Gene Structure and M20T Polymorphism of the Schistosoma mansoni Sm14 Fatty Acid-Binding Protein: Molecular, Functional and Immunoprotection Analysis. 1

rSm14 S.


Introduction
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 (S. mansoni, S. japonicum or S. haematobium).In America, only S. mansoni is found, 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 lipids supply.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).Since 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 cells 1 (Th1) 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 in order 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.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.

Experimental Procedures
Parasites -In the present 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 (Life Technologies, Rockville, MD, USA) was used for total RNA isolation from LE and BH schistosome strains.Two micrograms of total RNA were reverse transcribed by SuperScript II RT enzyme (Life Technologies) 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 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 (M20→A20) using the forward F A20 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 6xHis-tag.The obtained constructs were used to transform E. coli BL21 (DE3) strain (Novagen, Madison, WI, USA).One Liter of 2xYT medium (1.5% casein hydrolisate, 1% yeast extract, 0.5% NaCl; all w/v) was inoculated with 40ml of the overnight recombinant bacteria culture from single colonies and grown until the OD at 600 nm reached 0.6.The expression of the recombinant protein was induced with 1mM isopropyl-β-D-thiogalactoside (IPTG) and cultivated for additional 3 hours at 37 o C. The inclusion bodies containing the recombinant 6xHis-tagged protein were isolated from bacterial lysates and solubilized with 10 ml of buffer containing 8M urea, 0.05M Tris-HCl pH 8.0 and 0.005M 2-mercaptoethanol.The material was diluted 200x in the refolding buffer (0.005M imidazole, 0.5M NaCl, 0.05M Tris-HCl pH 8,0 and 0.005M 2-mercaptoethanol) and stirred for 16 hours 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, Pharmacia, Uppsala, Sweden).Subsequently, the column was washed with refolding buffer and then the elution was carried out with a buffer containing 0.5M NaCl, 0.5M imidazole, 0.05M Tris-HCl, pH 6.8.The eluted protein was dialyzed against PBS.
For the CD experiments, the protein was dialyzed against 10mM Na-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 ε 280 =12325 M -1 cm -1 , based on the expected amino acid sequence of the recombinant protein.
Spectroscopic and protein stability studies -Equilibrium unfolding as a function of temperature was monitored by CD while 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 0 C equipped with a Peltier unit for temperature control.Far-UV CD spectra were acquired using a 1mm 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 10µM in 10mM Naphosphate buffer as described above.The temperature range was from 15 to 75 0 C. The loss of secondary structure was followed by measuring the molar ellipticity [θ] at 216 nm.Fluorescence changes were followed with a SLM-AMINCO-Bowman Series II Luminescence Spectrometer (Spectronic Instruments, Garforth, Leed, UK), with 1 ml samples in a quartz cuvette.The protein concentration was 2µM and after each addition of urea the sample was equilibrated at 20 0 C before measurements were made.The intrinsic fluorescence of the rSm14 proteins, mainly due to 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.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 previously described (4,9).Briefly, 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 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
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 kbp genomic DNA segment was cloned and sequenced from two independent PCR clones (GenBank accession AY055467).The sequence analysis revealed the sites of exon-intron junctions.All intron-exon boundaries obey the GT/AG rule (10) (Figure 1A).The exon nucleotide sequences were identical to that previously reported for Sm14 cDNA cloned from the Puerto Rican strain of S. mansoni (11).
Like 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 was also determined by PCR and corresponded exactly to that observed here for S. mansoni (Figure 1B) (13).The comparison of helminth and mammalian FABPs genes showed that the sizes and positions of FABP exons are conserved among these organisms, whereas the size of the introns is quite variable (12) (Table I).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: aprox.1200 vs 674, aprox.850 vs 585 and aprox.70 vs 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 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 (Figure 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 (shown by an arrow in the Figure 1A).After alternative splicing of intron 1 using this putative splice site, the first 12 amino acids of exon 2 could be excised while all the remaining codons would remain in frame, exactly as observed in S. japonicum F25.This second splice site for intron 1, however, lacks the characteristic pyrimidine rich sequence in the intronexon 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 Figure 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).It is worth to note, however, that, in spite of this highly conserved gene organization, FABPs do present 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 freeliving nematode Caernorhabditis elegans do not have the intron 1 or possess additional introns (16).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 Chinese (18).In conclusion, our results indicate that Sm14 proteins in both BH and LE strains are less polymorphic than the orthologous Sj-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 lambda cDNA library clones (11) from Puerto Rican strain (unpublished results).
In order to understand the structural and functional features of these isoforms, we proceeded to verify if this amino acid position was conserved in the FABPs family using the primary sequence alignments previously reported (2, 12 and results not shown).The methionine at this position is changed for leucine in few FABP members or for valine only 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 to point 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 A-FABP and M18A in I-FABP) produced proteins characterized by a decrease of structural stability as well as of their affinity for fatty acids (17).Based on these evidences and since 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 analysis of Sm14 -The recombinant 6xHis-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 in order to better evaluate the importance of this amino acid residue in the Sm14 protein.Single protein bands with molecular masses about 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).Figure 5A (black line) shows that the three rSm14 proteins present a CD spectrum typical of a protein possessing a secondary structure containing mainly β-structural elements in agreement with the prediction based on its primary sequence (4).Overall, the CD spectra evidence 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.Figure 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 though, as also indicated by the CD spectra obtained after cooling down the protein, the transition is not completely reversible (Figure 5A, light grey 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 high temperature (Figure 5A, dark grey line) recorded when the ellipticity at 216 nm has reached a plateau, though they indicate a protein mainly unfolded, still present a negative shoulder at 222 nm, suggesting the presence of some residual helical determinants (Figure 5A).It is interesting to note that despite the fact that distinct fatty acid binding proteins have similar β-barrel structure, they may have different folding and unfolding intermediates (20).From the fitting of the CD traces recorded at increasing temperature (Figure 5B), the temperature of unfolding of each protein variant has been derived, showing that the rSm14-M20 isoform was more stable (Tm = 55.3 0 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).
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 move 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, while 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), they exhibit quite similar Kd values (0.63, 0.82 and 0.66 for rSm14-M20, rSm14-T20 and rSm14-A20, respectively)(Figure 6).In order to highlight 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 acid Vaccination experiments -The protection efficacy of the three rSm14 proteins against S.
mansoni cercariae was evaluated in outbred Swiss mice as previously described (4).As shown in Figure 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 WHO as a prerequisite for an antigen to be considered a potential vaccine candidate against human schistosomiasis.Exception is the rSm14-A20 mutant that turned out to be a poor protective antigen, reaching protection values around 20% of worm burden reduction.Interestingly, however, in the Sm14 the amino acid at position 20, as shown by molecular modeling (2 and results not shown), is buried within the interior of the molecule like in the other FAPBs (2, 12, 23 and 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, Figure 5).
The rSm14-M20 showed the highest secondary structure recovery (82.5%) after heating to 80 0 C, followed by rSm14-T20 (64.3%) (Figure 5A) and was more resistant to denaturation by urea as followed by intrinsic fluorescence of tryptophane (results not shown).These properties correlate well with the level of protective ability observed (Figure 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 take 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 the 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.

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 (Molecular Probes, Inc., Eugene, OR, USA).The excitation wavelength used for DAUDA was 345 nm.A stock solution of 10mM of DAUDA in ethanol, kept in the dark at -20 o C, was freshly diluted in PBS to 1mM or 0.1mM before use in the fluorescence experiments.The protein concentration was 2µM in 1 mL 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, USA) to a single non-competitive binding model to estimate the apparent dissociation constant (K d ) and maximal fluorescence intensity (F max ).Competitive experiments were designed to reveal a possible difference in affinity of the three rSm14 proteins for the various natural fatty acids.The miristic, palmitic, oleic and linoleic acids obtained from Sigma (Sigma, St. Louis, MO, USA) were stored and diluted as described above for DAUDA.In these experiments, the binding of 2µM of DAUDA to 2µM of protein was performed in the presence or absence of 2µM of each fatty acid.

Figure 3 .
Figure 3. Eight clones showed a sequence identical to the Sm14 protein cloned from Puerto by guest on November 19, 2017 http://www.jbc.org/Downloaded from

Figure 1 .
Figure 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

Figure 2 .
Figure 2. Intron positioning in the FABP gene family.The nucleotide and amino acid

Figure 3 .
Figure 3. Multiple sequence alignments of the S. mansoni Sm14 isoforms.Numbering is

Figure 4 .
Figure 4. Schematic representation of the Sm14 genomic organization and of its splicing

Figure 5 .
Figure 5. CD evaluation of the thermal stability of the rSm14 proteins.(A) CD spectra of the three Sm14 proteins measured at 15 0 C (black line), 80 0 C (dark grey line) and after cooling down the protein to 15 0 C (light grey line).Secondary structure recovery after heating the rSm14 proteins to 80 0 C and cooling down to 15 0 C was calculated for each protein considering the

Figure 6 .
Figure 6.Titration curves of DAUDA binding to rSm14 proteins.Increasing amounts of

Figure 7 .
Figure 7. Displacement of DAUDA by linoleic acid in the binding to rSm14-M20.

Figure 8 .Figure 6
Figure 8. Displacement of DAUDA binding to rSm14 proteins by natural fatty acids.The