Molecular and Biochemical Parasitology, 49 (1991) 177 180 © 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 A D O N I S 016668519100404T
177
MOLBIO 01627
Short Communication
Another 26-kilodalton glutathione S-transferase of Schistosoma mansoni M a r k D. W r i g h t ~, R o b e r t A. H a r r i s o n 1., A n g e l a M. M e l d e r 1, G e o r g e R. N e w p o r t 2 a n d G r a h a m F. Mitchell ~ I The Walter and Eliza Hall Institute o f Medical Research, Melbourne, Australia and 2School o f Public Health, University o f California, San Francisco, CA, U.S.A. Key words: Sch&tosoma mansoni; Glutathione S-transferase; Schistosomiasis
The glutathione S-transferases (GSTs) have potential as components of a vaccine against schistosomiasis [1]. Immunochemical analyses have revealed that both Schistosoma mansoni and Schistosoma japonicum have at least two GST isoenzymes, of 26 and 28 kDa (Sm26, Sm28, Sj26 and Sj28) [2]. Sm28 has induced impressive protection against schistosomiasis mansoni in a variety of hosts [3], whereas protection of mice against schistosomiasis japonica using Sj26 has been inconsistent [4]. Recently, two groups have independently reported the isolation of cDNA clones encoding the apparent S. mansoni homologue of Sj26 [5-6] (hereafter referred to as Sm26/1). Sm26/1 and Sj26 share an 81% identity at the amino acid level. Here we report the identification of a cDNA clone (Sm26/2) encoding a 26-kDa GST of S. mansoni distinct from Sm26/1. We also present Southern blot data which suggest Correspondence address: Graham F. Mitchell, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia. *Present address: Cairo, Egypt.
U.S. Naval Medical Research Unit No. 3,
Note: Nucleotide sequence data described in this paper have been submitted to the GenBank T M data base with the accession number M73624. Abbreviations: GST, Glutathione S-transferase; Sm28, Sj28, 28 kDa GST of S. mansoni and S. japonicum; Sm26/1, Sm26/2, Sj26, 26 kDa GST of S. mansoni and S. japonicum; SSC, saline sodium citrate.
that genes encoding both Sm26/1 and Sm26/2 are present within the genome of the S. mansoni isolate maintained in the Melbourne laboratory. Sm26/2 cDNA clones were identified in the San Francisco laboratory by screening an S. mansoni 2gtl 1 adult worm cDNA library using a cDNA probe encoding Sj26 [7]. The cDNA encoding Sm26/2 was isolated from purified 2gtll bacteriophage DNA by using the polymerase chain reaction [8] using 30-mer oligonucleotides complementary for sequences located 5' relative to the EcoRI sites on either 2gtll DNA strand. The resulting DNA fragment was subcloned into M13mpl0 for DNA sequencing [9]. The deduced protein sequence of Sm26/2 is shown in comparison with the published sequences of both Sm26/1 and Sj26 in Fig. 1. Sm26/1 and Sm26/2 are 82% identical whereas Sm26/1 and Sj26 share an 81% identity. Sm26/2 is 83% identical to the S. japonicum sequence. It is somewhat~ surprising that the two S. mansoni sequences (Sm26/1 and Sm26/2) are as closely related to the S. japonicum (Sj26) sequence as they are to each other. It is also apparent that the two potential sites for asparagine-linked glycosylation in Sm26/1 (amino acids 139-141 and 194-196) are not conserved in the Sm26/2 sequence. The question arises as to the relationship between Sm26/1 and Sm26/2; do these two distinct 26-kDa GSTs represent variants existing in different isolates of S. mansoni, or do
178
50 S
Sj26 SM26/2 Sm26/i
Sj26 Sm26/2 Sm26/i
ILGYWKIKGLVQPTRLLLEYLEEK
H
E
D
MAPKLGYWKIKGLVQPTRLLLEYLr~ER ~
G
Y W K~K
G L V Q P T R L L L E~L
K
K
V
E
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GLEF E E T]Y E E E RIAIY D R N E[I]D~AW~SN~JDIK F K L
70 80 GDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSR
90
100
YY
I
YY
I 120
IAYNKEFETLK
GDVKLTQSMAI~RYIADKHNMLGGCPKERAEISMLEGA~LDIRYGVSR
140
P N LP
1 i0
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~ K L T Q S M A I I R Y I A D K H N M L G ~ C P K E R A E I S M L E G A V L D I R ~ G V L R 130
60
EL
15
0
160
170
180
Sj26 Sm26/2 Sm26/1
LPGMLKMFEDRLSH
190 E~N L P ~ I
HPDFMLYDALDVVLYMDPK
TYLNGDKVT
IDFLNKLPGNLKMFEDRLS~KTYLNG
CVT 200
K N Y L N S~R
HPDFMLYDALDVVLYMD
N Q
E
210
Sm26/2
~5I
Sm26/I
K ~ C I~__~DLL P Q I K N Y L N S S R Y I K W P L Q G W D A T F G G G D T P P
Y I K W P L Q G WlSIA T F G G G D A P P
Fig. h Comparison of the predicted amino acid sequences of Sj26, Sm26/1, and Sm26/2. Amino acid residues conserved between the 26-kDa schistosome GSTs are boxed.
genes encoding both the Sm26/2 and Sm26/1 sequences exist within the one parasite isolate? S. mansoni genomic DNA was restricted with both EcoRI and HindIII and probed with the radiolabelled Sm26/1 and Sm26/2 cDNAs (Fig. 2). Sm26/1 strongly hybridised to a 2.4kb fragment and weakly hybridised to a fragment of 3.9 kb. Conversely, Sm26/2 hybridised strongly to the 3.9-kb fragment and less strongly to the 2.4-kb fragment. It is therefore likely that genes encoding both
A
B
3.92.4-
Fig. 2. Southern blot analysis of S. mansoni genomic D N A restricted with EcoRI and HindlII and probed with: (A) Sm26/ 2 eDNA; and (B) Sm26/1 cDNA. Filters were washed with 2 × SSC at 65°C.
Sm26/1 and Sm26/2 exist in the genome of the S. mansoni isolate maintained in the Melbourne laboratory. Thus, the 26-kDa GSTs, at least in S. mansoni, may be encoded by a multigene family. However, the possibility that Sm26/1 and Sm26/2 represent different alleles of a single gene locus can not be discounted without further genetic analyses using DNA obtained from single worms. In situ staining of adult worm GSTs, after electrophoretic separation, predicted two isoenzymes in S. japonicum and three in S. mansoni [10]. Whether the three isoenzymes detected in these previous studies represent Sm28, Sm26/1 and Sm26/2 is not known. The possible existence of two different 26kDa GSTs within the one S. mansoni parasite may have an important bearing on GSTinduced immunity. One explanation for the failure of Sm26/1 vaccination (Wright, Melder and Mitchell, submitted for publication), assuming a role for enzyme-neutralising antibodies, is that we have failed to elicit a neutralising response against the other 26kDa GST - Sm26/2. It will be interesting to determine whether a vaccine formulation that includes the three S. mansoni GSTs so far
179
discovered (i.e., Sm28, Sm26/2 and Sm26/1) will prove to be more successful than the experimental GST vaccines examined to date in the Melbourne laboratory.
Acknowledgements We wish to thank Vanessa Herrmann, Karen McLeod and Susan Wood for technical assistance and Kathy Davern for her administrative skills. Work in this laboratory is supported by the Australian National Health and Medical Research Council, the World Health Organisation (TDR)-Rockefeller Foundation Health Sciences for the Tropics Partnership in Research, the Australian International Development Assistance Bureau (AIDAB) and the Edna McConnell Clark Foundation.
References 1 Mitchell, G.F. (1989) Glutathione S-transferases. Potential components of anti-schistosome vaccines? Parasitol. Today 5, 3-437. 2 Tiu, W.U., Davern, K.M., Wright, M.D., Board P.G. and Mitchell, G.F. (1988) Molecular and serological characteristics of the glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni. Parasite lmmunol. 10, 693 706. 3 Balloul, J.M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J.M., Pierce, R.J., Carvallo, D., Lecocq,
J.P. and Capron, A. (1987) Molecular cloning of a protective antigen of schistosomes. Nature 326, 149153. 4 Mitchell, G.F., Garcia, E.G., Davern, K.M., Tiu W.U. and Smith, D.B. (1988) Sensitization against the parasite antigen Sj26 is not sufficient for consistent expression of resistance to Schistosoma japonicum in mice. Trans. R. Soc. Trop. Med. Hyg. 82, 885-889. 5 Henkle, K.J., Davern, K.M., Wright, M.D., Ramos A.J. and Mitchell, G.F. (1990) Comparison of the cloned genes of the 26- and 28-kilodalton glutathione Stransferases of Schistosoma japonicum and Schistosoma mansoni. Mol. Biochem. Parasitol. 40, 23 34. 6 Trottein, F., Kieny, M.P., Verwaerde, C., Torpier, G., Pierce, R.J., Balloul, J.M., Schmitt, D., Lecocq, J.P. and Capron, A. (1990) Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase. Mol. Biochem. Parasitol. 41, 35 44. 7 Smith, D.B., Rubira, M.R., Simpson, R.J., Davern, K.M., Tiu, W.U., Board, P.G. and Mitchell, G.F. (1988) Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase. Mol. Biochem. Parasitol. 27, 249 256. 8 Saiki, R.N., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuichi, R., Horn, G.J., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. 9 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 10 Smith, D.B., Davern, K.M., Board, P.G., Tiu, W.U., Garcia, E.G. and Mitchell, G.F. (1986) Mr 26000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione Stransferase. Proc. Natl. Acad. Sci. USA 83, 8703-8707.
177
MOLBIO 01627
Short Communication
Another 26-kilodalton glutathione S-transferase of Schistosoma mansoni M a r k D. W r i g h t ~, R o b e r t A. H a r r i s o n 1., A n g e l a M. M e l d e r 1, G e o r g e R. N e w p o r t 2 a n d G r a h a m F. Mitchell ~ I The Walter and Eliza Hall Institute o f Medical Research, Melbourne, Australia and 2School o f Public Health, University o f California, San Francisco, CA, U.S.A. Key words: Sch&tosoma mansoni; Glutathione S-transferase; Schistosomiasis
The glutathione S-transferases (GSTs) have potential as components of a vaccine against schistosomiasis [1]. Immunochemical analyses have revealed that both Schistosoma mansoni and Schistosoma japonicum have at least two GST isoenzymes, of 26 and 28 kDa (Sm26, Sm28, Sj26 and Sj28) [2]. Sm28 has induced impressive protection against schistosomiasis mansoni in a variety of hosts [3], whereas protection of mice against schistosomiasis japonica using Sj26 has been inconsistent [4]. Recently, two groups have independently reported the isolation of cDNA clones encoding the apparent S. mansoni homologue of Sj26 [5-6] (hereafter referred to as Sm26/1). Sm26/1 and Sj26 share an 81% identity at the amino acid level. Here we report the identification of a cDNA clone (Sm26/2) encoding a 26-kDa GST of S. mansoni distinct from Sm26/1. We also present Southern blot data which suggest Correspondence address: Graham F. Mitchell, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia. *Present address: Cairo, Egypt.
U.S. Naval Medical Research Unit No. 3,
Note: Nucleotide sequence data described in this paper have been submitted to the GenBank T M data base with the accession number M73624. Abbreviations: GST, Glutathione S-transferase; Sm28, Sj28, 28 kDa GST of S. mansoni and S. japonicum; Sm26/1, Sm26/2, Sj26, 26 kDa GST of S. mansoni and S. japonicum; SSC, saline sodium citrate.
that genes encoding both Sm26/1 and Sm26/2 are present within the genome of the S. mansoni isolate maintained in the Melbourne laboratory. Sm26/2 cDNA clones were identified in the San Francisco laboratory by screening an S. mansoni 2gtl 1 adult worm cDNA library using a cDNA probe encoding Sj26 [7]. The cDNA encoding Sm26/2 was isolated from purified 2gtll bacteriophage DNA by using the polymerase chain reaction [8] using 30-mer oligonucleotides complementary for sequences located 5' relative to the EcoRI sites on either 2gtll DNA strand. The resulting DNA fragment was subcloned into M13mpl0 for DNA sequencing [9]. The deduced protein sequence of Sm26/2 is shown in comparison with the published sequences of both Sm26/1 and Sj26 in Fig. 1. Sm26/1 and Sm26/2 are 82% identical whereas Sm26/1 and Sj26 share an 81% identity. Sm26/2 is 83% identical to the S. japonicum sequence. It is somewhat~ surprising that the two S. mansoni sequences (Sm26/1 and Sm26/2) are as closely related to the S. japonicum (Sj26) sequence as they are to each other. It is also apparent that the two potential sites for asparagine-linked glycosylation in Sm26/1 (amino acids 139-141 and 194-196) are not conserved in the Sm26/2 sequence. The question arises as to the relationship between Sm26/1 and Sm26/2; do these two distinct 26-kDa GSTs represent variants existing in different isolates of S. mansoni, or do
178
50 S
Sj26 SM26/2 Sm26/i
Sj26 Sm26/2 Sm26/i
ILGYWKIKGLVQPTRLLLEYLEEK
H
E
D
MAPKLGYWKIKGLVQPTRLLLEYLr~ER ~
G
Y W K~K
G L V Q P T R L L L E~L
K
K
V
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L GL[~]FPNLP
GLEF E E T]Y E E E RIAIY D R N E[I]D~AW~SN~JDIK F K L
70 80 GDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSR
90
100
YY
I
YY
I 120
IAYNKEFETLK
GDVKLTQSMAI~RYIADKHNMLGGCPKERAEISMLEGA~LDIRYGVSR
140
P N LP
1 i0
IAYNKE~ETLK
~ K L T Q S M A I I R Y I A D K H N M L G ~ C P K E R A E I S M L E G A V L D I R ~ G V L R 130
60
EL
15
0
160
170
180
Sj26 Sm26/2 Sm26/1
LPGMLKMFEDRLSH
190 E~N L P ~ I
HPDFMLYDALDVVLYMDPK
TYLNGDKVT
IDFLNKLPGNLKMFEDRLS~KTYLNG
CVT 200
K N Y L N S~R
HPDFMLYDALDVVLYMD
N Q
E
210
Sm26/2
~5I
Sm26/I
K ~ C I~__~DLL P Q I K N Y L N S S R Y I K W P L Q G W D A T F G G G D T P P
Y I K W P L Q G WlSIA T F G G G D A P P
Fig. h Comparison of the predicted amino acid sequences of Sj26, Sm26/1, and Sm26/2. Amino acid residues conserved between the 26-kDa schistosome GSTs are boxed.
genes encoding both the Sm26/2 and Sm26/1 sequences exist within the one parasite isolate? S. mansoni genomic DNA was restricted with both EcoRI and HindIII and probed with the radiolabelled Sm26/1 and Sm26/2 cDNAs (Fig. 2). Sm26/1 strongly hybridised to a 2.4kb fragment and weakly hybridised to a fragment of 3.9 kb. Conversely, Sm26/2 hybridised strongly to the 3.9-kb fragment and less strongly to the 2.4-kb fragment. It is therefore likely that genes encoding both
A
B
3.92.4-
Fig. 2. Southern blot analysis of S. mansoni genomic D N A restricted with EcoRI and HindlII and probed with: (A) Sm26/ 2 eDNA; and (B) Sm26/1 cDNA. Filters were washed with 2 × SSC at 65°C.
Sm26/1 and Sm26/2 exist in the genome of the S. mansoni isolate maintained in the Melbourne laboratory. Thus, the 26-kDa GSTs, at least in S. mansoni, may be encoded by a multigene family. However, the possibility that Sm26/1 and Sm26/2 represent different alleles of a single gene locus can not be discounted without further genetic analyses using DNA obtained from single worms. In situ staining of adult worm GSTs, after electrophoretic separation, predicted two isoenzymes in S. japonicum and three in S. mansoni [10]. Whether the three isoenzymes detected in these previous studies represent Sm28, Sm26/1 and Sm26/2 is not known. The possible existence of two different 26kDa GSTs within the one S. mansoni parasite may have an important bearing on GSTinduced immunity. One explanation for the failure of Sm26/1 vaccination (Wright, Melder and Mitchell, submitted for publication), assuming a role for enzyme-neutralising antibodies, is that we have failed to elicit a neutralising response against the other 26kDa GST - Sm26/2. It will be interesting to determine whether a vaccine formulation that includes the three S. mansoni GSTs so far
179
discovered (i.e., Sm28, Sm26/2 and Sm26/1) will prove to be more successful than the experimental GST vaccines examined to date in the Melbourne laboratory.
Acknowledgements We wish to thank Vanessa Herrmann, Karen McLeod and Susan Wood for technical assistance and Kathy Davern for her administrative skills. Work in this laboratory is supported by the Australian National Health and Medical Research Council, the World Health Organisation (TDR)-Rockefeller Foundation Health Sciences for the Tropics Partnership in Research, the Australian International Development Assistance Bureau (AIDAB) and the Edna McConnell Clark Foundation.
References 1 Mitchell, G.F. (1989) Glutathione S-transferases. Potential components of anti-schistosome vaccines? Parasitol. Today 5, 3-437. 2 Tiu, W.U., Davern, K.M., Wright, M.D., Board P.G. and Mitchell, G.F. (1988) Molecular and serological characteristics of the glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni. Parasite lmmunol. 10, 693 706. 3 Balloul, J.M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J.M., Pierce, R.J., Carvallo, D., Lecocq,
J.P. and Capron, A. (1987) Molecular cloning of a protective antigen of schistosomes. Nature 326, 149153. 4 Mitchell, G.F., Garcia, E.G., Davern, K.M., Tiu W.U. and Smith, D.B. (1988) Sensitization against the parasite antigen Sj26 is not sufficient for consistent expression of resistance to Schistosoma japonicum in mice. Trans. R. Soc. Trop. Med. Hyg. 82, 885-889. 5 Henkle, K.J., Davern, K.M., Wright, M.D., Ramos A.J. and Mitchell, G.F. (1990) Comparison of the cloned genes of the 26- and 28-kilodalton glutathione Stransferases of Schistosoma japonicum and Schistosoma mansoni. Mol. Biochem. Parasitol. 40, 23 34. 6 Trottein, F., Kieny, M.P., Verwaerde, C., Torpier, G., Pierce, R.J., Balloul, J.M., Schmitt, D., Lecocq, J.P. and Capron, A. (1990) Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase. Mol. Biochem. Parasitol. 41, 35 44. 7 Smith, D.B., Rubira, M.R., Simpson, R.J., Davern, K.M., Tiu, W.U., Board, P.G. and Mitchell, G.F. (1988) Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase. Mol. Biochem. Parasitol. 27, 249 256. 8 Saiki, R.N., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuichi, R., Horn, G.J., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. 9 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 5467. 10 Smith, D.B., Davern, K.M., Board, P.G., Tiu, W.U., Garcia, E.G. and Mitchell, G.F. (1986) Mr 26000 antigen of Schistosoma japonicum recognized by resistant WEHI 129/J mice is a parasite glutathione Stransferase. Proc. Natl. Acad. Sci. USA 83, 8703-8707.
