TwonovelMesocestoidesvogaefattyacid binding
proteins –functionalandevolutionary 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 acidbindingproteins (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 acidbinding 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 fattyacidbindingproteins (FABPs) identified
in the parasite platyhelminth Mesocestoidesvogae (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, fattyacidbinding 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 fattyacidbindingproteins (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. vogaeproteins 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 fattyacid 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 fattyacid 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 fattyacidbinding 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).
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