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

Abstract

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 the protein was dialyzed against 10 pH The protein was by by The of the recombinant proteins were from at an of based on the amino acid sequence of the recombinant unfolding as a function of was by the unfolding as a function of was by spectroscopy. CD were carried out on a at 20 with a for CD were using a cell at over the from to were for sample and from the The protein was at 10 in 10 buffer as The was from to °C. The of structure was by the of at were with a II with in a The protein was and after of urea, the sample was at 20 were The of the of the two was the at nm and the in the in the of The fatty acid of the three recombinant Sm14 proteins was by following the in by the of the fatty acid 11-(dansylamino)undecanoic acid (DAUDA) obtained from Molecular The used for DAUDA was A of 10 DAUDA in in the at was diluted in to 1 or in the The protein was in 1 ml of at 20 °C. data were for the without and by standard to a single binding to the and experiments were designed to a difference in affinity of the three proteins for the various natural fatty The and acids obtained from were and diluted as for In these the binding of DAUDA to protein was performed in the or of of fatty The three recombinant rSm14-T20, and were for vaccine efficacy in female as 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 M. Oliveira A. Katz N. Int. J. Parasitol. PubMed Scopus Google Scholar). The three with of recombinant proteins in in as were with The were with cercariae after the and The protection was by the is the of worms in and is the of worms in analysis was with The genomic sequence of Sm14 was by PCR using DNA isolated from a of adult worms of the Brazilian endemic S. mansoni BH The is than that from the of not The of genomic DNA was cloned and sequenced from two independent PCR clones The sequence analysis the sites of All of the the R. P. Biochem. PubMed Scopus Google Scholar). The nucleotide sequences were to that previously for Sm14 cDNA cloned from the strain of S. mansoni D. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). to the other of the gene the gene for Sm14 by three Simpson 1997; PubMed Scopus Google Scholar). Recently, the and position of the in the from S. were also by PCR and to that for S. mansoni 2000; PubMed Scopus Google Scholar). The of and showed that the and positions of are conserved among these the of the is Simpson 1997; PubMed Scopus Google Scholar). The of the japonicum, by of PCR products in agarose gels 2000; PubMed Scopus Google are than S. mansoni and for and 3 and S. mansoni, respectively. The 3 of S. mansoni is the of all of the found in the gene family Recently, a variant of the S. was characterized by cDNA 2000; PubMed Scopus Google Scholar) the for the first amino acids in were into the in nucleotide sequence of the of S. mansoni and S. japonicum, we that in S. japonicum, the of a second for the 1 at the of the the This same also be used to an S. mansoni variant After of 1 using this the first amino acids of be all of the as in S. However, this second for 1 the sequence in the R. P. Biochem. PubMed Scopus Google and as previously for S. 2000; PubMed Scopus Google we not this to of the and of the amino acids in the of acid sequence of the S. was by PCR The of the S. was by PCR 2000; PubMed Scopus Google Scholar). in a the obtained Sm14 genomic sequence was with the acid sequences of other of the gene the of of the three was found to be shown in the first a after first nucleotide in to the other which are after the PubMed Scopus Google Scholar). However, is that this conserved gene present a In the the gene not P. Biochem. Mol. Biol. 2001; PubMed Scopus Google Scholar) and protein from the not have the 1 or additional J. J. R. Mol. Biochem. Parasitol. 2000; PubMed Scopus Google Scholar). cDNA clones by independent reverse-transcribed PCR and cDNA from two Brazilian S. mansoni strains were A of the amino acid sequences for the coding regions of all of the obtained Sm14 cDNA clones total of and cDNAs from LE and BH is shown in clones showed a sequence to the Sm14 protein cloned from mansoni strain D. M. J. Biol. Chem. Full Text PDF PubMed Google the other clones presented a single mutation M20T change Interestingly, methionine at this position is conserved among the of protein In the and an important role for this amino acid residue in binding function was J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Two and the mutation and are considered to represent Sm14 Two clones as by with the genomic Sm14 sequence showed the of the amino acid sequence to the 3 and they were by the reverse-transcribed PCR analysis showed a single to the Sm14 open reading is to be a of the not clones → at the which is at of the first of the Sm14 gene not The clones had single in positions the amino acids were conserved and are by one These clones have been by PCR and for this reason, they were not in this the forms of Sm14 of the Sm14 genomic and of The the that in S. mansoni be for the expression of a reduced of Sm14 to all of the nucleotide sequences derived from mansoni LE strain showed methionine at position 20 and 1 in the case of the clones obtained from one BH strain (Instituto Butantan, São Paulo, clones had the threonine at this In addition, the cDNA library derived from the BH strain from de Quı́mica, Universidade de São Paulo, São Paulo, Brazil) clones with threonine and three clones with methionine at position The polymorphism of the protein was also characterized in strains of S. 2000; PubMed Scopus Google Scholar). In this the cDNA clones obtained had and of was to the previously cDNA from the strain of S. M.M. PubMed Scopus Google Scholar). In indicate that Sm14 proteins in both BH and LE strains are than the S. The sequence analysis of the obtained clones the existence of two isoforms mansoni (Sm14-M20 and Sm14-T20). This polymorphism, besides the PCR clones from BH and LE was also confirmed from isolated library clones D. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar) from R. R. M. M. A. L. T. M. and P. L. To the structural and functional of these we to this amino acid position was conserved in the family using the sequence previously not (2Esteves A. Joseph L. Paulino M. Ehrlich R. Int. J. Parasitol. 1997; 27: 1013-1023Crossref PubMed Scopus (32) Google Simpson 1997; PubMed Scopus Google Scholar). The methionine at this position is changed for in a or for in proteins of and These with the of residue D. P. J. Mol. Biol. PubMed Scopus Google Scholar) can be by or without the protein structure and These the for position 20 in the protein The is the is out that the methionine residue at this position appears to play an important role in the structural and functional of these In fact, the mutagenesis of this residue in two in and in proteins characterized by a of structural stability as as their affinity for fatty acids J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). on these evidence and the of the threonine is from the methionine in terms of and we to significant the two isoforms of The recombinant His6-tagged Sm14 proteins and were and purified using a affinity additional mutant was also constructed to better evaluate the importance of this amino acid residue in the Sm14 protein with of were for rSm14-M20 and rSm14-T20 to analysis as as for rSm14-A20 protein not that the three proteins present a CD of a protein a structure containing in with the based on 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). the CD in the activity of the three that a single mutation at position 20 their structural The stability of the protein was both by CD and spectroscopy. the of unfolding of the three The we are in the of a by the CD obtained after the the is not The structure was higher for the rSm14-M20 by rSm14-T20 and rSm14-A20 and The CD at a were the at nm had reached a they that a protein a at the of is to that the that fatty acid-binding proteins have a β-barrel they have and unfolding 2000; PubMed Scopus Google Scholar). the of the CD at an the of unfolding of protein variant has been that the rSm14-M20 isoform was more stable than rSm14-T20 isoform and rSm14-A20 mutant and The stability of the three has also been by following the change of their as a function of of The data confirmed that rSm14-M20 has a higher structural stability not The fatty acid binding of the three proteins was by using the fatty acid DAUDA that and on into binding proteins A. A. PubMed Scopus Google Scholar). The of DAUDA from to and nm for rSm14-T20, and not and the of of the of DAUDA into an PubMed Scopus Google confirmed the binding of the fatty acid to the three These are with the obtained from structurally as the and the to and Int. J. Biochem. Biol. 2001; PubMed Scopus Google Scholar). The and the a to and Int. J. Biochem. Biol. 2001; PubMed Scopus Google to is for S. S. J. Biochem. J. 2000; PubMed Scopus (32) Google Scholar). the in the that in are with and this their difference in affinity. Interestingly, the of the binding of DAUDA to the three a of to of the Simpson 1997; PubMed Scopus Google and the and for rSm14-T20, and To the in the binding of the three Sm14 proteins for other fatty acids, we performed competitive experiments using DAUDA as and natural fatty acids as The of and acids a affinity of the three for these by the in the of the of a competitive acid is used to DAUDA from The of fatty acid to DAUDA from the binding is shown in A of the of The data that the rSm14-M20 has the higher affinity for all of the natural fatty the indicate that the in the of DAUDA with the of both the fatty acid and the of Simpson 1997; PubMed Scopus Google Scholar) as for other the importance of the of the amino acid residue at position 20 in is to that in the case of the acid is not able to DAUDA from the other an of affinity of the mutant for this relatively fatty A to the and 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 structure of human that that fatty acids are able to with T. N. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar). on this we can that of DAUDA not be able to with residue that be present in position 20, thus the affinity exhibited by the three proteins for the fatty acid the other the experiments fatty acids have been used the existence of that are to the fatty acid and the residue at position 20 of the these indicate that the of the amino acid at position 20 is important for structure stability and for the binding to fatty of DAUDA by acid in the binding to binding experiments were carried out using DAUDA in the of acid and of DAUDA binding to proteins by natural fatty The of is The same as for were used for protein fatty acid in the of DAUDA The protection efficacy of the three proteins against S. mansoni cercariae was in as 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). shown in rSm14-T20 or rSm14-M20 proteins a significant protective response and with these proteins showed a in with to or higher than the by the as a for an antigen to be considered a vaccine candidate against human The is the rSm14-A20 mutant that out to be a protective protection of of Interestingly, in the Sm14, the amino acid at position 20 as shown by not (2Esteves A. Joseph L. Paulino M. Ehrlich R. Int. J. Parasitol. 1997; 27: 1013-1023Crossref PubMed Scopus (32) Google Scholar) is the of the in the other (2Esteves A. Joseph L. Paulino M. Ehrlich R. Int. J. Parasitol. 1997; 27: 1013-1023Crossref PubMed Scopus (32) Google Simpson 1997; PubMed Scopus Google Int. J. Biochem. Biol. 2001; PubMed Scopus Google T. N. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar) is not to be as an the data the importance of this residue in structural and functional of the lack of significant protective for rSm14-A20 is the of these than the change of the The rSm14-A20 was the more and Sm14 protein not The rSm14-M20 showed the structure after to by rSm14-T20 and was more to by urea as by of not These with the level of protective In fact, the rSm14-T20 the of the in a protective response to that for rSm14-A20 thus that for immune Sm14 has to the This is an important for the development of a vaccine (3Bergquist N.R. Colley D.G. Parasitol. Today. 1998; 14: 99-104Abstract Full Text Full Text PDF PubMed Scopus (184) Google and that a that into the structure of the protein has to be in the of In the data presented for the first time the genomic of the Sm14 protein and the existence of A of the structural and functional of of these has that the Sm14-M20 characterized by a higher structural stability and by a more affinity for natural fatty acids, appears to be the more antigen for the development of the schistosomiasis vaccine. This for the vaccine development. In particular, we that the stability of the protein be a can provide a better immune response to the of the We Dr. for the S. mansoni cDNA library and Dr. for the adult worms of the S. mansoni BH