Molecular and Biochemical Parasitology, 40 (1990) 23-34 Elsevier

23

MOLBIO 01307

Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni Kimberly J. Henkle, Kathy M. Davern, Mark D. Wright, Anthea J. Ramos and Graham F. Mitchell The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia

(Received 21 August 1989; accepted 31 October 1989)

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivityusing rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sin26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes. Key words: Glutathione S-transferase isoenzyme; Schistosomiasis; Mice; Immunogenicity; Sequence; Homology

Introduction The glutathione-S-transferases (GSTs) have p o t e n t i a l as c o m p o n e n t s o f a v a c c i n e against s c h i s t o s o m i a s i s [1-4]. B o t h Schistosoma ]aponic u m a n d S. m a n s o n i w o r m s c o n t a i n G S T s o f 26 Correspondence address: G.F. Mitchell, Immunoparasitology Unit, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia. Note: Nueleotide sequence data reported in this paper have been submitted to the EMBL, GenBank T M and DDBJ data bases with the accession numbers M26913 and M26914. Abbreviations: aa, amino acid; AWE, adult worm extract; FCA, Freund's Complete Adjuvant; GST, glutathione Stransferase; rSj26, near-native recombinant Sj26; rSm28, expressed recombinant Sm28; Sj26FP, Sj26-13-galactosidase fusion protein; Sm26, Sj26, Sm28, Sj28, 26 and 28-kDa glutathione S-transferases of S. japonicum and S. mansoni; WEHI, Walter and Eliza Hall Institute of Medical Research.

(Sj26 a n d Sm26) a n d 28 k D a (Sj28 a n d Sm28) which can b e r e s o l v e d b y p o l y a c r y l a m i d e gel electrophoresis. I n situ enzymatic staining o f adult w o r m extracts s e p a r a t e d b y e l e c t r o p h o r e s i s s h o w e d t h a t t h e r e a r e at least t w o i s o e n z y m e s o f G S T in S. j a p o n i c u m a n d at least t h r e e in S. mansoni [5]. W e h a v e f o u n d t h a t a l t h o u g h b o t h Sj26 a n d Sm26 can b e i m m u n o g e n i c d u r i n g schistos o m e infection in mice [5,6], t h e r e is an a p p a r e n t d i f f e r e n c e in t h e i m m u n o g e n i c i t y o f t h e 2 8 - k D a G S T s o f S. j a p o n i c u m a n d S. m a n s o n i w o r m s . Sm28 can b e a s t r o n g i m m u n o g e n in mice d u r i n g infection, w h e r e a s Sj28 is a w e a k i m m u n o g e n in these circumstances and antibody responses are b a r e l y d e t e c t a b l e . A n t i b o d i e s to Sj26 a n d Sm26 cross-react extensively, as d o antibodies to the 28kDa molecules, no cross-reactivity yet having b e e n d e t e c t e d b e t w e e n t h e two M r species [5]. W e h a v e i s o l a t e d c D N A s e n c o d i n g Sj28 a n d Sm26 a n d c o m p a r e d t h e i r n u c l e o t i d e s e q u e n c e s

0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

24

and predicted primary protein structure with published Sm28 and Sj26 sequences. Since some T cell epitopes have been located in Sm28 [7], these regions in particular were examined in Sj28 in order to gain insight into differential immunogenicity. Surface proteins of S. japonicum and S. mansoni adult worms were labeled to determine whether all GSTs have a surface location as has been reported for Sm28 [2]. In addition, the predicted protein sequences of the 4 GSTs were compared to determine whether there are domains in common that may play a role in shared GST functions such as glutathione binding. Materials and Methods

Parasites and antigen preparations. Adult S. japonicum (Philippines, Sorsogon strain) and S. mansoni (Puerto Rican strain) were collected from BALB/c mice by portal perfusion. These mice were infected with cercaria from the life cycles maintained at The Walter and Eliza Hall Institute (WEHI) [8]. S. japonicum and S. mansoni GSTs were purified from homogenates of adult worms by elution from glutathione (GSH)-agarose beads (Sigma, U.S.A.) [3,5]. Living S. japonicum and S. mansoni adult worms, collected from BALB/c mice by perfusion, were washed three times in cold PBS to remove debris and then rinsed in 0.1 M borate buffer, pH 8.5. These parasites were then iodinated using the Bolton and Hunter reagent (Amersham, U.K.) as previously described [9]. After the labeling procedure, the worms were checked microscopically and showed no evidence of gross damage. Labeled proteins were extracted in TNET buffer (0.5% Triton X-100 (Sigma, U.S.A.) in 0.05 M Tris-HC1, pH 8.0, 0.15 M NaC1, 0.005 M EDTA, 100 U m1-1 aprotinin) by homogenization for 5-10 min at 4°C followed by centrifugation at 10 000 x g. This supernatant was then used in the immunoprecipitation studies. Purified SjGST also used in such studies was dialyzed against 0.1 M borate buffer, pH 8.5, and iodinated using the BoRon and Hunter reagent. Radiolabeled proteins were incubated with various antisera at 4°C overnight, and the antibodyantigen complexes immunoprecipitated by the addition of protein A-bearing Staphylococcus au-

reus (Cowan 1 strain; Commonwealth Serum Laboratories, Australia). The complexes were then washed three times in TNET buffer before electrophoretic analysis. Samples were electrophoresed on 13% acrylamide slab gels under reducing conditions. Antisera. Rabbits were immunized with purified antigens in FCA, with at least two aqueous boosts at approximately one-month intervals. Sera were collected at 10-14 days after the boost. Antisera used in these studies were collected from rabbits immunized with S. japonicum adult worm extract (SjAWE); SjGST (Sj26 plus Sj28) purified from adult worms; Sj28, purified from adult worms after elution from GSH-agarose as fractions which were enriched for Sj28, and contained no detectable amounts of Sj26 by SDS-PAGE analysis and protein staining; recombinant Sj26 (rSj26) produced from the plasmid pSi5 (see below); recombinant Sm28 (rSm28) produced from kZAP phagemids (see below); SmGST (Sm26 plus Sm28); and control Plasmodium falciparum [3-galactosidase fusion protein kindly provided by R.F. Anders, WEHI. Mice were obtained from a specific pathogenfree facility at WEHI and then maintained conventionally. C57BL/6 mice were injected with either SjGST (Sj26 plus Sj28) or SmGST (Sm26 plus Sm28) purified from adult worms. Mice were immunized by both the subcutaneous and intraperitoneal routes with a total of 25 pog GST over 3 injections, the first emulsified in Freund's complete adjuvant (FCA), followed by two aqueous boosts at intervals of 26 days and 8 weeks. Sera were collected 9 days after the third injection. Preparation of RNA and DNA. Adult worms in liquid nitrogen were crushed into a powder with a chilled mortar and pestle. The powder was dissolved in 8 M guanidinium thiocyanate and layered on top of a step gradient of 5.7 M and 3.0 M CsCI and centrifuged at 29000 rev./min for 20 h. Genomic DNA was recovered from the interface between the CsC1 layers. The R N A in the pellet was extracted twice with butanol/chloroform (4:1), and ethanol-precipitated. Poly(A) + RNA was isolated by oligo(dT) chromatography [10,11].

25 RNA was prepared from adult S. japonicum worms obtained from Lowell University, MA, U.S.A. The S. rnansoni parasites were of Puerto Rican origin and maintained at the University of Iowa. The presumptive origin of the S. ]aponicure worms is the Leyte region of the Philippines. A S. ]aponicum cDNA library in hgtll was constructed [12], using reagents and enzymes purchased from Amersham. EcoRI-cleaved, dephosphorylated hgtll D N A (Vector Cloning Systems) was ligated to the linker-containing cDNA and the reaction products packaged into phage using Gigapack packaging extracts (Stratagene, La Jolla, CA, U.S.A.). Both a S. ]aponicum and a S. mansoni cDNA library in hZAP were constructed in a similar manner using reagents purchased from BRL and Stratagene. The libraries were introduced into Escherichia coli strains Y1090 or BB4 and immunoscreened [13-16]. Rabbit anti-Sj28 and anti-SmGST antisera were used at a dilution of 1:200 in Blotto (5% skim milk in phosphate-buffered saline, pH 7.3; PBS) followed by 125I-labeled protein A and subsequent autoradiography. Cloned restriction fragments or cDNAs used as hybridization probes were labeled using the random primer method [17] (Hexamer-primer labelling kit; Bresa, Adelaide, Australia). The hybridizations were conducted at 65°C for 16 h [11,18]. The filters were washed, then blotted dry and autoradiographed. The Sm26 cDNA was identified using 32p-labeled Sj26 insert purified from the plasmid pSj5 [19] by EcoRI digestion. Larger Sj28 cDNAs were isolated using 32p-labeled Sj28 insert from cDNAs identified by immunoscreening, as well as 32p-labeled genomic fragment (K.H., unpublished). The cDNA inserts in hgtll were excised by digestion with EcoRI, purified from a low-melting-point agarose gel and ligated into M13mpl8 and M13mpl9 for sequencing. Phagemids containing the Sj28, Sm28 and Sm26 cDNAs were recovered from hZAP by the automatic excision process as described by the manufacturers (Stratagene). Colonies expressing Srn28 were expanded, induced with 5 mM isopropyl 13v-thiogalactopyranoside (Sigma) and the rSm28 protein was purified from the bacterial pellet, after sonication, by affinity chromatography on GSH-

agarose. Due to the presence of internal EcoRI sites in Sj28, and the possibility of such site(s) in Sm26, the DNA purified from both the Sj28 and Sm26 cDNA phagemids was digested with SacI and KpnI. The purified insert was then directionally ligated to both SacI/KpnI M13mpl8 and M13mpl9. The standard dideoxy chain termination reactions were performed [20] using [35S]dATP and Sequenase (United States Biochemical Corporation, OH, U.S.A.).

9.4-11, 67~

43,.~ 30-~

20.~

14-~ 1

2

1

2

3

4

5

67 ~

43.~

30"~ 20"~

3

4

5

Fig. 1. AutoradiographyfollowingSDS-PAGE of immunoprecipitates of Bolton and Hunter labeled-SjGSTusing sera from five individual C57BL/6 mice immunized with SjGST (panel A) or SmGST (panel B). Molecularweights (xl0 -3) are indicated.

26

Results

Sj28 is weakly immunogenic relative to Sm28. To compare the immunogenicity of purified S. japonicum and S. mansoni GSTs, C57BL/6 mice were immunized with either GST preparation and sera collected for immunoprecipitation studies using Bolton and Hunter labeled SjGST. Fig. 1A shows the immunoprecipitation of [125I]SjGST with individual sera from five C57BL/6 mice immunized with SjGST. Although labeled Sj26 is immunoprecipitated by all mice, Sj28 is barely detectable. In a parallel experiment shown in Fig. 1B, the same labeled SjGST was immunoprecipitated with individual sera from five C57BL/6 mice immunized with SmGST. As predicted [5], sera from SmGST-immune mice contained cross-reactive anti-Sj26 antibodies. Moreover, 4 out of the 5 mice immunized with SmGST also produced anti-Sm28 antibodies which were cross-reactive with Sj28, as shown by immunoprecipitation resuits in Fig. lB. Data obtained with immunized WEHI 129/J mice were comparable to those presented above for C57BL/6 mice. From these and other experiments [5,21], the Sj26 and Sm26 appear to have similar immunogenicity in mice, while the Sj28 and Sm28 mole-

cules are different. We have consistently observed that it is difficult to obtain a significant antibody response to Sj28 in mice immunized with the homologous antigen. In contrast, antibodies against Sm28, which will bind to both Sm28 and Sj28, are induced readily. On occasions, antibodies to Sm26 can be difficult to elicit (e.g., see Fig. 2) but this is likely to reflect the low amounts present in S. mansoni adult worms compared with Sm28 (and Sj26 in S. japonicum) [5]. In these experiments, between 25 and 50 p~g of GST was administered over at least two injections. Whereas for S. japonicum the proportions of Sj26 and Sj28 were approximately equal, Sm26 was estimated to comprise 5-10% of the S. mansoni GST [5].

Sj28 is located at the surface of the adult schistosome. To determine whether Sj28 and Sm28 have a similar location in the parasite, living adult worms were iodinated using the Bolton and Hunter reagent. This procedure was chosen so as to preferentially label surface proteins, although internal molecules or those buried deep in the tegument may occasionally be iodinated. After the labeling procedure, the worms were checked microscopically and showed no evidence of gross tegumental damage. Labeled proteins were ex-

~94 94~

~67

67~

~43

43~

~30

30~ ~20

~14 A

B

C

D

E

F

G

H

I

J

K

L

Fig. 2. Immunoprecipitation of Bolton and Hunter-labeled living adult worms: $. japonicum (lanes A-F) or S. mansoni (lanes G-L). Lanes A and O show the whole labeled extract. Sera used for immunoprecipitation were rabbit anti-SjAWE (lane B), antiSmAWE (lane H), anti-SmGST (lanes C and I), anti-rSm28 (lanes D and J), anti-rSj26 (lanes E and K) and control anti-P, falciparum fusion protein (lanes F and L). Molecular weights (× 10-3) are indicated.

27 tracted from the S. ]aponicum and S. mansoni worms by homogenization and centrifugation and were analyzed by SDS-PAGE and autoradiography. The whole labeled extracts, used in the immunoprecipitation studies are shown in Fig. 2, lanes A (S. ]aponicum) and G (S. mansoni), as are immunoprecipitates using anti-SjAWE, antiSmAWE, anti-SmGST, anti-rSm28 and anti-rSj26 antisera. All four GSTs were detected in their respective labeled products following immunoprecipitation. However, whereas Sj26, Sm26 and Sm28 were only present in small amounts, Sj28 was a major labeled protein at the surface of (S. ]aponicum) adult worms. Anti-rSj26 antibodies did not recognize Sj28 or Sm28 in these extracts (lanes E and K). Similarly, anti-rSm28 antibodies failed to recognize Sj26 or Sm26 (lanes D and J) indicating a general lack of cross-reactivity between the 26K and 28K isoenzymes, as reported previously [5]. A control serum, anti-P, falciparum [3-galactosidase fusion protein, did not recognize any labeled protein. This result has also been confirmed using the chloramine T labeling method which has been reported to be 'surface-specific' in the labeling of many species of parasite [22,23] (results not shown). Surface label profiles are similar when worms are harvested from either intact or hypothymic nude mice (unpublished results).

Comparison of the nucleotide sequences of S]28 and Sm28. To identify a cDNA encoding the 28kDa GST of S. ]aponicum, a hgtll expression library was screened with rabbit antiserum raised against the purified protein. A large number of Sj28 cDNAs were identified and initially eight of these were further characterized. All of these hgtll clones were shown to have inserts of 670 bp, which terminated at an EcoRI site within the coding region. One of these cDNAs, termed Sj28109 cDNA, was completely sequenced and was used as a probe in some experiments. To identify larger Sj28 cDNAs, a probe was generated from an Sj28 genomic done. This probe contained only sequences found 5' of that in the Sj28-109 cDNA, but did not encompass the entire 5' coding region to the start methionine. This probe identified many cDNAs in a hZAP library. Six of these cDNAs were sequenced from each

end and the largest was termed Sj28-15. The nucleotide sequences of all of the Sj28 cDNAs examined were found to be identical. The Sj28-15 cDNA was characterized further and its sequence compared with the published Sm28 sequence (see below). Sm28 cDNAs were also identified by screening hZAP expression libraries with rabbit antiserum raised against SmGST. This antiserum recognizes Sm28 very well, with low levels of anti-Sm26 antibodies (see Fig. 2, lane I). This antiserum did not identify any clones corresponding to Sm26 in the cDNA library. One insert corresponding to Sm28 was characterized in detail and was found to have a sequence identical to that described by Balloul and colleagues [1]. However, this cDNA (Sm28--7) is incomplete, as it begins 8 nucleotides from the A of the translation start methionine codon of the published Sm28 sequence [1]. The Sj28-15 cDNA is 764 bp long. A comparison of its nucleotide sequence with that of Sm28 is shown in Fig. 3. The Sj28-15 cDNA does not extend to the start codon for translation. A comparison based on the Sm28 sequence indicates that it lacks the first 15 nucleotides of the coding sequence. The 5' untranslated regions of the GST mRNAs in schistosomes appear to be unusually small, approximately 20 nucleotides long (ref. 1, and D.B. Smith, personal communication). The 3' untranslated regions of Sj28--109 and Sj28-15 cDNAs are both three nucleotides larger than that for Sm28, and the greatest percentage of nucleotide homology between the two is achieved by assuming a 6-nucleotide insertion in the Sj28 sequence at position 680 and a 3 nucleotide insertion in the Sm28 sequence at position 741. This aligns the consensus polyadenylation signal sequences in both cDNAs. Another Sj28 cDNA examined contained three additional nucleotides after the T at position 785, before the poly(A) tail. Both Sj28 and Sm28 cDNAs contain a stretch of thymidines approximately 20 nucleotides upstream of the poly(A) signal sequence, which has also been observed in other schistosome cDNAs (unpublished results). The overall identity of the Sj28--15 and Sm28 cDNAs at the nucleotide level is 73% (559/761). However, the homology in the coding regions is greater than that in the 3' untranslated regions.

28 i0

20

30

40

50

70

60

80

90

100

CCGATCCGCA TGATTCTTGT **** ** * *** ******

GGCAGCGGGA ***~** **

GTAGAATTCG * * * * * * **

ACGGACGCGG

TCGATTCGGA

C-GCAGCTGGT GTAGACTACG

.....................

Sm28

TGCAAGAT__._~

Sj28

110 120 AAGATGAAAG GATTGAATTC ******* ** *** ***

130 CAAGATTGGC **~*******

CTAAAATCAA * ***~****

A~CCATA ****** **

170 160 CCTGGAC-GAA GATTGCCTAT ** ** ***

Sm28

AAGATGAGAG

CAAGAT~

CAAAAATCAA

ACCAACTATT

CCAGGCGGAC

Sj28

TAAAACAkATG TCAGAGAGTT *** *** * ********

TGGCTATTGC **********

240 ACGATTTATA *** * ***

250 260 GCGCCmAAAAC ACAACATGAT *** **** * * *****

~GATACA *** ** ***

GACGATGAGT ATTATATCAT AGAGAAGATG ***** ** * * **** * * * * * * **

Sm28

AAATGGATG

TGGCTATTGC

ACGGTATATG

GCGAAGAAAC

GGGTGAAACA

GACGAGC4%AT ACTATAGTGT

350

360

Sj28

ATTGGCCAAG TCGAAGATGT ***** ** * ********

TGAAAGTGAA *** ****

TACCATAAGA CTCTCATAAA ** ** ** * ** * ** **

GCCACCAGAA ***** ****

GAAAAAGAGA ** *******

AAATCTCCAA * ** ****

GGAGATATTG *********

AACGGTAAAG ***** ****

Sm28

ATTGGTCAGG

AGAACATGAA

TATCACAAAA

GCCACAAGAA

GAGAAAGAGA

AGATAACCAA

AGAGATATTG

A

Sj28

TTCCCATTCT **** ****

TCTCCA%AGCA ATTTGTGAAA **** * ** ** ***

Sm28

TTCCAGTTCT

TCTCAATATG

Sj28

TTCCATTGAT * *******

CATATAACTG ATTTGGATAA *** * **** ** *******

GGAATTTTTA ACTGGCAAGT * **** ** **********

Sm28

TGTCATTGAT

CATGTGACTG

AGGATTTCTA

Sj28

C~GAAAT *********

Sm28

TTC-GCGAAAT ATTTATCGAA

Sj28

CATTAGTGTG * ******

ATCTACTTAT * ** ***

GATTGTGTCT * ****

740 TTTACATCTT *** * *

750 ° . .T G T T T T T *******

770 780 790 800 760 CTTTCAAAAT AAACGTTAAT TTGTGCTTAA AAAAAAAAA TCTAAAACTG * * ***** *** * * * ** ** * ** **

Sm28

GACTAGTGTC

ACCTTTTTAC

AAGGATGTCA

TTTGTTTTAT

GGGTGTTTTT

TTCGCAATTG

210

310

410

510

610

710

GTGAAGCTT ATCTATTTCA . *** ** *****~**

ACGGACGTGG ACGTGCTGAA ******* ** **********

S j28

CTC-GCGAGCA TATCAAGGTT

AATTAGTTTC 220

TTAGAGAGTT

230

320

CTGAAGATGT

330

ATCTATTTTG 140

150

340

CTTTGATGAA

ACGTC-CTGAA

ATCATATGAT

GATTGCCTGC

TGACTCTTGT 180

200

ACAGACAAAA

~ T G T

AGTGAAAGTC

ACTGATGATC

ATGGGCACGT

270

280

370

380

TGAAAAGTTG

390

400

A

~

G

490

500

AATCTGACTG ** *** ~**

TCGGAGATAA GGTCACTCTG * ** ** ** ** *****

GCTGATGTAG ****** ***

TTCTGATTGC * ********

ATCTC-CGAAT CTCTGAAAGG

GTCGACAGGA

AAGCTGC-CTG

T ~ C A A

GCTGATTTAG

TCCTGATTGC

520

530

ATCTGGATAA

440

450

540

550

460

560 ATCCTGAGAT **********

ACTC-GCAAGT ATCCTGAGAT

480

300

GTCTACAGGT *** *****

430

470

290

CCTTAAAAGA * * ****

420

AGTAACTCTA

570

580

TCACAAACAT ** * * * * * *

CGTAAACATC ** ** ***

CCATAAACAT

CGAGAAA

CAGGCC~

ACTCCCTTCT 730

AAACCTATCA

ACAGAAAC-CT

CGGTGTAACG

TTATTAAAAT

590 TATTGGCCAC * ** ****

600 TTCACCAAAA ******

TC TGTTAC-CCAG TTCACCGCGT

670 680 620 630 640 650 660 AAAGAGGATG TGCTGGAAAT ACGGCATTTT AACACTGCCA ~.AAGAATAT ACTTATC.AGA GAGACATGCA * * * * ** * ** * ** ** ** ** ** * * ***** * ** * **** **

720

190

CGTGAAAATC

AGATTTAAG

TAACTTAGTT

690 700 TCGCCATATT GGTAATAACC ***** * ** ***

...... ATATT

TCCTGTTTAA

GATAGTAAAG

AAA ......

Fig. 3. Comparison of nucleotide sequences of cDNAs of Sm28 [1] and Sj28-15. Identities are marked ('). The initiation codon in the Sm28 sequence is underlined, as are the termination codons in both sequences and consensus polyadenylation signal sequences. Both cDNAs have been aligned to give the greatest identity, with 1 small break in each sequence.

When calculated separately, the coding region has 77% (481/621) identity and the 3' untranslated region has 56% (78/140) identity, assuming the nucleotide insertions in both sequences.

greater than that in the putative 3' untranslated region (54%; 36/67). Interestingly, neither Sm26 nor Sj26 appears to contain a stretch of thymidines within the 3' untranslated region, as is present in the 28-kDa sequences.

Identification of Sm26 cDNAs. Sm26 cDNAs were identified by screening the kZAP cDNA library with a Sj26 cDNA probe obtained by EcoRI

Comparison of the amino acid sequences of the glutathione S-transferases. The amino acid se-

digestion of the pSi5 plasmid [19]. One clone was characterized in detail, namely Sm26--21. A comparison of the Sm26 and Sj26 cDNA nucleotide sequences is shown in Fig. 4. Sm26-21 is 704 bp long and by comparison with the start codon of Sj26, it is probably lacking the first 21 nucleotides of coding sequence. The overall homology between the Sj26 and Sm26 cDNAs at the nucleotide level is 79% (554/703). Again, the identity in the coding regions (81%; 518/636) is

quences of the four GSTs were deduced from the nucleotide sequences of the cDNAs. The total amino acid (aa) length of Sm28 is 211. Of the 206 Sj28 amino acids compared with Sm28 (see Fig. 5), 159 are identical (77%). Of the 47 differences, 21 are conservative changes. The others are distributed throughout the protein including positions within regions known to contain T cell epitopes in Sm28 [7]. These regions are indicated in Fig. 5 (P1, P2 and P3). When the regions of Sm28

29 80 90 100 50 60 70 40 CTTCTTTTGG AACACCTTGAAGAAACTTAT TGG AAAGTCAAAGGCCTTGTACAACCAACTCGA *** * * * * ** * * * * * * * * * e * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * *

Sm26

10 20 30 .....................................

S~26

TTTAC~TAAC TTC~I~.AT_._GGT C C I ~ T A T A C T A~TTATT~.M~ A A A A T T A A G G G C C T T G T G C A A C C C A C T C G A

CTTCTTTTGGAATATCTTGAACIAAAAATAT

Sm26

110 120 130 ~CGTGCGTATGATCGC2UtTGAAATCGATGCCTGGA ** ** * * * * * * * * ** * * * * * * ***

150 160 170 GCAACGATAAATTTAAATTAGC-CCTGGAGT * * * * * ** *** **** ** ******

TCCCARATCT * ~* * * * * *

190 200 TCCTTATTAT ATTGATGGTG ********** **********

SJ26

GAAGAGCATT

GAAA~GTTTGAATTGGGTTTC-GAGT

TTCCCAATCT

TCCTTATTAT

Sm26

250 260 210 220 230 240 AC-CTGACARACACAACATGT ATTTTAAATTAACACAATCT ATGGCTATCATACGTTATAT ********** ** ******* ****** *** ***** **** ********** *********

S~26

ATGTTAAATT

Sm26

310 320 330 TGAAGGAGCGGTTTTGGACATTAGGATGGGTGTTTTAAGA **~******* ******** * **** ** *****

TGTATGAGCG

AACACAGTCT

CGATGAAGGT

A T ~ T C A

140 ***

GATAAATGGC

TACGTTATAT 340 ***

AC-C~GACA~

CACAAC~TGT

180

280 270 TC-GGGC-CTTG ~ C G T G C G C , * * * * * * * * * ***** **** * *** TGGGTGGTTG

TC~GAG

ATTGATGGTG

290 300 AAATTTCGATGCT ** * **** *****

CGTGCAGAGATTTCAATGCT

380 390 400 350 360 370 ATCC-CATACAATAADaGAATA TGAAACCCTC AAAGTTGATT TTCTCAACAAACTTCCTGGG ** **** ** * * * * * * * * * ** * ****** *** ********** **** * ***

TTCTTAC~%~T~T~

Sj26

TGAAC-GAGCG GTTTTGGATA

Sm26

410 AGGCTGAAAA * ********

480 490 5OO 450 460 470 420 430 440 ATTTGAACGGTAATTGTGTA A C T C A T C C T G ACTTTATGTT A T A ~ - . ~ C - C C CTTC~.'~'It'C~ TGTTCGAAGATCGTTTGTCT~CTT ** ** ** ******** * **** ** ******* **** ***** ********** ****** * * * * * * * * * * * * * ** * * * **

SJ26

ATGCTGAAAA

TGTTCGAAGA

Sm26

510 TTTTATACAT **********

580 590 600 550 560 570 520 530 540 ACTTAAATTC ATTAGTTTCT TTCAAAAAGT GTATTGAAGA TTTACCACAAATCAAGAACT GGACTCACAG TGCTTGAACG AGTTT~ * * ****** ** * * * * * * * * ** ** ********* **** ** * * * * ** * * *** *~*** ******** * ** *****

S~26

TTTTAT&CAT

G G A ~ T G

Sm26

610 620 630 TAC-CAGGTAC ATAAAATGGC CTC~TTGGGATGCT **** *** *** ***** ** **** ** ***

TTAGATACGG

TCGTTTATGT

TGCCTGGATG

TGTTTCGAGA

CATAAAACAT

CGTTCCCRAA 640 * **

SJ26

CAGCAAGTAT

ATAGCATC-G

Sm26

710 AAARAGCCTG * *

720 730 740 TTATATGTTT ACTAAATAAA AAATAACAAT *** * * ******** * ** *

Sj26

TATTTATCAC

,TTACAATTAA ACTAAATATA

CTTTGCAGGGCTGGCAAGCC

AATGTCGACA

ATTGCATATA

GT~CTT

ATTTAAATGGTGATCATGTA

ATTAGTTTGT

TTTAAAAAAC

TGAACTCTC

AAAGTTGATT

ACCCATCCTG

GTATTGAAGC

TATCCCACAAATTGATAAGTACTTGAAATC

680 690 650 660 670 ACGTTTGGTGGTGGAGATAC TCCTCCAAAA TAGATTAACAGTAAATCTGG * * * * * * * * * * * * * * ** * * * * * * * * * * ** * * * * * * * * * *

700 TAAATAACAT ** *****

ACGTTTGGTG

TAGTAAACAT

GTGGCGACCA

TCCTCCAAAA

TAAATTAAGA

ATGATTGTTT

750

.........

Fig. 4. Comparison of nucleotide sequences of cDNAs of Sj26 [3] and Sm26-21. Identities are marked ('). Initiation methionine of Sj26 as well as termination codons in both sequences are underlined. The greatest alignment between cDNAs requires no breaks in sequence,

corresponding to the synthetic peptides studied by Auriault and colleagues [7,24] are compared with the primary sequence of the analogous regions of Sj28, several amino acid changes are observed. In the region P1 (aa24--43) there is a conservative change from a glutamic acid in Sj28 to an aspartic acid in Sm28 at aa 30, a hydrophobic, nonpolar phenylalanine at aa 31 in Sj28 to a polar tyrosine in Sm28, and a negatively charged glutamic acid at aa 37 to an uncharged, polar serine residue in Sm28. In the second peptide region P2, (aa 115-131) there are two amino acid changes. At aa 116 in Sj28, a proline is a polar glutamine in Sm28, and at aa 124, there is a conservative change of serine to threonine. Finally when comparing the Sj28 to the third peptide region P3 (aa140-153), there are four changes. A conservative threonine at 142 in Sj28 is a serine in Sm28, a glutamic acid at 145 in Sj28 is a glycine in Sm28,

an asparagine at 149 in Sj28 is a lysine in Sm28, and a threonine at 151 in Sj28 is replaced by an alanine in Sm28. Fig. 5 also shows a comparison of the amino acid sequences of Sm26 and Sj26 deduced from their cDNA nucleotide sequences. The total amino acid length of Sj26 is 218, and Sm26-21 has 211 amino acids. The amino acid sequences are 81% identical, (170/211) and of the 41 amino acids which are different, 8 of these are conservative changes. It is interesting to note that the amino acid length of Sj26 and the predicted length of Sm26 is greater than the length of Sm28 and the predicted length of Sj28 (218 vs. 211). However, the calculated molecular weight of Sj26 is 26 217 and that of Sm28 is 27 620, both being close to the observed molecular weights. The amino acid sequences (Fig. 5) of Sj28 and Sm28 were compared with those of Sj26 and Sm26

30

Sj26

MSPILGYWKIKGLVQPT

Sm26

RLLLEYLEEK YE.__EHLYERDE GD_KWRNKKFE LGLEFPNLPYY

WKVKGLVQPT RLLLEHLEET YEERAYDRNE

IDAWSNDKFK LGLEFPNLPYY

sj28,

VKLIY FNGRG_RAEPI RMILVAAGVE FEDERIEFQD WPKIKPTIPG GRL-PIVKITD

s~8,,

MAGEHIKVIY FDGRG_RAESI RMTLVAAGVD YEDERISFQD WPKIKPTIPG GRL-PAVKVTD P1

Sj26

IDGDVKLTQ- SMAIIRYIAD ~ G G C P K

Sm26

IDGDFKLTQ- SMAIIRYIAD KHNMLGACPK E_RAEISMLEG AVLDIRMGVL RIAYNKEYET

si28

KRGDVKTMSE SLAIARFIAR KHNMMGDTDD E_YYIIEKMIG Q_VEDVESEYH KTLIKPPEEK

sin28

DHGHVKWMLE SLAIARYMAK KHHMMGETDE E_YYSVEKLIG QAED_VEHEYH KTLMKPQEEK

Sj26

LKVDFLS--- KLPEMLKMFE DRL_CHKT_YLN --GDHVTHPD FMLYDALD_V~_ LYMD_PMC_LDA

Sm26

L_KVDFLN--- KLPGRLKMFE DRL_SNKT_YLN --GNCVTHPD FML_YDALDVV LYMD_SQCLNE

Sj28

EKISKEILNG KVPILL_QAIC ETL_KESTGNL TVGDKVTLAD VVLIASID_HI TDLDKEFLTG

Sm28

EKITKEILNG KVPVLLNMIC ESL_KGST_GKL AVGDKVTLAD LVL_IAVIDHV TDLD_KGFLTG P2

E_RAEISMLEG AVLDIRYGVS RIAYSKDFET

P3

sj26

FPKLVCF _KKR IE_AIPQIDKY L_KSS_KYIA_WP L_QGWQAT___FGG GDHPPK

sm26

FPKLVSFKK__C IE_DLPQIE~TY _LNSS__RYIKWP L_QGWDAT___FGG GDTPPK

Sj28

K Y PEIHKHRKH_ L LATSPK _IA~KY LSERHAT_._~

Sm28

K_Y PE_IHKHRENL L A S S P ~ Y

_LSNRPAT__.PF

Fig. 5. Comparison of amino acid sequences of Sj26, Sm26, Sj28 and Sm28 deduced from the nucleotide sequence of cDNAs. Identities between the two 26-kDa species and between the two 28-kDa species are indicated by (') and amino acids common to 3 or more species are underlined and printed in bold type. Regions corresponding to peptides known to contain T cell epitopes in Sm28 are indicated (P1, P2 and P3).

to highlight any common domains in these isoenzymes. Overall, there is very little homology between the 26- and 28-kDa molecules, only around 20% identity, even when allowing gaps in the sequences to produce a better alignment. However, there is a region of significant homology 61 and 63 amino acids from the putative start methionine in Sj26 and Sj28, respectively (Fig. 6). Over the next 28-aa stretch there is 57% identity between the two S. japonicum GSTs, and slightly

less between Sj26 or Sm26 and Sm28. Several mammalian sequences have been included to highlight the striking number of conserved residues within this region of many GSTs. In fact, of the 28 amino acids, 7 are conserved, assuming the glycine at position 62 of human GST2 and rat GSTYc aligns with glycine residue 61 in Sj26. As well as 7 completely conserved residues, there are many amino acids common to several sequences, e.g., with respect to the Sj26 sequences, aspartic

31 Sj26 Sm26 Sj28 Sm28 HUM2 RATYC RATYB3 MUS

61 a

63 61 61 66 66

GDVKLTQ-SMAIIRYIADKHNMLGGCPKE GD*KLTQ-SMAIIRYIADKHNMLG*CPKE GDVK****S*AI*R*IA*KHNM*G****E **************************** DG*KL*Q-**AI**YIA*K*NL*G***KE DG*KL*Q-**AI**YIA*K*NL*G***KE G**K*TQ-S*AI*RY***KHNL*G****E G**K*TQ-S*AI*RY*A*KH*L*G****E

Fig. 6. Amino acid sequences of regions of glutathione Stransferases compared to Sj26, from Sm26, Sj28 and Sm28, as well as mammalian GSTs; human GST2, rat GSTYC, rat GSTYB3 and mouse GST. The mammalian sequences were obtained from the GenBank TM data base. Residues conserved in all sequences are indicated in bold type, but, as ifldicated in the text, there are numerous other residues common to many, but not all, sequences in this region. Residues different from that in the Sj26 sequence are indicated by ('). The position of the amino acid from the start methionine is denoted (a). To obtain the best alignment with the schistosome 28-kDa GSTs, a gap ( - ) has been included in the other sequences.

acid at position 62, glutamine at position 67, serine at position 68, arginine at position 73, tyrosine at position 74, alanine at position 76, histidine at position 79, asparagine at position 80 and leucine at position 82. Discussion

We report that the 28-kDa GST of S. japonicure is weakly immunogenic in mice relative to its homologue in S. rnansoni, that is a vaccine candidate antigen [1,2,7]. Since these proteins are (a) abundant in schistosomula and adult worms, (b) located at least transiently at or near the worm surface, and (c) likely to have an essential role in worm physiology, they are attractive ingredients of a multi-component vaccine [4]. Differences in the two proteins that lead to differences in immunogenicity are important, because components of a schistosomiasis vaccine will presumably have to be highly immunogenic and, from all current indications, should induce both T and B cell immunity. We have compared the primary structure of these two proteins, particularly in regions defined as T cell epitopes in the S. mansoni molecule [7,24]. Three synthetic peptides from different positions of the Sm28 molecule were tested for their ability to stimulate T cells (and to bind antibody) (P1, P2 and P3 in Fig. 4) [24]. In P1, there are three amino acid differences between the two

proteins, while in P2 there are two and in P3 there are four, indicating that there are differences between the molecules in regions known to contain T cell epitopes. The NH 2 terminal region of P1 was shown to be most significant in stimulating mouse T cells [7]. The three amino acid differences between Sj28 and Sm28 in the region of P1 may alter this T cell epitope such that adequate help for B cells in production of anti-S j28 antibodies is not provided, at least for those mouse strains that have been tested to date. Antibodyindependent cellular mechanisms are considered by many to be primarily responsible for protective immunity in mice. If the Sj28 molecule does lack T epitopes (and it is not assumed that the three identified in Sm28 to date represent the total complement of T epitopes), it would suggest that this molecule may not be a 'host-protective immunogen' for many mouse strains. Sm28 and Sj28 cross-react extensively in both directions at the antibody level, particularly when hyperimmune rabbit sera are used. Although the two 28-kDa GSTs of S. japonicum and S. mansoni are 77% identical at the amino acid level, evolution has maintained functionality of the proteins while their immunogenicity diverged. In unpublished results, differences have been observed in hybridization patterns for the two cDNAs encoding these proteins that are due to nucleotide changes, resulting in restriction enzyme recognition site changes within the coding region as well as outside the gene. The 26-kDa GSTs of schistosomes are also potential vaccine components. Resistance against S. japonicum in mice induced by Sj26 vaccination has generally been low although it has on occasions conferred up to 50% protection [3,25]. This molecule is particularly well-recognized by 129/J mice that can be highly resistant to chronic infection with S. ]aponicum although it now appears that portal system peculiarities contribute to this resistance [26-29]. The S. mansoni homologue of Sj26, namely Sm26, is also well recognized by 129/J mice, particularly early in infection [6]. From the results presented in this paper, the antibody cross-reactivity reported for Sj26 and Sm26 [5] is readily explained by the 82% identity between these two antigens at the amino acid level.

32 In situ staining of adult worm GSTs, after electrophoretic separation, predicted two isoenzymes in S. japonicurn and three in S. mansoni [5]. Whether the third isoenzyme in S. mansoni is a heterodimer between Sm26 and Sm28, or a yet to be described GST isoenzyme, remains to be seen. Preliminary partial sequence data, kindly provided by R. Harrison, G. Newport and N. Agabian, UCSF, from a cDNA clone of Sm26 (Puerto Rican strain) isolated in their laboratory (but not yet shown to be encoding a functional GST), indicate significant differences from the sequence of Sm26 presented here. In this study, the Bolton-Hunter reagent was used to demonstrate that the 28-kDa GST is among the surface proteins of S. japonicum. This technique has been widely used to label surface proteins of various parasites [22,23] and has been observed to be primarily surface-specific. It has been found on occasions to label internal somatic tissues and this could be due to labeling of free amino groups [30]. A surface location for Sj28 was confirmed using the chloramine T labeling method which has been shown to label only the surface of many parasites [22]. Assuming in the case of S. japonicum that this method has been surface-specific, Sj28 has been identified at the surface of the adult parasite in relatively large amounts. Sj26 was also found to be present at the surface but only in modest amounts, as were both Sm26 and Sm28 in S. mansoni adult worms. A surface location is consistent with the localization of the Sm28 to the tegument of S. mansoni adult worms using immunofluorescence techniques. The molecule was also located in the parenchyma and the excretory epithelial cells by immunogold electron microscopy [2]. In addition, these studies showed the presence of Sm28 in the tegument of the schistosomula as well as in granules of the head gland from which tegumental components can be derived. However, another group has used indirect immunofluorescence to show that the S. mansoni GSTs are restricted only to the parenchyma, and are not found within the epidermis or tegument [31]. This controversy remains unresolved, but it is possible that different methods of handling adult worms after perfusion from infected animals, or perhaps differences in the intensity of surface im-

mune attack within donor animals, could influence the amounts of GST detectable at the surface. The GSTs are certainly not integral membrane proteins [32]. By comparing putative amino acid sequences of all 4 GSTs (i.e., Sm26, Sj26, Sm28 and Sj28), the only region with substantial similarity was around amino acids 60 to 90 (Fig. 6). The conservation of certain residues in the region extends to sequences of mammalian GSTs in sequence data banks. There are some mammalian sequences which show little similarity in this region, however the majority of sequences in the data base have a marked degree of identity. Moreover, whereas the degree of identity at the amino acid level between different classes of GSTs is approximately 20%, the percentage homology in this region is almost 50%. There is even some similarity in this region to sequences from plant GSTs (P.G. Board, personal communication). Domains responsible for common GST activities such as GSH binding remain totally undefined and this region, as distinct from N-terminal regions, has not been considered previously as a GSHbinding site (P.G. Board, personal communication). It will be of interest to determine whether induced antibodies to peptides corresponding to this region not only affect enzyme function, but show cross-reactivity between isoenzymes which to date have not been shown to be immunologically cross-reactive.

Acknowledgements We thank Vanessa Hermann for excellent technical assistance, Susan Wood and Karen McLeod for maintaining the parasite life cycles, and Drs. Mark Rogers, Wilfred Tiu and Philip Board for helpful discussion. We also thank Drs. John Donelson and David Moser for their participation in cDNA library construction whilst K.J.H. was at the University of Iowa, U.S.A. This work was supported by the National Health and Medical Research Council of Australia, the Rockefeller Foundation Great Neglected Diseases Network and the John D. and Catherine T. MacArthur Foundation.

33

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