From the Instituto Butantan, Avenida Vital Brasil
1500, 05503-900, São Paulo, Brazil, § Instituto de
Química, §§ Instituto Biociências,
Universidade de São Paulo, Avenida Prof. Lineu Prestes 748, 05508-900, São Paulo, Brazil,
Centro de Biologia Molecular
Estrutural, Labotarório Nacional de Luz Síncrotron,
Caixa Postal 6192, 13084-971 Campinas, Brazil,
** Departamento de Helmintologia, Instituto Oswaldo
Cruz-Fiocruz, Avenida Brasil 4365, 21045-900, Rio de Janeiro,
Brazil, and
Department of Experimental
Medicine, University of Parma, 43100 Parma, Italy
Received for publication, November 4, 2002, and in revised form, January 3, 2003
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ABSTRACT |
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The Schistosoma mansoni Sm14
antigen belongs to the fatty acid-binding protein family and is
considered a vaccine candidate against at least two parasite
worms, Fasciola hepatica and S. mansoni. Here
the genomic sequence and the polymorphism of Sm14 have been
characterized for the first time. We found that the conserved
methionine at position 20 is polymorphic, being exchangeable with
threonine (M20T). To evaluate the function of the amino acid residue at
this position, we have also constructed the mutant Sm14-A20 besides the
two native isoforms (Sm14-M20 and Sm14-T20). The three purified
recombinant His6-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant Schistosomiasis is the second major parasitic disease in the world
after malaria and afflicts over 200 million people. This disease is
caused by blood flukes belonging to the genus Schistosoma (Schistosoma mansoni, Schistosoma japonicum, or
Schistosoma hematobium). In America, only
S. mansoni is found, which had been introduced from Africa
in the colonial time (1). These parasites lack the
oxygen-dependent pathways required for the synthesis of
sterols and fatty acids, thus being entirely dependent on their hosts for these and other complex lipid supplies. In fact, the intracellular fatty acid-binding protein Sm14 is particularly important for schistosomes in the uptake, transport, and compartmentalization of
host-derived fatty acids (2). Because these proteins play a vital role
in their physiology and survival, they can represent an ideal target
for vaccine development. Indeed, this parasite antigen is considered a
promising vaccine candidate for human schistosomiasis by the World
Health Organization (3). The S. mansoni Sm14 showed already
a protective activity against two parasite worms, Fasciola
hepatica and S. mansoni (4). Recently, the T helper
cell 1-mediated immune response elicited by Sm14 has been associated
with the resistance to schistosomiasis in individuals from endemic
regions of Brazil (5). For this reason, the study of the S. mansoni gene structure and polymorphism of Sm14 is of pivotal
importance to identify the isoform better suited to devise an efficient
vaccine. The data presented in this work indicate that the sequence of
the various Sm14 proteins is relatively conserved among the
American strains of S. mansoni and provide the first
experimental evidence for the existence of a reduced polymorphism,
especially with respect to S. japonicum
FABPs.1 In particular, the
single mutation M20T is the principal example in terms of recurrence.
Interestingly, this change induces important structural and functional
modifications in the protein that can have direct consequence in the
development of a schistosomiasis vaccine based on this FABP. In fact,
the more structurally stable Sm14-M20 isoform appears to be the better
vaccine candidate.
Parasites--
In this work, we used both the male and female
adult specimens of Brazilian endemic S. mansoni strains:
LE has been provided by the Laboratory of Helminthology
(FIOCRUZ, Rio de Janeiro, Brazil) and BH by the Laboratory of
Parasitology (Instituto Butantan, São Paulo, Brazil).
Sm14-encoding Sequences--
The genomic DNA was isolated from
the S. mansoni worms following the standard protocols (6).
The TriZOL reagent (Invitrogen) was used for total RNA isolation from
LE and BH schistosome strains. Two micrograms of total RNA were
reverse-transcribed by SuperScript II reverse transcriptase
enzyme (Invitrogen) using an Oligo(dT)18 primer. The first
cDNA strands were used as template in the PCR reaction using the
same sense and antisense primers designed for genomic Sm14 coding
sequence: forward Sm14 primer,
5'-ACCTCGAGGATATCCATATGTCTAGTTTCTTGG-3', and reverse Sm14 primer,
5'-TTTCCTTTTGCGGCCGCACGCGTGAATTCGAGGCGTTAGGATAGTCGTT-3'. The restriction sites for XhoI, EcoRV, and
NdeI in the forward primer and NotI,
MluI, and EcoRI in the reverse primer are
underlined, respectively. In boldface are the
sequences derived from the Sm14 open reading frame. In addition, a
sample from one cDNA library of S. mansoni BH strain
kindly provided by Dr. S. Verjovski-Almeida (Instituto de
Química, Universidade de São Paulo, São Paulo, Brazil) was also used as template. A total of three independent PCR
amplifications from reverse-transcribed cDNAs were analyzed in this
study. PCR products were purified from agarose gels after electrophoretic separation using the In Concert Rapid Gel Extraction system kit (Invitrogen), cloned into pGEM-T vector (Promega, Madison, WI), and sequenced using the T7 and SP6 promoter primers. The nucleotide sequence analysis of Sm14 cDNAs were performed on the ABI PRISM 377 sequencer using the ABI PRISM Big Dye Terminator cycle
sequencing kit.
PCR in Vitro Mutagenesis--
To create site-directed mutation
in the cDNA encoding for Sm14, we used the PCR in vitro
mutagenesis method (7). The amino acid residue at position 20, in this
case methionine, was changed to alanine (Met-20 Expression and Purification of the Recombinant Sm14
Proteins--
The cDNA clones of the two native Sm14 isoforms
(Sm14-M20 and Sm14-T20) and the mutant variant (Sm14-A20) cDNA
clones were isolated from agarose gels after digestion with
XhoI and MluI and ligated to the pAE expression
vector (8) previously digested with the same enzymes. This vector
allows the expression of recombinant proteins with a minimal N-terminal
His6 tag. The obtained constructs were used to transform
Escherichia coli BL21 (DE3) strain (Novagen, Madison, WI).
One liter of 2× YT medium (1.5% casein hydrolisate, 1% yeast
extract, 0.5% NaCl; all w/v) was inoculated with 40 ml of the
overnight recombinant bacteria culture from single colonies and grown
until the optical density at 600 nm reached 0.6. The expression of the
recombinant protein was induced with 1 mM
isopropyl-1-thio- Spectroscopic and Protein Stability Studies--
Equilibrium
unfolding as a function of temperature was monitored by CD, whereas the
equilibrium unfolding as a function of denaturant concentration was
studied by fluorescence spectroscopy. CD measurements were carried out
on a Jasco J-810 Spectropolarimeter at 20 °C equipped with a Peltier
unit for temperature control. Far-UV CD spectra were acquired using a
1-mm path length cell at 0.5-nm intervals over the wavelength range
from 190 to 260 nm. Five scans were averaged for each sample and
subtracted from the blank average spectra. The protein concentration
was kept at 10 µM in 10 mM sodium-phosphate
buffer as described above. The temperature range was from 15 to
75 °C. The loss of secondary structure was followed by measuring the
molar ellipticity of [ Fatty Acid Binding--
The fatty acid dissociation constant of
the three recombinant Sm14 proteins was determined by following the
changes in fluorescence by increasing the concentration of the
fluorescent fatty acid analogue 11-(dansylamino)undecanoic acid (DAUDA)
obtained from Molecular Probes (Eugene, OR). The excitation wavelength
used for DAUDA was 345 nm. A stock solution of 10 mM DAUDA
in ethanol, kept in the dark at Vaccination Experiments--
The three recombinant proteins,
rSm14-M20, rSm14-T20, and rSm14-A20, were evaluated for vaccine
efficacy in 4-6-week-old female outbred Swiss mice as described
previously (4, 9). The mice received three subcutaneous inoculations
with 7-day intervals of recombinant proteins in PBS emulsified in
aluminum hydroxide as adjuvant. Control mice were injected with PBS
plus adjuvant. The animals were further challenged percutaneously with
100 cercariae (LE strain)/mouse 60 days after the last immunization
dose and perfused 45 days later. The overall protection efficiency was calculated by the equation [(C Sm14 Genomic Sequence and Organization--
The genomic sequence
of Sm14 was amplified by PCR using DNA isolated from a pool of adult
worms of the Brazilian endemic S. mansoni BH strain. The
amplified product is larger than that amplified from RNA, indicating
the presence of introns (data not shown). The amplified 1.7 kilobase
pair of genomic DNA segment was cloned and sequenced from two
independent PCR clones (GenBankTM accession AY055467). The
sequence analysis revealed the sites of exon-intron junctions. All of
the intron-exon boundaries obey the GT/AG rule (Fig.
1A) (10). The exon nucleotide
sequences were identical to that previously reported for Sm14 cDNA
cloned from the Puerto Rican strain of S. mansoni (11).
Similar to the other members of the FABP gene family, the gene for Sm14
contains four exons separated by three introns (12). Recently, the
presence and position of the introns in the related FABP
gene from S. japonicum were also determined by PCR and
corresponded exactly to that observed here for S. mansoni (Fig. 1B) (13). The comparison of helminth and mammalian FABP genes showed that the sizes and
positions of FABP exons are conserved among these organisms, whereas
the size of the introns is quite variable (Table
I) (12). The size of the FABP introns of
S. japonicum, estimated by migration of PCR products in
agarose gels (13), are larger than S. mansoni introns: ~1200 versus 674; ~850 versus 585; and ~70
versus 42 nucleotides for introns 1, 2, and 3 of
S. japonicum and S. mansoni,
respectively. The intron 3 of S. mansoni is the smallest of
all of the related introns found in the FABP gene family so
far. Recently, a deletion variant of the S. japonicum FABP
called F25 was characterized by cDNA cloning (13) where the codons
for the first 12 amino acids located in exon 2 were absent (Fig.
1B). Taking into account the high identity in nucleotide
sequence of the FABP genes of S. mansoni and
S. japonicum, we speculated that in S. japonicum, the presence of a second 3'-acceptor site for the
intron 1 at the beginning of the exon 2 would explain the generation of
S. japonicum F25 transcript. This same alternative splicing
could also be used to generate an orthologous S. mansoni F25
variant (Fig. 1A, arrow). After alternative
splicing of intron 1 using this putative splice site, the first 12 amino acids of exon 2 could be excised while all of the remaining
codons would remain in-frame, exactly as observed in S. japonicum F25. However, this second splice site for intron 1 lacks
the characteristic pyrimidine-rich sequence in the intron-exon junction
(10), and therefore as previously postulated for S. japonicum F25 (13), we do not expect this event to occur
frequently.
When the obtained Sm14 genomic sequence was aligned with the nucleic
acid sequences of other members of the FABP gene family, the
positioning of each of the three introns was found to be conserved. As
shown in Fig. 2, the first intron always
divides a codon after its first nucleotide in contrast to the other
exon/intron borders, which are located after the entire codon (14).
However, it is noteworthy that despite this highly conserved gene
organization, FABPs do present a few exceptions. In the
desert locust Schistocerca gregaria, the orthologous gene
does not contain intron 2 (15) and several putative lipid-binding
protein genes from the free-living nematode Caernorhabditis
elegans do not have the intron 1 or possess additional introns
(16).
Sm14 Polymorphism--
Several cDNA clones generated by
independent reverse-transcribed PCR reactions and cDNA libraries
from two Brazilian S. mansoni strains were analyzed. A
multiple alignment of the deduced amino acid sequences for the coding
regions of all of the obtained Sm14 cDNA clones (a total of 30 cDNAs, 13 and 17 cDNAs from LE and BH strains, respectively) is
shown in Fig. 3. Eight clones showed a
sequence identical to the Sm14 protein cloned from Puerto Rican S. mansoni strain (11), whereas the other 14 clones
presented a single mutation M20T change (ATG
Overall, almost all of the nucleotide sequences derived from the
S. mansoni LE strain showed methionine at position 20 (12 Met-20 residues and 1 Thr-20 residue), whereas in the case of the
clones obtained from one BH strain (Instituto Butantan, São Paulo, Brazil), seven clones had the threonine at this position. In
addition, the cDNA library derived from the another BH strain (obtained from Instituto de Química, Universidade de São
Paulo, São Paulo, Brazil) yielded seven clones with threonine and
three clones with methionine at position 20.
The polymorphism of the orthologous FABP protein was also characterized
in Philippine strains of S. japonicum (13). In this case,
the cDNA clones obtained had different sequences, and none of them
was identical to the previously reported FABP cDNA from the Chinese
strain of S. japonicum (18). In conclusion, our results indicate that Sm14 proteins in both BH and LE strains are less
polymorphic than the orthologous S. japonicum FABPs.
Significance of Sm14 M20T Polymorphisms--
The sequence analysis
of the obtained clones indicates the existence of two main isoforms of
S. mansoni FABP (Sm14-M20 and Sm14-T20). This polymorphism,
besides the PCR clones from BH and LE strains, was also confirmed from
isolated Structural Analysis of Sm14--
The recombinant
His6-tagged Sm14 proteins (rSm14-M20 and rSm14-T20) were
expressed and purified using a metal affinity chromatography. An
additional mutant form (rSm14-A20) was also constructed to better
evaluate the importance of this amino acid residue in the Sm14 protein.
Single protein bands with molecular masses of ~16 kDa were observed
for rSm14-M20 and rSm14-T20 when subjected to SDS-PAGE analysis as well
as for rSm14-A20 protein (data not shown). Fig.
5A, black line,
shows that the three rSm14 proteins present a CD spectrum typical of a
protein possessing a secondary structure containing mainly
Fatty Acid Binding Analysis--
The fatty acid binding capability
of the three rSm14 proteins was examined by using the
environment-sensitive fluorescent fatty acid analogue DAUDA that alters
its fluorescence emission spectra and intensities on entry into binding
proteins (21). The blue shift of DAUDA fluorescence maximum from 543 to
538, 530, and 528 nm for rSm14-M20, rSm14-T20, and rSm14-A20,
respectively (results not shown), and the concomitant increase of its
fluorescence emission (both indicative of the entry of DAUDA into an
apolar environment (22)) confirmed the binding of the fluorescent fatty acid to the three rSm14 variants. These shifts are comparable with the
results obtained from structurally related FABPs such as the heart
FABP, brain FABP, and adipocyte FABP where the fluorescence emission
moves to 536, 531, and 530 nm, respectively (23). The liver FABP and
the intestinal FABP produce a shift to 500 and 496 nm, respectively
(23), values closer to what is observed for S. japonicum F10
FABP (24). At large, the observed variability in the emission
wavelength indicates that in each FABP, there are local contacts with
specific residues and this might justify their difference in affinity.
Interestingly, the stoichiometry of the binding of DAUDA to the three
rSm14 variants shows a molar ratio of 1:1 similar to most of the
FABPs (12), and the rSm14 variants exhibit quite similar
Kd values (0.63, 0.82, and 0.66 for rSm14-M20,
rSm14-T20, and rSm14-A20, respectively) (Fig.
6). To highlight the possible differences
in the binding affinities of the three Sm14 proteins for other fatty
acids, we performed competitive experiments using DAUDA as tracer and
some natural fatty acids as competitors. The addition of myristic, palmitic, oleic, and linoleic acids indeed revealed a diverse affinity
of the three mutants for these substrates represented by the different
extents in the stoichiometric reversal of the fluorescence enhancement
of DAUDA. Fig. 7 shows a typical
competitive curve where linoleic acid is used to displace DAUDA from
rSm14-M20. The efficiency of each fatty acid to displace DAUDA from the
rSm14 binding pocket is shown in Fig. 8.
A value of 100% indicates the complete displacement of DAUDA. The data
show that the rSm14-M20 has the higher affinity for all of the natural
fatty acids. Overall, the results indicate that the efficiency in the
displacement of DAUDA increases with the increase of both the fatty
acid length and the number of insaturations (12) and, as observed for
other FABPs, confirm the importance of the nature of the amino acid residue at position 20 in Sm14. It is interesting to observe that in
the case of rSm14-A20, the myristic acid is not able to displace DAUDA
evidencing differently from the other rSm14 proteins, an absence of
affinity of the mutant for this relatively short fatty acid. A possible
key to interpret the similarities and differences in affinity exhibited
by the three Sm14 proteins with respect to DAUDA and to the various
fatty acids can be derived by the analysis of the crystallographic
structure of human brain FABP that reveals that only long chain fatty
acids are able to interact with Met-20 (25). Based on this observation,
we can hypothesize that because of its short chain, DAUDA (only 11 carbon atoms) will not be able to interact with any residue that might
be present in position 20, thus justifying the equivalent affinity
exhibited by the three proteins for the fluorescent fatty acid probe
(Fig. 6). On the other hand, the competition experiments (Fig. 8) where longer fatty acids have been used revealed the existence of distinct affinities that very likely are attributed to selective contacts between the fatty acid and the distinct residue at position 20 of the
rSm14 proteins. Altogether, these results indicate that the nature of
the amino acid at position 20 is very important for structure stability
and for the binding features to fatty acids.
Vaccination Experiments--
The protection efficacy of the three
rSm14 proteins against S. mansoni cercariae was evaluated in
outbred Swiss mice as described previously (4). As shown in Fig.
9, rSm14-T20 or rSm14-M20 proteins
stimulated a significant protective response (44 and 67%,
respectively). Animals vaccinated with these proteins showed a
reduction in mean worm burden with values close to or higher than the
40% reduction value defined by the WHO as a prerequisite for an
antigen to be considered a potential vaccine candidate against human
schistosomiasis. The exception is the rSm14-A20 mutant that turned out
to be a poor protective antigen, reaching protection values of around
20% of worm burden reduction. Interestingly, however, in the Sm14, the
amino acid at position 20 as shown by molecular modeling (results not
shown) (2) is buried within the interior of the molecule like in the
other FAPBs (2, 12, 23, 25) and, therefore, is not expected to be
exposed as an epitope. Nonetheless, the data described here demonstrate
the importance of this residue in modulating some structural and
functional features of Sm14. Thus, the lack of significant protective
ability for rSm14-A20 is probably the result of these properties rather than the change of the epitope exposure. The rSm14-A20 was the more
unstable and less structured Sm14 protein (data not shown) (Fig. 5).
The rSm14-M20 showed the highest secondary structure recovery (82.5%)
after heating to 80 °C followed by rSm14-T20 (64.3%) (Fig.
5A) and was more resistant to denaturation by urea as
followed by intrinsic fluorescence of tryptophan (results not shown).
These properties correlate well with the level of protective ability
observed (Fig. 9). In fact, heating the rSm14-T20 just before the
immunization of the animals resulted in a poor protective response
similar to that observed for rSm14-A20 mutant, thus suggesting that for
immune protection, Sm14 has to possess the correct fold. This is an
important observation for the development of a Sm14-based vaccine (3),
and it implies that a quality control that takes into account the
structure integrity of the protein has to be included in the production
of rSm14.
In conclusion, the data presented here show for the first time the
genomic organization of the Sm14 protein and the existence of
polymorphism. A preliminary evaluation of the structural and functional
features of some of these mutants has revealed that the Sm14-M20
isoform, characterized by a higher structural stability and by a more
pronounced affinity for natural fatty acids, appears to be the more
suitable antigen for the development of the schistosomiasis vaccine.
This observation shall contribute for the Sm14-based vaccine
development. In particular, we believe that the enhanced stability of
the protein may be a key property because it can provide a better
immune response due to the presence of the proper fold.
-barrel structure as
shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other
forms (rSm14-M20>rSm14-T20>rSm14-A20). All of the Sm14
proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA)
without substantial difference in the binding affinity. However,
rSm14-M20 exhibited a higher affinity for natural fatty acids than the
rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments
against DAUDA (rSm14-M20>rSm14-T20>rSm14-A20). The rSm14-M20 or
rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce
significant protection against S. mansoni cercariae
challenge in immunized mice. The level of protection efficacy
correlates with the extent of structure stability of the recombinant
Sm14 isoforms and mutant.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Ala-20) using the
forward (F) A20 5'-gctgtcGCgtcaaagctag-3' and the reverse (R)
A20 5'-agctttgacGCgacagcatc-3' primers (the nucleotide positions
that were targets for mutagenesis are in uppercase) in
appropriate combination with forward and reverse Sm14 primers. The
mutation was confirmed by DNA sequence analysis.
-D-galactopyranoside and cultivated for
an additional 3 h at 37 °C. The inclusion bodies containing the
recombinant His6-tagged protein were isolated from bacterial lysates and solubilized with 10 ml of buffer containing 8 M urea, 0.05 M Tris-HCl, pH 8.0, and 0.005 M 2-mercaptoethanol. The material was diluted 200 times in
the refolding buffer (0.005 M imidazole, 0.5 M
NaCl, 0.05 M Tris-HCl, pH 8.0, and 0.005 M 2-mercaptoethanol) and stirred for 16 h at room temperature. After the refolding procedure, the recombinant protein was purified by
metal-affinity chromatography in Chelating-Sepharose Fast Flow resin
(5-ml resin bed, 1-cm-diameter column, Amersham Biosciences). Subsequently, the column was washed with refolding buffer and then the
elution was carried out with a buffer containing 0.5 M
NaCl, 0.5 M imidazole, and 0.05 M Tris-HCl, pH
6.8. The eluted protein was dialyzed against PBS. For the CD
experiments, the protein was dialyzed against 10 mM
sodium-phosphate buffer, pH 7.4. The protein purity was monitored by
SDS-PAGE followed by Coomassie Brilliant Blue staining. The
concentrations of the soluble recombinant proteins were estimated from
absorbance at 280 nm, considering an extinction molar coefficient of
280 = 12,325 M
1 cm
1 based on the expected amino acid sequence of the
recombinant protein.
] at 216 nm. Fluorescence changes were
followed with a SLM-AMINCO-Bowman Series II Luminescence Spectrometer
(Spectronic Instruments, Garforth, Leeds, United Kingdom) with 1-ml
samples in a quartz cuvette. The protein concentration was 2 µM, and after each addition of urea, the sample was
equilibrated at 20 °C before measurements were made. The intrinsic
fluorescence of the rSm14 proteins, mainly because of the two Trp
residues, was recorded setting the excitation wavelength at 285 nm and
monitoring the shift in the emission maximum in the range of 330-355 nm.
20 °C, was freshly diluted in PBS
to 1 mM or 0.1 mM before use in the
fluorescence experiments. The protein concentration was 2 µM in 1 ml of PBS at 20 °C. Fluorescence data were
subtracted for the blank values (samples without proteins) and fitted
by standard non-linear regression techniques (ORIGIN software version
6.1, Origin Lab Corporation, MA) to a single non-competitive binding
model to estimate the apparent dissociation constant
(Kd) and maximal fluorescence intensity
(Fmax). Competitive experiments were designed to
reveal a possible difference in affinity of the three rSm14 proteins
for the various natural fatty acids. The myristic, palmitic, oleic, and
linoleic acids obtained from Sigma were stored and diluted as described
above for DAUDA. In these experiments, the binding of 2 µM DAUDA to 2 µM protein was performed in
the presence or absence of 2 µM of each fatty acid.
V)/C] × 100, where C is the average number of worms in control
animals and V is the average number of worms in vaccinated
animals. Statistical analysis was done with Student's t
test (p < 0.05).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequence of the Sm14 gene
and alignment of the deduced protein with the S. japonicum
F25 and F10 FABPs. A, sequence of the Sm14
gene. The complete sequence is in the GenBankTM data base
under accession number AY055467. The canonical GT/AG sequences for
intron/exon boundaries are in boldface. The internal
EcoRI site is underlined, and an arrow
indicates the 3'-alternative acceptor splice site for the intron 1. In
this alternative splicing, a smaller Sm14 protein of the size of the
S. japonicum F25 can be generated. B, amino acid
sequence alignment of FABPs encoded by the S. mansoni
Sm14 gene and by the S. japonicum FABP cDNA
clones F10 and F25. The alignment shows the position of introns 1, 2, and 3. In the case of S. japonicum F25, the gap ( )
represents the observed amino acid deletion.
Sizes of the introns and of the amino acids in the exons of several
FABPs
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Fig. 2.
Intron positioning in the FABP
gene family. The nucleotide and amino acid sequences of a
number of FABPs have been aligned to obtain the maximal positional
codon identity. The position of the three introns falls at exactly the
same points in each gene. Asterisk, the numbering of LBP-3
corresponds to the putative mature protein and the reported introns
corresponding to introns 3, 4, and 5 of lbp-3 gene.
ACG). Interestingly,
methionine at this position is strongly conserved among the members of
FABP protein family. In the rat intestinal and adipocyte FABPs, an important role for this amino acid residue in ligand binding function was demonstrated (17). Two clones, Sm14-M20-L114 and Sm14-T20-L114, displayed the synonymous mutation M114L and are considered to represent
true polymorphic Sm14 proteins. Two clones as deduced by comparison
with the genomic Sm14 sequence (Fig. 1A) showed the deletion
of the amino acid sequence corresponding to the entire exon 3 (Sm14-
E3 clones), and they were probably generated by alternative
splicing. Nevertheless, because the reverse-transcribed PCR analysis
showed a single amplification product corresponding to the Sm14
full-length open reading frame, alternative splicing is expected to be
a rare misprocessing of the mRNA precursor (results not shown).
Five clones displayed silent mutations (CTA
CTG) at the Leu-23
codon, which is located at 3' end of the first exon of the Sm14 gene
(data not shown). The remaining clones had Met-20 plus single changes
in positions where the amino acids were less conserved and are
represented by only one clone. These clones may have been generated by
PCR mutagenesis, and for this reason, they were not further studied in
this work. Fig. 4 summarizes the possible
splicing forms of Sm14 proteins.
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Fig. 3.
Multiple sequence alignments of the
S. mansoni Sm14 isoforms. Numbering is
according to the Sm14 sequence. ( ) indicates identical amino acid;
dots have been introduced to allow the best sequence
alignment and to represent amino acid gap. M = Sm14-M20; T = Sm14-T20. The number of sequenced clones
of each isoform is in parenthesis. Predictions of the
secondary structure features are also shown below the sequences. The
alignment includes the sequences of three S. japonicum FABPs
(Sj-FABPs): Sj-FABP, Sj-F10, and Sj-F25 (13).
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Fig. 4.
Schematic representation of the Sm14 genomic
organization and of its splicing forms. The asterisk
denotes the possible alternative splicing site that in S. mansoni would be responsible for the expression of a reduced form
of Sm14 similar to Sj-F25.
-cDNA library clones (11) from Puerto Rican
strain.2 To understand the
structural and functional features of these isoforms, we
proceeded to verify whether this amino acid position was conserved in
the FABPs family using the primary sequence alignments reported
previously (results not shown) (2, 12). The methionine at this position
is changed for leucine in a few FABP members or for valine in
testis lipid-binding proteins of rat and mouse. These changes agree
with the rule of "safe" residue substitutions (19) where Met can be
substituted by Leu or Val without perturbing the protein structure and
function. These mutations matched the changes observed for position 20 in the FABP protein family. The only exception is the Sm14-T20 isoform.
It is worth pointing out that the methionine residue at this position
appears to play an important role in modulating the structural and
functional properties of these proteins. In fact, the mutagenesis of
this residue in two different FABPs (M20A in adipocyte FABP and M18A in
intestinal FABP) produced proteins characterized by a decrease of
structural stability as well as their affinity for fatty acids
(17). Based on these evidence and because the nature of the threonine
is very different from the methionine in terms of hydrophobicity and
chain size, we expect to observe significant differences between the two main isoforms of Sm14.
-structural elements in agreement with the prediction based on its
primary sequence (4). Overall, the CD spectra show some
differences in the chiroptical activity of the three proteins,
suggesting that indeed a single mutation at position 20 influences
their structural features. The stability of the protein was assessed
both by CD and fluorescence spectroscopy. Fig. 5B shows the
trace of thermal unfolding of the three rSm14 proteins. The sigmoidal
shape indicates we are in the presence of a two-state transition. As
indicated by the CD spectra obtained after cooling down the protein,
the transition is not completely reversible (Fig. 5A,
light gray line). The secondary structure recovery was
higher for the rSm14-M20 followed by rSm14-T20 and rSm14-A20 (82.6, 64.4, and 56.8%, respectively). The CD spectra at a high temperature
(Fig. 5A, dark gray line) were recorded when the
ellipticity at 216 nm had reached a plateau, although they indicated
that a protein mainly unfolded still presents a negative shoulder at
222 nm, suggesting the presence of some residual helical determinants
(Fig. 5A). It is interesting to note that despite the fact
that distinct fatty acid-binding proteins have a similar
-barrel
structure, they may have different folding and unfolding intermediates
(20). From the fitting of the CD traces recorded at an increasing
temperature (Fig. 5B), the temperature of unfolding of each
protein variant has been derived, showing that the rSm14-M20 isoform
was more stable (Tm = 55.3 °C) than rSm14-T20
isoform and rSm14-A20 mutant (Tm = 44.6 and 45.8, respectively). The folding stability of the three rSm14 variants has
also been investigated by following the change of their fluorescence
emission maximum as a function of increasing concentration of urea. The
data confirmed that indeed rSm14-M20 has a higher structural stability
(results not shown).
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Fig. 5.
CD evaluation of the thermal stability of the
rSm14 proteins. A, CD spectra of the three Sm14
proteins were measured at 15 °C (black line) and 80 °C
(dark gray line), and after cooling down the protein, they
were measured at 15 °C (light gray line). Secondary
structure recovery after heating the rSm14 proteins to 80 °C and
cooling down to 15 °C was calculated for each protein considering
the change of the wavelength value when [ ] = 0. The corresponding
wavelength value considered for random structure was 191.5 nm.
B, variation of the molar ellipticity [
] measured at
216 nm throughout the temperature range of 15-75 °C for the three
recombinant Sm14 variants. Tm values for each of the
rSm14 proteins were calculated as the inflection point of each curve
according to Boltzman's function using the Origin program.
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[in a new window]
Fig. 6.
Titration curves of DAUDA binding to rSm14
proteins. Increasing amounts of DAUDA were added to a 2 µM solution of the protein in PBS. The enhancement of
DAUDA fluorescence upon binding to the rSm14 variants was obtained by
subtracting the fluorescence of the same concentration of DAUDA in the
absence of the protein. All of the data were analyzed by a non-linear
regression model according to Hill's function using the Origin
program.
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[in a new window]
Fig. 7.
Displacement of DAUDA by linoleic acid in the
binding to rSm14-M20. Competitive binding experiments were carried
out using 2 µM DAUDA in the presence of 2 µM linoleic acid and 2 µM rSm14-M20.
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Fig. 8.
Displacement of DAUDA binding to rSm14
proteins by natural fatty acids. The percentage of displacement is
indicated above each bar. The same conditions as described
for Fig. 6 were used here for each rSm14 protein (2 µM)
versus each fatty acid (2 µM) in the presence
of DAUDA (2 µM).
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Fig. 9.
Protection of vaccinated animals against
S. mansoni. Protection was measured as the mean
of worm burden in each group of animals and was expressed in percentage
of protection as explained under "Experimental Procedures." Animals
were immunized with only adjuvant (Adj) or adjuvant plus
rSm14-T20 (T) or plus rSm14-M20 (M) or plus
rSm14-A20 (A). One group of animals was immunized with
adjuvant plus rSm14-T20 previously heated to 90 °C for 5 min just
before the inoculation ( T). *, p < 0.05 when compared with the group of animals immunized with only Adj; **,
p < 0.05 when compared with the group of animals
immunized with non-heated rSM14-T20.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Sérgio Verjovsky-Almeida for providing us the S. mansoni cDNA library and Dr. Toshie Kawano for the adult worms of the S. mansoni BH strain.
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FOOTNOTES |
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* This work was supported by grants from the Brazilian agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Butantan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY055467, AF492389, and AF492390.
¶ Recipient of CAPES fellowship for this study.
¶¶ To whom correspondence should be addressed: Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, CEP 05503-900, São Paulo, SP, Brazil. Tel.: 55-11-37267222, ext. 2083; Fax: 55-11-37261505; E-mail: hoplee@butantan.gov.br.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211268200
2 C. R. R. Ramos, M. M. Vilar, A. L. T. O. do Nascimento, M. Tendler, I. Raw, and P. L. Ho, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: FABP, fatty acid-binding protein; DAUDA, 11-(dansylamino)undecanoic acid; PBS, phosphate-buffered saline.
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