Báo cáo khoa học: Two novel Mesocestoides vogae fatty acid binding proteins – functional and evolutionary implications doc

Two novel Mesocestoides vogae fatty acid binding proteins – functional and evolutionary implications Gabriela Alvite, Lucı ´a Canclini, Ileana Corvo and Adriana Esteves Biochemistry Section, Cellular and Molecular Biology Department, Faculty of Sciences, University of the Republic, Montevideo, Uruguay Fatty acid binding proteins (FABPs) are small (14– 15 kDa) cytosolic proteins that bind non-covalently to hydrophobic ligands, mainly fatty acids. These proteins are members of the calycin superfamily, which includes lipocalins and avidins [1]. Several tissue-specific FABP types have been identified in vertebrates, each named after the tissue in which they are predominantly expressed [2,3]. FABPs are involved in lipid metabolism, specifically in the transport of fatty acids from the plasmalemma to intracellular sites of conversion. In addition, several members have been implicated in cell-growth modula- tion and proliferation. The precise function of each FABP type remains poorly understood, as sub-special- ization of functions is suggested by the specific tissue and temporal expression, in addition to ligand prefer- ences [4–7]. Parasitic platyhelminths FABPs are interesting mole- cules to study for a better understanding of the biology of these organisms. First, these parasites are unable to synthesize de novo most of their own lipids, in particu- lar long-chain fatty acids and cholesterol [8]; conse- quently these molecules are obtained from the host, and delivered by FABPs to specific destinations within the cells. Second, they are promissory vaccine candidates. The first platyhelminth FABP described was Sm14, identified in the trematode Schistosoma mansoni [9]. Sm14 was isolated as a highly immunogenic peptide Keywords fatty acid binding proteins; introns; Mesocestoides vogae; parasites; platyhelminths Correspondence A. Esteves, Facultad de Ciencias, Seccio ´ n Bioquimica, Igua ´ 4225, P3 Anexo Norte, CP 11400, Montevideo, Uruguay Fax: +598 2 525 8617 Tel: +598 2 525 2095 E-mail: aesteves@fcien.edu.uy Database Nucleotide sequences have been submitted to the GenBank database under the accession numbers EF488508 (MvFABPa mRNA), EF488509 (MvFABPb mRNA), EF488510 (MvFABPb gene) and EF488511 (MvFABPa gene) (Received 4 August 2007, revised 29 October 2007, accepted 5 November 2007) doi:10.1111/j.1742-4658.2007.06179.x This work describes two new fatty acid binding proteins (FABPs) identified in the parasite platyhelminth Mesocestoides vogae (syn. corti). The corre- sponding polypeptide chains share 62% identical residues and overall 90% similarity according to clustalx default conditions. Compared with Cestoda FABPs, these proteins share the highest similarity score with the Taenia solium protein. M. vogae FABPs are also phylogenetically related to the FABP3 ⁄ FABP4 mammalian FABP subfamilies. The native proteins were purified by chromatographical procedures, and apparent molecular mass and isoelectric point were determined. Immunolocalization studies determined the localization of the expression of these proteins in the larval form of the parasite. The genomic exon–intron organization of both genes is also reported, and supports new insights on intron evolution. Consensus motifs involved in splicing were identified. Abbreviation FABP, fatty acid binding protein. FEBS Journal 275 (2008) 107–116 ª 2007 The Authors Journal compilation ª 2007 FEBS 107 that presented significant protective activity against experimental infections in an animal model [10]. Homologous proteins from Schistosoma japonicum (SjFABPc) [11], Fasciola hepatica (Fh15) [12] and Fas- ciola gigantica (FgFABP) [13] also induce protection from challenge infection [13–15]. In addition to those from Trematoda, proteins of this family have been isolated from Cestoda members [16–18]. One of them, expressed in salmonella as a TetC–rEgDf1 fusion, is under evaluation as a potential vaccine against echinococcosis [19,20]. Mesocestoides vogae (syn. corti; Cestoda: Cyclo- phyllidea), despite not being a public health threat, is an important model organism as it shares similarities with taenia that are of public health interest. This par- asite is easy to maintain in the laboratory by intraperi- toneal passages through male mice, producing a very large number of larvae (tetrathyridia). The parasitic material obtained with this procedure is more homoge- nous, from a genetic point of view, than that derived from natural infections [21]. Likewise, the method of propagation in experimental animals allows the possi- bility of proteomic studies of particular genes, thus contributing to the elucidation of FABP functions in cestode parasites. In the present work, we report the isolation of the first two FABPs from M. vogae, their amino acid sequences, evolutionary relationship, tissue expression and genomic organization, showing that they are actu- ally encoded by two genes. Results Cloning and sequence analysis The coding sequences of two M. vogae genes, referred as Mvfabpa and Mvfabpb, were identified as being simi- lar to those for fatty acid binding proteins (Fig. 1A,B). They share 65% identity at the nucleotide level and 62% identity at the amino acid level (Fig. 1C). Two additional sequences showed differences compared with the clone Mvfabpa (Fig. 1A). The nucleotide at posi- tion 170 was C rather than T in one clone, and the nucleotide at position 299 was A rather than G, result- ing in a change in the encoded amino acids from L to S and G to D, respectively. These nucleotide differ- ences may represent a polymorphism or a PCR arte- fact. As a consensus FABP 5¢-end primer was used, this coding region sequence is uncertain, and it was not considered in any of the analyses performed. A conserved polyadenylation motif (AATAAA) is absent in both clones. However, putative signals are present in Mvfabpa (TATAAA) and Mvfabpb (ATTA C B A Fig. 1. Mvfabp sequences. (A) Mvfabpa nucleotide and correspond- ing amino acid sequences. The putative polyadenylation motif is underlined; polymorphisms are shaded dark grey; the sequences of primers used for genomic analysis are shaded in grey. (B) Mvfabpb nucleotide and corresponding amino acid sequences. The putative polyadenylation motif is underlined; sequences of primers used for genomic analysis are shaded light grey. The numbering of the nucleotide and amino acid sequences (based on known FABPs) is shown on the right. (C) Alignment of M. vogae FABP sequences. Two levels of shading show residues that are 100% conserved (dark grey) and 80% conserved (light grey). The numbering at the top indicates amino acid positions based on known FABPs. M. vogae FABPs G. Alvite et al. 108 FEBS Journal 275 (2008) 107–116 ª 2007 The Authors Journal compilation ª 2007 FEBS AA) (Fig. 1A,B). A similar signal was found in the Echinococcus granulosus fapb1 gene [16]. The initial blastx search (default conditions) at National Center for Biotechnology Information (NCBI) showed that the proteins encoded by the MvFABPa and MvFABPb clones have the greatest number of hits with Taenia solium FABP (score 137, E value 3e )31 and score 136, E value 5e )31 , respec- tively), indicating that the cDNA clones encode FABP proteins. Alignment to representative plathyhelminth FABP sequences using clustalw revealed a higher identity with Cestoda proteins (39%) than with Trema- toda proteins (13%), indicating several amino acids shared by cestodes FABPs that could represent markers of this class. A multiple alignment is shown in Fig. 2. Exon–intron structure In order to analyse the exon–intron structure in Mvfabpa and Mvfabpb genes, PCR products obtained using genomic DNA as template were sequenced. Only one intron was identified in each gene (Fig. 3A,B). The identified introns have the same position as the second intron of vertebrate FABPs. The Mvfabpa and Mvfabpb introns are 79 and 90 bp long, respectively (Fig. 3C). Bioinformatic analysis revealed several cis signals involved in RNA processing. Using nsplice predictor software, consensus sequences, including GT–AG splice junctions, were found. We also found the typical polypyrimidine tract in the 3¢ intron region. esefinder analysis revealed two putative binding sites for the splicing regulator ASF (a member of the SR family of splicing factors) in each gene [22]. Both cis-acting ele- ments are located at the same position in each clone, upstream and downstream of the corresponding intron (Fig. 3A,B). A similar sequence to the consensus branch site for animal genes [CT(A ⁄ G)A(C⁄ T)], with the essential adenine in the correct position, was found in the Mvfabpb gene (Fig. 3B). Phylogenetic analysis The rooted phylogenetic tree shown in Fig. 4 was con- structed to assess the relationship between M. vogae Fig. 2. Alignment of platyhelminth FABP sequences. Sequences from the following species were aligned: MvFABPa and MvFABPb from M. vogae; TsFABP from T. solium (ABB76135); EgFABP1 (AAK12096) and EgFABP2 (AAK12094) from E. granulosus; SbFABP (AAT39384) from S. bovis; Sm14 (AAL15461) from S. mansoni; Fh15 (Q7M4G0), FASHE2 (Q7MAG2) and FASHE3 (Q9UIG6) from F. hepatica. Two levels of shading show residues that are 100% conserved when comparing M. vogae FABPs with Cestoda FABPs (light grey) and M. vogae FABPs with Trematoda FABPs (dark grey). Numbers on the right indicate the protein sequence length; the numbering at the top indicates the position of each amino acid relative to the amino terminal end. G. Alvite et al. M. vogae FABPs FEBS Journal 275 (2008) 107–116 ª 2007 The Authors Journal compilation ª 2007 FEBS 109 FABPs and other members of the family, including vertebrate FABPs. It strongly supports the inclusion of M. vogae proteins in the same clade as FABP3 and FABP4 from other species (bootstrap value = 1000), suggesting that M. vogae fabp genes are orthologous to vertebrate fabp3 ⁄ 4 genes. M. vogae FABPs sequences were consistently a sister group to the cluster of cestodes FABPs. Protein purification After chromatographic procedures, two major protein bands with apparent molecular masses of 15.4 kDa (I) and 14.7 kDa (II) were identified by SDS–PAGE (Fig. 5A). An antibody raised against the E. granulosus recombinant protein GST–EgFABP1 also recognized these proteins (Fig. 5B). MALDI-TOF analysis of each electro-eluted band (I and II) revealed peptide mass fingerprints in accordance with predicted tryptic diges- tions of MvFABPb and MvFABPa, respectively, with more than 40% coverage (data not shown) [23]. In addition, sequencing of the 15.4 kDa (I) peptide indi- cated that it is MvFABPb. The reported M. vogae protein sequences do not con- tain N-terminal residues because we used a degenerate A B C Fig. 3. Exon–intron structure of Mvfabp genes. (A) Mvfabpa nucleo- tide sequence. (B) Mvfabpb nucleotide sequence. Numbers on the right indicate the sequence length. Lower case letters indicate the intronic sequence; boxes indicate gt ⁄ at splice sites and the stop codon; (—), consensus splice sequence; ( ), putative branch site; (- - - -), putative ASF binding site; bold letters indicate the polypyrimi- dine tract. (C) Intron position comparison of FABPs genes. Horizon- tal lanes represent translated genes sequences. Inverted triangles indicate intron positions. Numbers below the line indicate the codon position based on vertebrate FABPs; numbers above the line indi- cate intron length. FABP3, heart type; FABP1, liver type; FABP2, intestinal type; Hs, Homo sapiens; Mv, Mesocestoides vogae; Eg, Echinococcus granulosus; Sm, Schistosoma mansoni. Fig. 4. Phylogenetic relationships between vertebrate and platyhel- minth FABPs. Rooted tree derived from neighbour-joining analysis using platyhelminth sequences and selected representative verte- brate FABPs. Hs, Homo sapiens; Dr, Danio rerio; Xl, Xenopus laevis; Gg, Gallus gallus; Rn, Rattus norvegicus; Eg, Echinococcus granulosus; Mv, Mesocestoides vogae; Sm, Schistosoma mansoni; Sj, Schistosoma japonicum; Sb, Schistosoma bovis; Ts, Taenia soli- um; Fh, Fasciola hepatica; Fg, Fasciola gigantica; Mt, Mycobacte- rium turberculosis. Bootstrap values (1000 replicates) are shown alongside the branches. Branch lengths are proportional to the genetic distances, as indicated by the scale bar representing 0.2 substitutions per site. M. vogae FABPs G. Alvite et al. 110 FEBS Journal 275 (2008) 107–116 ª 2007 The Authors Journal compilation ª 2007 FEBS primer to amplify them from cDNA, so the molecular masses of native forms are greater than those calculated from the sequences. The molecular masses and lengths of known FABPs are 14.1–15.5 kDa and 128–133 residues, respectively. It is worth men- tioning that calculated molecular mass generally differs from experimental determinations using SDS–PAGE. Post-translation modifications cannot be excluded either. When a chromatographically purified fraction con- taining putative FABPs was subjected to 2D electro- phoresis, three spots were evident, two acidic forms (pI 5.5 and 5.9) with the same mass, and a heavier basic one (pI 7.7) (Fig. 5C). As the reported sequences do not contain N-terminal residues, comparisons between calculated and experimental pI cannot be made. These results indicate that the apparent molecu- lar mass of MvFABPa is 14.7 kDa, with a pI of 5.5 or 5.9, while the apparent molecular mass of MvFABPb is 15.4 kDa, with a pI of 7.7. The third spot may be attributable to a contaminant protein. Alternatively, it may be one of the polymorphic forms reported or a new isoform. Expression studies A polyclonal antibody raised against the homologous FABP from E. granulosus (EgFABP1) that recognizes both MvFABPs was employed to analyse tetrathyridia M. vogae FABPs expression using laser confocal microscopy. The most intense staining was observed in the tegument and the region surrounding the calcare- ous corpuscles, and control sections were unstained. Homogenous low-level fluorescence labelling was also observed in the parenchymal region. No signal was observed inside the corpuscles (Fig. 6). A B C Fig. 5. FABP purification. (A) 15% SDS–PAGE. Lane 1, M. vogae whole extract; lane 2, purified fraction from Sephacryl chromato- graphy; the molecular mass in kDa is indicated (MM). (B) Western blot of chromatographically purified fraction containing putative MvFABPs. The primary antibody was anti-GST–EgFABP1, and the western blot was developed using the alkaline phosphatase reac- tion. (C) Partial image of 2D electrophoresis of a gel filtration-eluted fraction (10 lg of total protein) containing putative FABPs. The molecular mass (kDa) and pH gradient are indicated. Cc Tg 50.0µm Fig. 6. Immunolocalization of expression. Laser confocal microgra- phy of immunolabelled M. vogae tetrathyridia sections using a poly- clonal antibody against EgFABP1. Tg, tegument; Cc, calcareous corpuscles. The inset at the top left corner shows a differential interference contrast image; the inset at the bottom right corner shows a control section treated without primary antibody. G. Alvite et al. M. vogae FABPs FEBS Journal 275 (2008) 107–116 ª 2007 The Authors Journal compilation ª 2007 FEBS 111 Discussion Two M. vogae FABPs with high protein sequence identity scores are reported. blast searching, multiple sequence alignments and their apparent molecular mass confirm that these proteins belong to the FABP family. The presence of two highly similar FABPs in a platyhelminth parasite as well as in other invertebrates is not surprising; E. granulosus, Caenorhabditis elegans and Manduca sexta FABPs are good examples [24]. The observed high expression of MvFABPs at the tegumental level has interesting implications in platy- helminth biology and parasite control. It suggests that FABPs could be involved in fatty acid uptake through the tegument surface from the host, as cestodes are unable to synthesize their own long-chain fatty acids and cholesterol [9]. In addition, the tegument is a major source of antigens, which are released into the host circulation and elicit the host’s immune response. Because of their high expression, FABPs are a promis- ing candidate antigen for vaccines against diseases caused by platyhelminth parasites [13–15,19,20]. Early evolutionary studies analysing vertebrate FABPs distinguished major subfamilies (FABP3 ⁄ FABP4 ⁄ FABP8, FABP2, FABP1 ⁄ FABP6 and CRABPI/CRABPII/cRBPI) derived by gene duplica- tion from a common ancestor close to the verte- brate ⁄ invertebrate split [25,26]. As the number of known FABP sequences increased, the basic subfamily organization in vertebrates was maintained, despite the fact that more complex relationships appeared. How- ever, progressive inclusion of invertebrate members, which do not share extensive sequence motifs with ver- tebrate FABPs, blurred the dendrogram topologies [27]. Relationships between the invertebrate and vertebrate members of the FABPs family have shown that platy- helminth FABPs cluster with the FABP3 and CRABP subfamilies, whereas nematodes and arthropods are dis- tributed among the FABP1, FABP2 and FABP3 sub- families [24]. The fact that M. vogae FABPs share high homology with FABP3 and FABP4 supports this obser- vation. Almost all invertebrate FABPs present the high- est pairwise sequence identity with this subfamily. Its members have great structural diversity, favouring a variety of binding arrangements and suggesting func- tional diversity [24,28]. Assuming that the ancestral Cestoda FABP gene has suffered a gene duplication to produce Mvfabpb and other cestoda FABPs, our data support the hypothesis that M. vogae is a basal group and perhaps external to Cyclophyllidea [29,30]. A significant issue is the relative importance of intron loss and gain through eukaryotic history. Introns are often found at exactly the same positions in orthologous genes of widely divergent eukaryotic species [31–33], suggesting intron-rich eukaryotic ancestors and massive recurrent intron loss along diverse lineages [34,35]. The genomic organization of all vertebrate fabp genes is remarkably conserved, with three interrupting introns of varying sizes inserted in analogous positions along the coding sequence [24]. The position of each intron in M. vogae fabp genes is correlated with that of intron II of vertebrate fabp genes. The fact that vertebrate genes have not gained introns in the last 600 million years [35], and that the same structure has been reported for the tobacco hornworm [36] and S. mansoni [37] FABP genes, makes it conceivable that this exon–intron orga- nization represents the FABP gene ancestral structure [26,38]. Analysis of insect, nematode and platyhelminth genes show that this organization is generally not con- served in invertebrate FABP genes [24,26], indicating lineage-specific trends for intron loss and gain. The question of why two similar FABPs are expressed in the same stage of Cestoda parasites, as well as in many invertebrates, remains open [24]. Co- expression of several members of this protein family in a given tissue was also reported in vertebrates, suggest- ing specific functions and regulation processes [39,40]. Functional specialization must be the result of subtle changes in the internal cavity or on the surface, favour- ing interaction with specific targets. Subtle but consis- tent conformational and surface changes as putative markers for differential targeting of protein–lipid com- plexes within the cell have been reported previously in a study of two FABPs expressed in the adipocyte [40]. A recent proposal by Gutman and co-workers suggests that separate regions on the FABP surface could be free to interact with cellular components [41]. Recently, the Golgi apparatus and mitochondria were suggested as putative liver FABP (FABP1) targets, but there are no reports concerning these interactions or residues involved in these interactions [42]. As the lipid compo- sition of intracellular organelles varies, the composition of M. vogae organelles should be investigated. Pronounced variations in the electrostatic surface around specific FABPs have been reported previously, suggesting that they interact with different moieties [43]. A relationship between the surface electrostatic potential and the fatty acid transfer mechanism has also been suggested [44]. The possible effects of changes in the positive electrostatic ridge across the region around the helix-turn-helix motif of adipocyte lipid binding protein have been addressed in studies by Storch and co-workers, which show that adipocyte lipid binding protein, heart FABP and intestinal FABP, but not liver FABP, transfer fluorescent fatty acids to the phospholipid bilayer predominantly via M. vogae FABPs G. Alvite et al. 112 FEBS Journal 275 (2008) 107–116 ª 2007 The Authors Journal compilation ª 2007 FEBS collisional interaction with the membranes [45–47]. The lysine at position 31 (21 in the reported sequence) may play an important role in governing ionic interac- tions between FABPs and membranes [48]. This may be of relevance in M. vogae, as glutamine is present at position 31 in MvFABPa, while lysine is present at position 31 in MvFABPb. Further investigations are required to elucidate whether these residues lead to functional differences between the M. vogae proteins. Future work is also required to elucidate the puta- tive ligands of MvFABPs, their equilibrium dissocia- tion constants and pH dependence, 3D structure, subcellular localization, and the mechanism of fatty acid transfer between FABPs and phospholipid bilay- ers (collisional or diffusional). A more comprehensive understanding of biochemical differences between these proteins may provide clues as to the role of fatty acid binding proteins in platyhelminth parasites. Experimental procedures Parasite material M. vogae tetrathyridia were maintained by intraperitoneal passage through male CD1 mice (3 months old) and used to set up in vitro cultures in modified RPMI-1640 medium as previously described [49]. Parasites were harvested by peritoneal aspiration, and extensively washed using Hank’s balanced salt solution (Sigma, St Louis, MO, USA). Cloning strategies Total RNA was extracted from M. vogae tetrathyridia, using Tri-reagent (Sigma) according to the manufacturer’s instructions, with a tetrathyridia ⁄ Tri-reagent ratio of 1 : 10. Retrotranscription was performed using Super- script II retrotranscriptase (Sigma), with CDS primer (5¢-AAGCAGTGGT AACAACGC AGAGTACT 30 NN-3¢;BD Biosciences ⁄ Clontech, Basingstoke, UK) and 1 lg of total RNA. The reaction product was kept at ) 20 °C until use. PCR was performed using a forward primer containing the 5¢ consensus coding region of FABPs (5¢-TTIKTIGG NMMNTGGAARTT-3¢), the SMART III reverse primer (5¢-AAGCAGTGGTAACAACGCAGAGT-3¢; Clontech) and M. vogae cDNA as template. The following conditions were used: initial denaturation at 94 °C for 5 min, followed by 40 DNA denaturating cycles at 94 °C for 30 s and 40 °C for 30 s for primer annealing, and DNA synthesis elongation at 72 °C for 30 s. A final elongation step was performed at 72 °C for 5 min. The reaction was performed in 25 ll total volume, with 1.25 units of recombinant Taq polymerase (Fermentas, Hanover, MD, USA). PCR prod- ucts were fractionated by 2% agarose gel electrophoresis, excised from the gel, purified using a GFX gel band purification kit (GE Healthcare, formerly Amersham Bio- sciences, Piscataway, NJ, USA), and ligated into pGEM-T Easy vector (Promega Life Sciences, Madison, WI, USA) to transform the XL1 Escherichia coli strain. Twenty recombinant colonies were selected for sequencing. Exon–intron structure The conserved 5¢-end of the sequenced M. vogae fabps (5¢-TTTCGACGAGGTGATGC-3¢) was used to design the forward primer, and specific sequences of the 3¢-ends of Mvfabpa and Mvfabpb (5¢-TGTGTGTCCACGCTAAACG CC-3¢ for Mvfabpa;5¢-GATATTCGCGTTGCAACCTCT- 3¢ for Mvfabpb) were used to design the reverse primers to amplify corresponding genomic DNA sequences. The designed primers are 5¢ and 3¢ to conserved introns I and III, respectively, of reported fabp genes (see Fig. 1A for pri- mer locations). The following conditions were used: initial denaturation at 94 ° C for 5 min, followed by 35 cycles of 94 °C for 45 s for DNA denaturation, 58 °C for 45 s for primer annealing, and DNA synthesis elongation at 72 °C for 45 s. A final elongation step was performed at 72 °C for 5 min. PCR products were fractionated by 2% agarose gel electrophoresis. The bands were excised from the gel, purified using a GFX gel band purification kit (Amersham Biosciences), and sequenced. Sequence analysis DNA sequencing was performed using automatic methods (ABI PRISM) at the ‘CTAG’ Service (Faculty of Sciences, Montevideo, Uruguay). Both strands were sequenced in all cases. nsplice version 0.9 (http://www.fruitfly.org) [50] and esefinder release 2.0 [51] algorithms were also employed. Sequences were submitted to blastx 2.2.15 analysis [52] against the complete GenBank, European Molecular Biol- ogy Laboratory (EMBL), DNA Bank of Japan (DDBJ) and Protein Data Bank (PDB) databases (nonredundant protein sequences), including all organisms. DNA and pro- tein sequence alignment was performed using the clustalw algorithm under default conditions: DNA weight matrix IUB; protein weight matrix Gonnet PAM 250, score plot scale = 5; residue exception cut-off = 5; minimum length of segments = 1 [53]. Phylogenetic analysis A rooted phylogenetic tree using programs from the mega (version 3.1) package and amino acid data sets for platy- helminths and representative vertebrate FABPs was con- structed. Sequence alignment was performed using the clustalw algorithm under the same conditions as described above. The topology and branch lengths of the phylogenetic tree were estimated using the neighbour- G. Alvite et al. M. vogae FABPs FEBS Journal 275 (2008) 107–116 ª 2007 The Authors Journal compilation ª 2007 FEBS 113 joining method based on the number of amino acid substi- tutions per site (Poisson-correction distance method, com- plete-deletion option for gap sites). The significance of branching points was assessed by bootstrapping with 1000 pseudoreplicates. We included the following proteins from the GenBank and SwissProt databases: T. solium FABP (ABB76135); EgFABP1 (formerly EgDf1) (AAK12096) and EgFABP2 (AAK12094) from E. granulosus; Sm14 (AAL15461) from S. mansoni; FABPc (AAG50052) from S. japonicum; SbFABP (AAT39384) from Schistosoma bovis; FABP3 (Q9U1G6) from F. hepatica; FgFABP (AAB06722) from F. gigantica; FABP1 (AAK58094), FABP2 (AAP13101), and FABP4 (AAL30743) from Gallus gallus; FABP1 (AAH32801), FABP2 (AAH69637), FABP3 (AAP36511) and FABP4 (AAP36447) from Homo sapiens; FABP1b (AAI07840), FABP2 (AAP93851) and FABP3 (AAH49060) from Danio rerio; FABP2 (AAC38012) and FABP3 (AAH56855) from Xenopus laevis; FABP4 (AAH84721) from Rattus norvegicus; CRABP1 (AAH22069) from H. sapiens ; CRABP1 (CAA72930) from G. gallus; CRABP1 (AAO85530) from D. rerio; CRABP1 (AAB32580) from X. laevis. As an external group, Rv0813c (CAA17619), a fatty acid binding protein-like protein from Mycobacterium tuberculosis, was included [54]. Identification of native proteins Native M. vogae FABPs were purified using chromato- graphic procedures. Tetrathyridia (10 mL) were extracted from infected mice, washed with NaCl ⁄ P i , and homoge- nized in 10 mL 50 mm Tris ⁄ HCl, pH 8, 0.15 m NaCl, 180 lgÆmL )1 phenylmethylsulfonyl fluoride, 10 lLÆmL )1 Triton X-100, and protease inhibitors leupeptin (3 lgÆmL )1 ) pepsatin (3 lgÆmL )1 ), Pefabloc (120 lgÆmL )1 ), EDTA-Na 2 (2 lgÆmL )1 ) and aprotinin (0.3 lgÆmL )1 ) (Roche Molecular Biochemicals, Mannheim, Germany). After clarification (11 000 g, 30 min, 4 °C), NH 4 (SO 4 ) 2 fractionation was per- formed (70% saturation). After dialysis against starting buffer (30 mm Tris ⁄ HCl, pH 8.3), the supernatant was con- centrated by ultra-filtration and applied to a Sephacryl HR-100 column (2.5 · 44 cm) (Sigma) with a flow rate of 0.6 mLÆmin )1 . Collected fractions were concentrated and analysed by 15% SDS–PAGE [55]. The fraction containing putative FABPs was submitted to Western blot using anti- serum raised against a recombinant E. granulosus FABP (GST–EgFABP1), and 2D electrophoresis [56]. For further identification, putative FABPs were cut from SDS–PAGE gels and eluted using a Hoefer GE-200 gel apparatus (Har- vard Apparatus, Holliston, MA, USA) according to the manufacturer’s instructions. Each eluted band was submit- ted to tryptic digestion and MALDI-TOF peptide mass fingerprinting (Faculty of Sciences Service, UdelaR, Uru- guay). The heavier electro-eluted band was submitted to tryptic digestion in order to perform peptide sequencing (LANAIS-PRO, Conicet-UBA, Bueunos Aires, Argentina). Expression studies To localize FABP expression, immunohistochemical studies were performed using a polyclonal antibody raised against EgFABP1 protein. Tetrathyridia were fixed in 4% parafor- maldehyde in 0.1 m NaCl ⁄ P i overnight at 4 °C, and then extensively rinsed in the same buffer. After gradual dehydration, the material was embedded in LR-White resin. Sections 0.5 lm thick were used for post-embedding immu- nostaining and laser confocal analysis. The sections were incubated for 30 min at room temperature in 0.1% Tween- 20 in PHEM buffer, pH 7.5 (25 mm Hepes, 60 mm Pipes, 10 mm EGTA, 2 mm MgCl 2 ), and then incubated overnight at 4 °C with the primary antibody (anti-EgFABP1) in blocking solution (50 mm glycine, 0.1% Tween-20, 10% normal goat serum in PHEM buffer, pH 7.5). After wash- ing with PHEM buffer, the sections were incubated with goat anti-rabbit Alexafluor 647 (Molecular Probes, Invitro- gen Labeling and Detection, Eugene, OR, USA) in block- ing solution overnight at 4 °C. The control sections were treated without primary antibody. Sections were viewed using an Olympus BX61 scanning laser confocal micro- scope, and the images were processed with fluoview 300 software, version 4.3. Acknowledgements The authors thank Dr C. Martı ´ nez (Seccio ´ n Bio- quı ´ mica, Facultad de Ciencias, UdelaR, Montevideo, Uruguay) and Dr I. Noguera (Department of Cell Biology and Kaplan Cancer Center, and the Raymond and Beverly Sackler Foundation Laboratory, New York University Medical Center, NY, USA) for criti- cal reading of this manuscript, Dr A. Kun (Instituto de Investigaciones Biolo ´ gicas Clemente Estable, Mon- tevideo, Uruguay) for her assistance with laser scan- ning confocal microscopy, and Q. F. J. Saldan ˜ a and Q. F. L. Domı ´ nguez (Departamento de Quı ´ mica y Farmacia, Facultad de Quı ´ mica, Montevideo, Uru- guay) for parasite provision. This work was supported by a grant from Comisio ´ n Sectorial de Investigacio ´ n Cientı ´ fica (Uruguay). References 1 Flower DR (1993) Structural relationship of streptavi- din to the calycin protein superfamily. 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