Molecular and Biochemical Parasitology, 48 (1991) 67 76 ~?, 1991 Elsevier Science Publishers B.V. / 0166-6851/91/$03.50 ADONIS 016668519100285A

67

MOLBIO 01582

Further characterisation of the Schistosoma japonicum protein Sj23, a target antigen of an immunodiagnostic monoclona[ antibody K a t h l e e n M. D a v e r n , M a r k D. Wright, Vanessa R. Herrrriann a n d G r a h a m F. Mitchell The Walter and Eli:a Hall Institute of Medical Research, Melbourne, Victoria, Australia Received 22 January 1991; accepted 5 April 1991)

Sj23, the 23-kDa target antigen m Schistosoma japonicum adttlt worms of the hybridoma monoclonal antibody (mAb) I-134, has been identified and cloned from cDNA libraries, mAb I-134 has been successfully used in immunodiagnostic assays to detect S. japonicum infection in Philippine patients. Sequence analysis has shown that Sj23 is the homologue, with 84% amino acid identity, of Sm23, a 23-kDa molecule from S. mansoni worms previously described from our laboratory. The domain structures of Sj23 and Sm23 are strikingly similar to the human membrane proteins ME491, CD37, CD53 and TAPA1, which may suggest a functional role for the schistosome rfi6~eett~e~ in cellular proliferation. Key words: Schistosoma japonicum; Integral membrane protein; lmmunodiagfiosis; eDNA clone; Growth factor: Receptor

Introduction

Previous work from this laboratory identified a monoclonal antibody (mAb), 1-134, that was diagnostic for Schistosoma japo~tiet, m (Philippines) infection in humans. Sera from over 90% of Philippine individuals with proven schistosomiasis japonica were positive when used in competitive immunoassays with labelled 1-134 [1-4]. The monoclonal antibody recognises a 23-kDa protein of adult worms, termed Sj23 [5], which is an integral membrane protein [6]. More recently, we identified and cloned a 23-kDa integral membrane protein of Correspondence address: Graham F. Mitchell, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M63706. Abbreviations: BSA, bovine serum albumin diluted in PBS: mAb, monoclonal antibody; NEPHGE, non-equilibrium pH gradient electrophoresis; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

S. mansoni (Sm23) which resembles a human tumour-associated antigen, ME491 [7,8]. A schistosome molecule identified using a mAb M2, raised against membrane extracts of schistosomula [9] is now known to be the same molecule as Sm23 (D. Harn, personal communication). Here we report the cloning and further characterisation of Sj23, and by sequence analysis provide evidence that this molecule is the S. japonicum homologue of Sm23.

Materials and Methods

Antigens and antisera. A Philippine (Mindoro) strain of S. japonicum and a Puerto Rican strain of S. mansoni were maintained in the laboratory according to previously published methods [10]. Parasites were stored fresh frozen at - 7 0 " C until used for preparation of antigens. The integral membrane protein fraction of adult worms was prepared according to published methods [6]. Briefly, 0.5 ml of adult schistosomes were homogenised in an equal volume of ice-cold phosphate-buffered

68

saline (PBS), 5 ml of cold, precondensed Triton X-II4 (Fluka AG, Switzerland) at a concentration of 0.5% was added and mixed gently then held on ice for 90 rain with gentle mixing every 10 min. After removal of detergentinsoluble material by several rounds of centrifugation for 15 min at 10000 × g, the supernatant was carefully loaded onto a 5-ml 6% sucrose cushion and incubated at 3T~C for 5 rain. The tube was then centrifuged at 950 x g for 5 rain at 37°C, and the upper detergentdepleted fraction and the detergent-rich pellet were recovered separately. The pellet was resuspended in 1 ml cold PBS, applied to a second sucrose cushion and re-extracted. The rabbit antisera used in these experiments were from a rabbit (D129) infected with 300 S. japonicum cercariae and bled 129 days later; a rabbit (R792) exposed to approximately 5000 S. mansoni cercariae on 2 occasions at 7 months' interval and serum collected 18 days later (our Puerto Rican isolate of S. mansoni does not infect rabbits, although mice are entirely permissive; ref. 11); and a rabbit (R306) which was repeatedly immunised with extracts of S. japonicum adult worms. The production of a mouse antiserum specific for the C-terminus of Sm23 has been previously described [7]. Hybridoma monoclonal antibodies 1-134 and control were purified from ascites by elution from protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) and concentrated to 1 mg ml ~ [1,2].

AJfinity purification of specific

antibodies.

Antibodies specific for recombinant schistosome proteins were purified from antisera by low pH elution from nitrocellulose filters impregnated with purified fusion protein in the pGEX vector (see below) having first been preincubated on filters impregnated with purified recombinant Sj26 (rSj26) to remove any anti-GEX antibodies.

Electrophoresis and Western blotting. Twodimensional electrophoresis using non-equilibrium pH gradient electrophoresis (NEPHGE) in the first dimension and standard SDSPAGE under reducing conditions was per-

formed as previously described [12]. Schistosome antigens separated by two-dimensional electrophoresis were transferred to nitrocellulose filters and probed with antibody and ~25Ilabelled protein A following previously described methods [13,14].

Identification of Sj23 clones in the S.japonicum cDNA library. The production of both a S. japonicum c D N A expression library in the 2ZAP vector (Stratagene, La Jolla, CA) and in 2gtl 1 using reagents and enzymes purchased from Amersham (U.K.) have been described elsewhere [15]. Cloned purified insert from Sm23 [7] or Sj23 used as a hybridisation probe was labelled using the random primer method [16] (Hexamer-primer labeling kit; Bresa, Adelaide, Australia). Hybridisations were conducted at 65°C for 16 h [17,18]. Phagemids were recovered from positive ).ZAP clones by the automatic excision process as described by the manufacturers (Stratagene).

DNA sequencing. Sj23 clones were identified in the S. japonicum 2gtl 1 c D N A library using 32p-labeled Sm23 purified insert. Sj23 insert was purified using polymerase chain reaction (PCR) [19]. Briefly, a plug of agarose picked from a plate of phage containing Sj23 insert was mixed in a tube containing in a total volume of 100 lal 10 mM Tris-HC1 pH 8.3/50 mM KC1/1.5 mM MgCI2/0.1 mg m l - gelatin/ 0.25 mM dNTP/10 pmol each oligonucleotide/ 2.5 U Taq polymerase (Cetus, CA, U.S.A.), overlaid with paraffin oil and subjected to 35 rounds of temperature cycling: 95°C 1 min, 47°C 2 rain and 68°C 2 min. Oligonucleotides used corresponded to 2gtll forward and reverse sequences flanking the insert site and were kindly provided by R. Simpson of this Institute. After cycling, Sj23 insert of approximately 1.2 kb was purified from a low-melting point agarose gel. The c D N A fragments were then ligated into SmaI M13mpl8 and M l 3 m p l 9 for D N A sequencing. The standard dideoxy chain termination reactions were performed using [35S]dATP and sequenase (United States Biochemical Corporation, OH, U.S.A.) [20].

69

start methionine) in pGEX-2T were expanded in L-broth containing 50 #g m l - ] ampicillin, induced by the addition of 1 mM IPTG (Sigma) and the resultant fusion protein was purified from the sonicated bacterial pellet by affinity chromatography on glutathione-agarose (Sigma) [21].

Expression of fragments of Sj23 in pGEX. Sj23 cDNAs were identified in a S. japonicum ~ZAP library using the 32P-labelled Sj23 insert identified above, recovered from phageraids by automatic excision following the manufacturer's instructions and restricted with RsaI and DraI. Fragments of 307 and 332 bp (predicted from the sequence data) were purified from a low melting point agarose gel and ligated into SmaI pGEX-2T and pGEX3X [21] for antigen expression.

ELISA assays. Plates were coated with purified recombinant antigens at 5 #g ml-~, blocked with 1% Bovine Serum Albumin in PBS (BSA), rinsed and antibodies diluted in BSA containing 0.05% Tween 20 were added and allowed to incubate at room temperature for 3 4 h, washed and horseradish peroxidaseconjugated protein A was then added and

Purification of Sj23-pGEX fusion proteins. Colonies containing the Sj23 C-terminal 332 bp fragment in pGEX-3X and the 307 bp fragment (from amino acids 6 to 107 from the

&

B

94~

.,q94 ~ .e67 443

~J~

67~ 43~

430

Q

30~"

~ 420

20~"

4114

141~

C

-494

94D,. 67~

F~

•

43~ 430 30D,-

O 420 .414

Fig. 1. Western blot following 2-dimensional SDS-PAGE of integral m e m b r a n e proteins of S. mansoni (panels A and B) and S. japonicum (C and D) adult worms probed with antisera from a rabbit exposed to S. mansoni (A), a rabbit infected with S. japonicum (C), mouse antisera specific for the C-terminus of Sm23 (B) and the m A b 1-134 (D). Approximate molecular weights ( × 10 3) are indicated. Arrows indicate Sm23 and Sj23 molecules. Acidic proteins are to the right.

70

incubated for 1 h. Substrate (2,2'-azino-bis(3ethylbenzthiazoline-6-sulfonic acid), Sigma) was added after a final rinse.

Results

Western blotting following two-dimensional SDS-PAGE of integral membrane proteins of adult worm extracts from S. japonicum and S. mansoni identified similarities between the previously characterised Sm23 [7] and the 23kDa protein from S. japonicum (Sj23) recognised by the mAb 1-134 [5]. As can be seen in Fig. 1, antibodies directed to the C-terminal portion of Sm23 expressed as a fusion protein in pGEX [7] recognise a molecule of 23 kDa molecular weight in the S. mansoni TX-114 soluble material (Fig. 1B). The hybridoma antibody 1-134 recognises a molecule of strikingly similar molecular weight and mobility in the S. japonicum integral membrane proteins (Fig. 1D). Each of these molecules, as well as many others, are identified respectively using antisera from a rabbit (D129) infected with S. japonicum (Fig. 1C) and a rabbit (R792) multiply exposed to cercariae from S. mansoni (Fig. 1A). Due to the similarities between the molecules, Sm23 and Sj23, a radio-labelled c D N A

of Sm23 was used to probe a S. japonicum library in 2gtll. Several clones were identified, each approximately 1 1.2 kb in size. From 11 clones identified (and subsequently from 48 clones identified in a S~ japonicum 2ZAP library), none were found to be expressing protein when immunoassays were performed using either hyperimmune rabbit antisera or the monoclonal antibody 1-134. Using oligonucleotides from )ogtll flanking regions, the polymerase chain reaction (PCR) was employed to produce large amounts of Sj23 c D N A for sequencing from one of the 1.2 kb clones. The complete nucleotide sequence of this clone is shown in Fig. 2. The predicted initiation and termination codons are underlined indicating an expressed molecule of 218 amino acids. The expected molecular weight of this molecule, determined from its constituent amino acids, is 23 700. It is interesting to note that at a position 6 nucleotides before and in frame with the start ATG, is a termination codon, TGA. The position of this codon may be an explanation for our failure to detect expression in any Sj23 clones. Fig. 3 shows a comparison of the deduced Sj23 protein sequence with the sequence of Sm23 [7], and also with the recently described family of human membrane proteins [8,22 25], The Sj23 and Sm23 molecules are very similar

M A T L G T G M R C L K S C V F I L N I T G G T A A T G G T A G C G A C C G G C GC T C A G C T G G A A T T C C C T C G G A G TC T A T T T A T TCT T G A A A A A T G G C G A C TT T G G G TAC T G G G A T G A G G T G T C T G A A G A G T T G T G T G T T C A T A T T G A A C A T I0 20 30 40 50 60 70 80 90 i00 ii0 120 I C L L C S L V L I G A G A Y V E V K • S Q Y • A N L H K V W Q A A P I A I I V TAT C T G T C T GT TA TG T TC CC T T G T A T T A A T A G G C G C T C ~ T G C A T A T G T A G A A G T T A A A T T C A G C C A G T A T G A G G C T A A T T T A C A T A A A G T C T G G C A G G C G G C T C C C A T C G C A A T T A T TGT 130 140 150 160 170 180 190 200 210 220 230 240 V G V V I L I V S F L G C C G A I K E M V C M L T M Y A F F L I V L L I A • L W GG T T G G A G T GG T A A T T C T C A T A G T A A G C T T C T T G G G C T G T T G T G G A G C T A T A A A G G A A A A C G T C T G C A T G C T T T A C A T G T A T G C A T TT T T C C T T A T T G T C C T T C T G A T T G C T G A G T T G G T 250 260 270 280 290 300 310 320 330 340 350 360 A A I V A V W Y K D K I D D • I N T L M T G A L • ~ P N E E I T A T M D K I Q T C C-CCGCCAT T G T TGCGGTC~3TG T A C A A G G A T A A A A T C G A T G A C G A A A T T A A T A C A C T A A T G A C T G G T G C T C T G G A A A A T C C A A A C G A G G A A A T A A C G G C A A C C A T ~ A T ~ T A ~ C 370 380 390 400 410 420 430 440 450 460 470 480 S F S C C G v K G P D D Y K G S V P A S C K • G Q • V • v Q G C L S v F S A • L A T CA T T C C A T T G T TG T G G A G T C A A A G G T C C A G A C G A T TA T A A A G G G A A T G T G C C A G C A T C A T G T A A A G A A G G G C A A G A A G T T T A T G T T C A G O G T T G T C T A T C T G T C T T T A G T G C A T TC TT 490 500 510 520 530 540 550 560 570 580 590 600 K R N L I I V A C V A F G V C F F Q L L S I V I A C C L G Q R I R D Y Q N V *

20

60

I00

140

180

218

AAAACGCA.ACTTAATAATTGTTGCCTGTGTG~CATTCGGTGTATGCTTCTTCCAACTGCTAAGCATCGTTATAGCTTGTTGTTTGGGTCAACGAATACACGATTATCAGAATGTTT~T 610 620 630 640 650 660 670 680 690 700 710 TCAAAGAGTGTAGTGTGTGTGGACTACTCATTTCTGTTTCCTTGTTTTTCCTTTTTGGTTTTTATTCAATGATGGCTTTTTATTGCCTAGATAATTGTGCCTTGGTTACTATTATTTATG 730 740 750 760 770 780 790 800 810 820 830 TATTCGATTTCATCGATGATACTCTGTTGGATACCATAATCTCATCGTTTCACAATTTACCATTATTAcAAAATAAACCGCATTGATTTACTTGATATATATTCTCACAAATGTGATGAA 850 860 870 880 890 900 910 920 930 940 950 TCATCCTCATTAATCTTGTGGTGTATATCTCATTAATAACTTTGAATGTTCACAATTGCCGTTTACATATAATTTTAAGCCTTTGATACC.CCTTTTATCTAACACTATGCTTATTTTGCA 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 GCTACAACATCGTTAGACACGTGTATAAATCAATGTTGTTCGGAATTCCGCCGATACTGACGGGCTCCAGGAGTCGTC lO9O 11oo 111o 112o 113o 114o 115o

720 840 960 1080

Fig. 2. N u c l e o t i d e s e q u e n c e with a m i n o acid t r a n s l a t i o n s of c D N A c l o n e o f ~ 2 3 . P r e d i c t e d i n i t i a t i o n a n d t e r m i n a t i o n c o d o n s a r e underlined.

71

Sj23

MATLGTGMRCLKSCVFILNIICLLCSLVLIGAGA

...... YVEVKFSQYEANLHKVWQAAPI--AIIWGVVI

65

Sm23

MATLGTGMRCLKSCVFVLNIICLLCSLVLIGAGA

...... YVEVKFSQYGDNLHKVWQAAPI--AIIVVGVII

65

ME491

M-AVEGGMKCVKFLLYVLLLAFCACAVGLIAVGV

...... GAQLVLSQTIIQGATPGSLLPV--VIIAVGVFL

TAPA-I

M-GVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYLELGDKPAPNTFYVGIYILIAVGAVM

72

CD37

MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILID-KTSFVSFVGLAFVP

68

CD53

MGM--SSLKLLKYVLFFFNLLFWICGCCILGFGI

Sj23

LIVSFLGCCGAIKENVC/MLYMYAFFLIVLLIAELVAAIVAVVYKDKIDDEINTLMTGALENPNEEI

..... TA

133

Sm23

LIVSFLGCCGAIKENVCMLYMYAFFLVVLLIAELAAIVAVVYKDRIDSEIDALMTGALDKPTKEI

..... TE

133

ME491

FLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHT---AS

134

TAPA-I

MFVGFLGCYGAIQESQCLLGTFFTCLVILFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAWDDDANNAKA

145

CD37

MGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEE

141

CD53

MVVAFLGCMGSIKENKCLLMSFFILLLIILLAEVTLAILLFVYEQKLNEYVAKGLTDSIHRYHSDNST---KA

133

Sj23

TMDKIQTSFHCCGV

....... KGPDDYKGNV

..... PASCK ................................

162

Sm23

FMNLIQSSFHCCGA

....... KGPDDYRGNV

..... PASCK ................................

162 180

.... LQINSKVLAISGIFT

...... YLLIHNNFGVLFHNLPSLTLGN--VFVIVGSII

64

63

ME491

I LDRMQADFKCCGAANYTDWEKIPSMSKNRV

..... PDSCCINVTVGCGIN

TAgA- 1

VVKTFHETLDCCGSSTLTALTTSV-LKNNLC

..... PSGS .................................

CD37

SWDYVQKQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSA

214

CD53

AWDSIQSFLQCCGINGTSDWTSGP

161

S j 23 Sm2 3

...... EGQEVYVQGCL SVF SAF LKRNL I IVACVAFGVCFFQLLS ...... EENLTYT EGCVSVFGAFLKRNLVIVACVAFGVCFFQLLS

ME491

..... FNEKAI HKEGCVEKI

TAPA-I

..... NII SNLFKEDCHQK

CD37

D I CAVPAE SH IYREGCAQGLQKWLHNNLI

CD53

..... P SDRKV--EC-CYAKARLWFH

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

GGWLRKNVLVVAAAALGI

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

PASC ................................. IVIACCLGQRIHDYQNV IVIACCLGRQIKEYENV

AFVEVLGIVFACCLVKS

IDDLF SGKLY L IGIAAIVVAVIMI

FEMI LSMVLC-CG

S IVGI CLGVGLLELGFMT

I RSGYEVM I RNS SVY

LS I FLCRNLDH---VYNRLARYR

S N F L Y I GI I T I C V C V I E V L G M S F A L T L N C Q I D K T

S QT IGL

180

218 218 238 237 281 219

Fig. 3. Comparison of the deduced amino acid sequence of Sj23 with Sm23 [7], ME491 [8], TAPA-I [24], CD37 [23] and CD53 [25]. Amino acid residues conserved between Sj23 and the other 5 molecules are printed in bold type.

having 183/218 amino acids identical (84%). There are no potential N-linked glycosylation sites in the Sj23 sequence whereas Sm23 has one site [7]. We have previously reported a striking similarity between Sm23 and the melanoma-associated antigen ME491 [7,8]. It is now clear that Sj23, Sm23 and ME491 are members of a family of membrane proteins which includes the human lymphocyte surface proteins CD37 [22,23] and TAPA-1 [24], and the pan-leukocyte surface marker CD53 [25]. In the alignment shown in Fig. 3, Sj23 shows a 34% identity to ME491 and a somewhat lesser similarity to CD53 (28%), TAPA-1 (20%) and CD37(19%). A hydropathy analysis of the Sj23 sequence (Fig. 4) predicts a molecule with 3 N-terminal transmembrane domains, a hydrophilic domain and a fourth transmembrane domain at the C-terminus. A similar domain structure has been predicted for Sm23 [7], ME491 [8], CD37 [23], TAPA-1 [24] and CD53 [25]. The Sj23 c D N A produced by PCR was used

to probe a S. japonicum 2ZAP c D N A library and 48 clones were identified. One clone, 1.2 kb in size, was used for expression of fusion protein. Initially, D N A was digested with the restriction enzymes RsaI and Dral selected 3 85

£

% ~00

250 ~

2

1 8 Ai1~113oQ£1dposl~9on

Fig. 4. Hydropathy analysis of the Sj23 sequence, indicating the domain structure of the molecule, using the method of Kyte and Doolittle [26].

Q

3O

2O

2

2O

from the predicted sequence to produce fragments of the molecule which were expected to be easier to clone and express, and which also avoided the stop codon present prior to the start ATG. Purified fragments corresponding to amino acids 6-107 and 109-218 were ligated into the pGEX vector and expressing clones (Sj23-6-107-GEX and Sj23CT-GEX respectively) were identified using the hyperimmune anti-S, japonicum rabbit antiserum. This antiserum binds to several molecules in S. japonicum adult worm TX-114 soluble material on Western blots following two-dimensional SDS-PAGE (Fig. 5, Panel 1). Antibodies affinity-eluted from filters impregnated with Sj23CT-GEX, bound to a group of molecules at 23 kDa molecular weight (Fig. 5, Panel 2) which closely resemble Sj23 recognised by the 1-134 mAb (Fig. 5, Panel 3). Moreover, I-134 bound to Sj23CT-GEX but not to Sj23-6-107-GEX nor to rSj26 (which is equivalent to GEX) in a direct binding ELISA assay (Table I). A control IgG2a hybridoma TABLE I Direct binding of monoclonal antibody I-134 to Sj23 fusion proteins

3O

2O

Fig. 5. Autoradiograph of Western blot following 2-dimensional SDS-PAGE of S. japonicum adult worm integral membrane proteins probed with hyperimmune rabbit antiserum (panel 1), rabbit antibodies affinity eluted from filters impregnated with Sj23CT-GEX (panel 2) and mAb 1-134 (panel 3). Acidic proteins are to the left.

Sj23CT-GEX

Sj23-6-107-GEX

rSj26

1-134

125 25 5 1

1.41 1.40 1.34 1.16

0.02 0 0 0

0.02 0 0 0

7-1-3

125 25 5 1

0.18 0.05 0.03 0.02

0.25 0.06 0.04 0.01

0.22 0.09 0.03 0

D129

1/20 1/100 1/500 1/2500

1.90 1.37 0.69 0.18

1.74 0.32 0.08 0

1.07 0.21 0.05 0

NRS

1/20 1/100 1/500 1/2500

0.37 0.11 0.04 0

0.33 0.078 0.02 0

0.49 0.07 0.02 0

A44o results of ELISA assay with Sj23CT-GEX, Sj23-6-107GEX or rSj26 as antigen. Antibodies assayed were mAb 1-134 or isotype matched control mAb 7-1-3 and infected rabbit antiserum D129 or control uninfected rabbit serum. The readout was protein A. mAbs were used at 125, 25, 5 and I/zg ml ~. Rabbit antisera were assayed at dilutions of I/20-1/2500 as indicated.

73 antibody, 7-1-3 (kindly provided by G. Morahan of this Institute), did not bind. Antibodies in serum from the infected rabbit, D129, bound to G E X alone and to both G E X fusion proteins at a higher titre.

Discussion

Previously, we have described a 23-kDa molecule, Sj23, of S. japonicum adult worms [5], which is an integral membrane protein [6]. A mAb, 1-134, directed to Sj23, has immunodiagnostic potential for S. japonicum infection in Philippine patients. Competitive radioimmunoassays with labelled 1-134 antibody and patients' sera enabled the correct diagnosis of greater than 90°/; of infected individuals with no false positives [14]. Recently we have cloned Sm23, a 23-kDa molecule from S. mansoni adult worms which is also an integral membrane protein [7]. In this paper we provide evidence to show that Sj23 is the S. japonicum homologue of the Sm23 molecule. Sj23 and Sm23, both integral membrane proteins of the same approximate molecular weight, also have similar migration characteristics following two-dimensional S D S - P A G E (see Fig. 1). Many clones corresponding to Sj23 were identified in S. japonicum c D N A libraries using Sm23 c D N A as a probe. Sequence analysis of one of these clones showed that Sj23 and Sm23 have 84% identity at the amino acid level. Moreover the molecules are similar in domain structure when analysed for predicted hydrophobicity [26] as can be seen in Fig. 4. This analysis predicted 4 transmembrane domains in both molecules, 3 at the N-terminus followed by a relatively hydrophilic region and a fourth transmembrane region at the C-terminus. The Sm23 has one predicted N-glycosylation site [7] whereas Sj23 has none. The molecular weight of Sj23, calculated from lhe constituent amino acids, is 23 700, very close to that observed on SDSP A G E under both reducing and non-reducing conditions [5]. From the calculated molecular weight it seems likely that Sj23 is only lightly glycosylated, if at all.

Although the degree of sequence identity between the schistosome molecules and the human molecules ME491, CD37, CD53 and TAPA-1 is relatively low, the amino acid residues which are shared between the molecules are not random, but in general are common to all (Fig. 3). Moreover, there is a striking similarity between these molecules when domain structures are compared. Hydropathy analyses predict 3 consecutive transmembrane domains at the N-terminus followed by a hydrophilic domain and a fourth transmembrane region towards the Cterminus for all of these membrane proteins [7,8,22 25]. The functions of the human membrane proteins ME491, CD53, CD37 and TAPA-I are not known. However, there is some evidence that 3 of these molecules play a role in cellular proliferation. ME491 has been postulated to be a cell surface receptor for an unknown ligand involved in growth regulation [27], whereas mAbs directed against CD37 and TAPA-I can modulate cellular proliferation [24,28]. Therefore, it is possible that the schistosome molecules are also involved in cell proliferation, acting as growth factor receptors to control development of specific specialised tissues of the parasites. Alternatively, the presence of multiple transmembrane domains is somewhat suggestive of transport molecules [25]. The Sj23 molecule is highly immunogenic to individuals infected with the Philippine strain of S. japonicum as greater than 90% have detectable antibodies to the molecule. Whether the same is true for Sm23 is yet to be tested. However, in the limited number of S. mansoni patient sera tested to date, antibodies to Sm23 were detected. For immunodiagnosis under field conditions there have been problems associated with a competitive radio-immunoassay, and a competitive ELISA [3]. These assays utilised crude adult worm extracts as antigen and specificity was conferred by using the I- 134 mAb. It should now be possible to convert the immunodiagnostic assay to a direct binding ELISA using recombinant Sj23, or an expressed fragment as the coating antigen. Already we know that the target epitope of 1-134 is in

74

the C-terminal fragment of the Sj23 molecule. Given that the Sj23 and Sm23 sequence identity breaks down within the hydrophilic domain, and as the mAb 1-134 does not bind to S. mansoni adult worm extract, this area of the Sj23 molecule is a likely candidate for the 1-134 target epitope.

Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, U N D P / W o r l d B a n k / W H O - T D R / Rockefeller Foundation Health Sciences for the Tropics - Partnerships in Research Program, Australian International Development Assistance Bureau, Edna McConnell Clark Foundation and the John D. and Catherine T. MacArthur Foundation.We thank Angela Melder, Susan W o o d and Karen McLeod for excellent technical assistance.

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