genes is similar, containing four exons and three introns. The introns are present at similar positions, although varying markedly in size. Echinococcus genes present only one intron, at the position of the third intron of the other members of the family. These results suggest that the first and the second introns in FABP genes were acquired later in evolution. The extent of identities shared by both EgFABPs genes and their genomic organisation suggests that these genes proceed from a recent duplicative event. The function of FABPs has remained elusive. Specialised functions for each member have been proposed mainly according to their tissue-specific locations [1,3]. Nevertheless, it was reported that different members of the family are co-expressed in a given tissue, suggesting different functions and regulation processes [50,53]. It is unclear why two similar FABPs are expressed in the same stage of E. granulosus. EgFABP1 and EgFABP2 may play different functions in the parasite or be under differential expression control. Differences in biochemical properties, tertiary structure, gene expression and the precise spatial and temporal expression of both E. granulosus proteins are under investigation. Identification of active regulatory elements in E. granulosus FABPs may contribute to elucidate the role of these proteins in the metabolism and host –parasite relationships.

Acknowledgements This work was supported by PEDECIBA (Uruguay), CSIC (Uruguay), CONICYT (Uruguay, project no. 4043) and SAREC (Sweden). The authors wish to thank M. Portela for skilful technical assistance and Dr. M. Marı´n for helpful discussions.

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