Portal de Periódicos da CAPES

Artigo Acesso aberto Produção Nacional Revisado por pares

BIBTEX RIS

Gene Structure and M20T Polymorphism of theSchistosoma mansoni Sm14 Fatty Acid-binding Protein

2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês

10.1074/jbc.m211268200

1083-351X

Celso Raul Romero Ramos, Rita C. R. Figueredo, Thelma A. Pertinhez, Mônica Magno Vilar, Ana L. T. O. Nascimento, Mı́riam Tendler, Isaı́as Raw, Alberto Spisni, Paulo Lee Ho,

Peroxisome Proliferator-Activated Receptors

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 β-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. 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 β-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. fatty acid-binding protein 11-(dansylamino)undecanoic acid phosphate-buffered saline 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, orSchistosoma hematobium). In America, onlyS. mansoni is found, which had been introduced from Africa in the colonial time (1Despres L. Imbert-Establet D. Monnerot M. Mol. Biochem. Parasitol. 1993; 60: 221-229Crossref PubMed Scopus (55) Google Scholar). 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 (2Esteves A. Joseph L. Paulino M. Ehrlich R. Int. J. Parasitol. 1997; 27: 1013-1023Crossref PubMed Scopus (32) Google Scholar). 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 (3Bergquist N.R. Colley D.G. Parasitol. Today. 1998; 14: 99-104Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The S. mansoni Sm14 showed already a protective activity against two parasite worms, Fasciola hepatica and S. mansoni (4Tendler M. Brito C.A. Vilar M.M. Serra-Freire N. Diogo C.M. Almeida M.S. Delbem A.C. Da Silva J.F. Savino W. Garratt R.C. Katz N. Simpson A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 269-273Crossref PubMed Scopus (189) Google Scholar). 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 (5Brito C.F. Caldas I.R. Coura Filho P. Correa-Oliveira R. Oliveira S.C. Scand. J. Immunol. 2000; 51: 595-601Crossref PubMed Scopus (38) Google Scholar). 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. japonicumFABPs.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. 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). The genomic DNA was isolated from the S. mansoni worms following the standard protocols (6Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 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 areunderlined, 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 Quı́mica, 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. To create site-directed mutation in the cDNA encoding for Sm14, we used the PCR in vitromutagenesis method (7Krajewski J.L. Dickerson I.M. Potter L.T. Mol. Pharmacol. 2001; 60: 725-731PubMed Google Scholar). The amino acid residue at position 20, in this case methionine, was changed to alanine (Met-20 → 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. 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 withXhoI and MluI and ligated to the pAE expression vector (8Junqueira de Azevedo I.L.M. Farsky S.H.P. Oliveira M.L.S. Ho P.L. J. Biol. Chem. 2001; 276: 39836-39842Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) 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 transformEscherichia 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 mmisopropyl-1-thio-β-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 8m urea, 0.05 m Tris-HCl, pH 8.0, and 0.005m 2-mercaptoethanol. The material was diluted 200 times in the refolding buffer (0.005 m imidazole, 0.5 mNaCl, 0.05 m Tris-HCl, pH 8.0, and 0.005 m2-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 mNaCl, 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 mmsodium-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 ε280 = 12,325 m−1 cm−1 based on the expected amino acid sequence of the recombinant protein. 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 [θ] 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. 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 −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. 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 (4Tendler M. Brito C.A. Vilar M.M. Serra-Freire N. Diogo C.M. Almeida M.S. Delbem A.C. Da Silva J.F. Savino W. Garratt R.C. Katz N. Simpson A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 269-273Crossref PubMed Scopus (189) Google Scholar, 9Tendler M. Pinto R.M. Oliveira Lima A. Gebara G. Katz N. Int. J. Parasitol. 1986; 16: 347-352Crossref PubMed Scopus (49) Google Scholar). 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 − 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 ttest (p < 0.05). 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) (10Breathnach R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Crossref PubMed Scopus (3292) Google Scholar). The exon nucleotide sequences were identical to that previously reported for Sm14 cDNA cloned from the Puerto Rican strain of S. mansoni (11Moser D. Tendler M. Griffiths G. Klinkert M.Q. J. Biol. Chem. 1991; 266: 8447-8454Abstract Full Text PDF PubMed Google Scholar). Similar to the other members of the FABP gene family, the gene for Sm14 contains four exons separated by three introns (12Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Annu. Rev. Nutr. 1997; 17: 277-303Crossref PubMed Scopus (195) Google Scholar). Recently, the presence and position of the introns in the related FABPgene from S. japonicum were also determined by PCR and corresponded exactly to that observed here for S. mansoni (Fig. 1B) (13Scott J.C. Kennedy M.W. McManus D.P. Biochim. Biophys. Acta. 2000; 1517: 53-62Crossref PubMed Scopus (19) Google Scholar). 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 (TableI) (12Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Annu. Rev. Nutr. 1997; 17: 277-303Crossref PubMed Scopus (195) Google Scholar). The size of the FABP introns ofS. japonicum, estimated by migration of PCR products in agarose gels (13Scott J.C. Kennedy M.W. McManus D.P. Biochim. Biophys. Acta. 2000; 1517: 53-62Crossref PubMed Scopus (19) Google Scholar), are larger than S. mansoni introns: ∼1200 versus 674; ∼850 versus 585; and ∼70versus 42 nucleotides for introns 1, 2, and 3 ofS. 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 (13Scott J.C. Kennedy M.W. McManus D.P. Biochim. Biophys. Acta. 2000; 1517: 53-62Crossref PubMed Scopus (19) Google Scholar) 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 ofS. 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 (10Breathnach R. Chambon P. Annu. Rev. Biochem. 1981; 50: 349-383Crossref PubMed Scopus (3292) Google Scholar), and therefore as previously postulated for S. japonicum F25 (13Scott J.C. Kennedy M.W. McManus D.P. Biochim. Biophys. Acta. 2000; 1517: 53-62Crossref PubMed Scopus (19) Google Scholar), we do not expect this event to occur frequently.Table ISizes of the introns and of the amino acids in the exons of several FABPsGeneAmino acid sequence length/exonIntron length1234123bpSm142358351767458542FABP-Sj1-aThe intron sizes of the S. japonicum FABP was estimated by PCR (13).23583516120085070Murine KLBP265834172300300500Rat L-FABP2258311614501220610Human I-FABP2258361612001020440Mouse mP22458341612002001300Mouse CRBPII24603416120018004001-a The intron sizes of the S. japonicum FABP was estimated by PCR (13Scott J.C. Kennedy M.W. McManus D.P. Biochim. Biophys. Acta. 2000; 1517: 53-62Crossref PubMed Scopus (19) Google Scholar). Open table in a new tab 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 (14Matarese V. Stone R.L. Waggoner D.W. Bernlohr D.A. Prog. Lipid Res. 1989; 28: 245-272Crossref PubMed Scopus (128) Google Scholar). 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 (15Wu Q. Andolfatto P. Haunerland N.H. Insect Biochem. Mol. Biol. 2001; 31: 553-562Crossref PubMed Scopus (29) Google Scholar) and several putative lipid-binding protein genes from the free-living nematode Caernorhabditis elegans do not have the intron 1 or possess additional introns (16Plenefisch J. Xiao H. Mei B. Geng J. Komuniecki P.R. Komuniecki R. Mol. Biochem. Parasitol. 2000; 105: 223-236Crossref PubMed Scopus (40) Google Scholar). 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 RicanS. mansoni strain (11Moser D. Tendler M. Griffiths G. Klinkert M.Q. J. Biol. Chem. 1991; 266: 8447-8454Abstract Full Text PDF PubMed Google Scholar), whereas the other 14 clones presented a single mutation M20T change (ATG→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 (17Richieri G.V. Low P.J. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1998; 273: 7397-7405Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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-ΔE3 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.Figure 4Schematic representation of the Sm14 genomic organization and of its splicing forms. The asteriskdenotes 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.View Large Image Figure ViewerDownload (PPT) Overall, almost all of the nucleotide sequences derived from theS. 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 Quı́mica, 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 (13Scott J.C. Kennedy M.W. McManus D.P. Biochim. Biophys. Acta. 2000; 1517: 53-62Crossref PubMed Scopus (19) Google Scholar). 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 (18Becker M.M. Kalinna B.H. Waine G.J. McManus D.P. Gene (Amst.). 1994; 148: 321-325Crossref PubMed Scopus (74) Google Scholar). In conclusion, our results indicate that Sm14 proteins in both BH and LE strains are less polymorphic than the orthologous S. japonicum FABPs. The sequence analysis of the obtained clones indicates the existence of two main isoforms ofS. mansoni FABP (Sm14-M20 and Sm14-T20). This polymorphism, besides the PCR clones from BH and LE strains, was also confirmed from isolated λ-cDNA library clones (11Moser D. Tendler M. Griffiths G. Klinkert M.Q. J. Biol. Chem. 1991; 266: 8447-8454Abstract Full Text PDF PubMed Google Scholar) from Puerto Rican strain. 2C. R. R. Ramos, M. M. Vilar, A. L. T. O. do Nascimento, M. Tendler, I. Raw, and P. L. Ho, unpublished results. 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) (2Esteves A. Joseph L. Paulino M. Ehrlich R. Int. J. Parasitol. 1997; 27: 1013-1023Crossref PubMed Scopus (32) Google Scholar, 12Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Annu. Rev. Nutr. 1997; 17: 277-303Crossref PubMed Scopus (195) Google Scholar). 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 (19Bordo D. Argos P. J. Mol. Biol. 1991; 217: 721-729Crossref PubMed Scopus (334) Google Scholar) 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 (17Richieri G.V. Low P.J. Ogata R.T. Kleinfeld A.M. J. Biol. Chem. 1998; 273: 7397-7405Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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. 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 β-structural elements in agreement with the prediction based on its primary sequence (4Tendler M. Brito C.A. Vilar M.M. Serra-Freire N. Diogo C.M. Almeida M.S. Delbem A.C. Da Silva J.F. Savino W. Garratt R.C. Katz N. Simpson A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 269-273Crossref PubMed Scopus (189) Google Scholar). 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 β-barrel structure, they may have different folding and unfolding intermediates (20Dalessio P.M. Ropson I.J. Biochemistry. 2000; 39: 860-871Crossref PubMed Scopus (76) Google Scholar). 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). 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 (21Kennedy M.W. Brass A. McCruden A.B. Price N.C. Kelly S.M. Cooper A. Biochemistry. 1995; 34: 6700-6710Crossref PubMed Scopus (89) Google Scholar). 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 (22Macgregor R.B. Weber G. Nature. 1986; 319: 70-73Crossref PubMed Scopus (223) Google Scholar)) 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 (23Zimmerman A.W. van Moerkerk H.T. Veerkamp J.H. Int. J. Biochem. Cell Biol. 2001; 33: 865-876Crossref PubMed Scopus (88) Google Scholar). The liver FABP and the intestinal FABP produce a shift to 500 and 496 nm, respectively (23Zimmerman A.W. van Moerkerk H.T. Veerkamp J.H. Int. J. Biochem. Cell Biol. 2001; 33: 865-876Crossref PubMed Scopus (88) Google Scholar), values closer to what is observed for S. japonicum F10 FABP (24Kennedy M.W. Scott J.C. Lo S. Beauchamp J. McManus D.P. Biochem. J. 2000; 349: 377-384Crossref PubMed Scopus (32) Google Scholar). 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 (12Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Annu. Rev. Nutr. 1997; 17: 277-303Crossref PubMed Scopus (195) Google Scholar), and the rSm14 variants exhibit quite similarKd 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 (12Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Annu. Rev. Nutr. 1997; 17: 277-303Crossref PubMed Scopus (195) Google Scholar) 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 (25Balendiran G.K. Schnutgen F. Scapin G. Borchers T. Xhong N. Lim K. Godbout R. Spener F. Sacchettini J.C. J. Biol. Chem. 2000; 275: 27045-27054Abstract Full Text Full Text PDF PubMed Google Scholar). 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.Figure 7Displacement 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.View Large Image Figure ViewerDownload (PPT)Figure 8Displacement 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).View Large Image Figure ViewerDownload (PPT) The protection efficacy of the three rSm14 proteins against S. mansoni cercariae was evaluated in outbred Swiss mice as described previously (4Tendler M. Brito C.A. Vilar M.M. Serra-Freire N. Diogo C.M. Almeida M.S. Delbem A.C. Da Silva J.F. Savino W. Garratt R.C. Katz N. Simpson A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 269-273Crossref PubMed Scopus (189) Google Scholar). 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) (2Esteves A. Joseph L. Paulino M. Ehrlich R. Int. J. Parasitol. 1997; 27: 1013-1023Crossref PubMed Scopus (32) Google Scholar) is buried within the interior of the molecule like in the other FAPBs (2Esteves A. Joseph L. Paulino M. Ehrlich R. Int. J. Parasitol. 1997; 27: 1013-1023Crossref PubMed Scopus (32) Google Scholar, 12Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Annu. Rev. Nutr. 1997; 17: 277-303Crossref PubMed Scopus (195) Google Scholar, 23Zimmerman A.W. van Moerkerk H.T. Veerkamp J.H. Int. J. Biochem. Cell Biol. 2001; 33: 865-876Crossref PubMed Scopus (88) Google Scholar, 25Balendiran G.K. Schnutgen F. Scapin G. Borchers T. Xhong N. Lim K. Godbout R. Spener F. Sacchettini J.C. J. Biol. Chem. 2000; 275: 27045-27054Abstract Full Text Full Text PDF PubMed Google Scholar) 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 (3Bergquist N.R. Colley D.G. Parasitol. Today. 1998; 14: 99-104Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), 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. 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.