Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

MOLECULAR AND

ELSEVIER

Molecular and Biochemical Parasitology 64 (1994) 11 23

BIOCHEMICAL PARASITOLOGY

Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction Volker T. Heussler, Dirk A.E. Dobbelaere* University of Berne, Institute of Parasitology. Ldnggass-Strasse 122, CH-3012 Berne, Switzerland Received 19 July 1993; accepted 20 November 1993

Abstract Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcpl-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state m R N A expression for some of these proteases. The 5' and the 3' regions of Fcpl were amplified using the rapid amplification of c D N A ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a ~.gtl0 cDNA library using Fcpl as a probe. The Fcpl cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcpl fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the antiGST:Fcpl antiserum, indicating stage-specific gene expression or processing of Fcpl. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDA, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme. Key words'." Fasciola hepatica; Cysteine protease; RACE, RT-PCR; Stage-specific gene expression

*Corresponding author. Tel.: +41 31-631 2111; Fax: +41 31631 26 35; e-mail: dobbelaere(a itpa.unibe.ch. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession numbers Z22763 Z22770. Abbreviations: PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; GST, glutathione-S-transferase; S-Ag, somatic antigen; E/S-Ag, excretory/secretory antigen; NEJ, newly excysted juveniles. 0166-6851/94/$7.00 ,"~ ~(~ 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 6 - 6 8 5 1 ( 9 3 ) E 0 1 9 8 - H

1. Introduction Fasciola hepatica, a p a r a s i t i c t r e m a t o d e o f w o r l d w i d e d i s t r i b u t i o n , is c a p a b l e o f infecting a wide range o f different m a m m a l s , p a r t i c u l a r l y cattle a n d sheep. F o r this reason, the p a r a s i t e can be the cause o f i m p o r t a n t e c o n o m i c losses in livestock p r o d u c t i o n . A f t e r ingestion o f m e t a c e r c a r iae by the host, the juvenile flukes are released.

12

V. 7". Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11-23

These newly excysted juveniles (NEJ) penetrate the intestinal wall, migrate through the liver and transform into immature forms. Finally the parasites pass the bile duct wall and develop into the mature forms that live in the microenvironment of the bile ducts. The penetration and migration steps and invasion of the bile ducts by the immature flukes are probably supported by a collagenase released by the parasite [1]. During the migration through the various tissues, the parasite is in direct contact with the host's circulation and therefore with its immune system. Different immune evasion mechanisms have been proposed for F. hepatica, including the secretion of antibody-cleaving proteolytic enzymes [2,3]. Furthermore, NEJ have been shown to secrete a cathepsin L-like protease which prevents the attachment of eosinophils to their surface [4]. Cysteine protease activities with immune-suppressive effects, have also been described for other parasites [6 8] (reviewed in [5]) and proteases of parasitic protozoa also seem to play a critical role in the survival and/or the virulence of the parasite [9-11]. Biochemical analysis has already indicated that adult and immature F. hepatica contain and release a considerable number of different proteases [2,3,12,13], but little is known about the molecular nature, genomic organisation and expression in various life-cycle stages of the genes encoding these proteases. In order to create the reagents that allow these points to be addressed, we decided to attempt to clone F. hepatica proteases. Degenerate primers, based on conserved structural motifs of cysteine proteases, have been shown to be powerful tools for the cloning of parasite protease genes or cDNA fragments of different organisms by PCR [10,14,15]. In this work, we used the same approach and describe the cloning by reverse transcription polymerase chain reaction (RTPCR) of cDNA fragments representing seven different F. hepatica cysteine proteases. One of these proteases was studied in more detail, using antibodies raised againts a GST-fusion protein.

2. Materials and methods

Parasites and bacterial strains.

Adult F. hepatica

were obtained from the bile ducts of infected cattle presented at the local slaughterhouse. The parasites were washed briefly in phosphate-buffered saline (PBS) and either stored at 4°C in PBS overnight prior to D N A or protein isolation, or used immediately for RNA preparation. Immature parasites were collected from the liver parenchyma of infected cattle. Rediae and cercariae were obtained from naturally infected snails. The metacercariae were a generous gift from C. Gaasenbeck (Centraal Diergeneeskundig Instituut, Lelystad, The Netherlands). NEJ were obtained from metacercariae by in vitro stimulation as described by Carmona et al. [4]. All different stages were homogenised in PBS in a glass homogeniser. The homogenates were centrifuged at 14 000 rev. rain-1 for 10 rain and supernatants stored at - 2 0 ° C . The E. coli strain XL1-Blue was used for cloning and also for expression of fusion proteins. E. coli strain Y1088 was used for growth and lysogeny of )~.

RNA purification. Adult F. hepatica flukes were disrupted in 4 M guanidine isothiocyanate by vigorous homogenisation using a Polytron homogeniser. Total RNA was extracted following the protocol of Chomczynski [16] with the exception that the RNA pellet was washed 3 times in 75% ethanol instead of once to improve solubility. The RNA pellet was finally dissolved in diethyl pyrocarbonate-treated H20 instead of 0.5% SDS. Poly(A) + RNA for cDNA synthesis was selected by oligo(dT) chromatography as described before [17]. Reverse transcription polymerase chain reaction and cloning of cysteine protease specific cDNA J~agments. Polyadenylated RNA isolated from adult flukes (1 #g) was converted into single-stranded cDNA using a oligo d(T)17 primer as described previously [17]. A 500 bp cDNA fragment was amplified by PCR from the single-stranded cDNA using two degenerate primers which correspond to highly conserved regions of eukaryotic cysteine proteases: A A dP5 (5' A C C G A A T T C C A G GGICAG T G cT G G TG T IAclTGc~-TGG 3') and dP3 (5' A C C G A A T T C C -

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11-23 GTA T A A CAIcAGTTcTTIACGATCCA~TA 3'); I = i n o s ine. EcoRI sites (underlined), which were used for cloning and 3 additional bases were added to the 5' ends of the primers. PCR using the temperature cycle profile described by Eakin et al. [15] did not yield fragments of a well-defined size. DNA fragments of the expected size were obtained, however, using the following temperature cycle: 95°C, 50 s; 50°C, 2 rain; 72°C, 3 min repeated 30 times followed by an extension of 72°C for 10 min. The Taq polymerase used was obtained from B6hringer, Mannheim. The PCR products were phenolextracted, precipitated with ethanol, digested with EcoRI and DNA fragments separated by electrophoresis in a 1.2% agarose gel. The 500 bp fragments were isolated by the Whatman paper method [18] and cloned into the EcoRI site of pUC 19 for sequence analysis.

Cloning of Fcpla by 3' RACE-PCR. cDNA synthesis and RT-PCR were carried out as described [19] using 1 #g polyadenylated RNA of adult F. hepatica and a hybrid d(T)lv-adapter primer as the starting material. The degenerate primer dP5 was used as gene-specific primer. The PCR product was extracted with phenol and chloroform, ethanol-precipitated and digested with EcoRI and SaII. D N A fragments were separated by electrophoresis and cDNA fragments contained in a single band of approx. 750 bp were isolated [18] and cloned into pUC 19. Cloning of Fcplb by 5' RACE-PCR. PCR was carried out following the protocol for nested 5' RACE-PCR described by Frohman [20]. cDNA was prepared from 1 #g poly(A) ~ RNA using the degenerate antisense primer dP3. The tailing reaction was performed in 0.5 × PCR buffer (Perkin Elmer Cetus) in order to limit the length of the synthesised poly(A) tails [21]. 1 #1 of the undiluted tailing reaction was used for PCR. The first extension step in the 5' RACE-PCR protocol described by Frohman [21] was reduced from 40 rain to 20 min The gene-specific primers P1 (5' GTCA C T T T G G C A T A T 3') and P2 (5' CCGGAATT C G T A T T G A C A C G G A C C 3') (see Fig. 4) were derived from the sequence obtainted from Fcpla. The amplification product of 750 bp was cloned

13

directly from the PCR reaction, using the TA cloning kit of Invitrogen. Positive clones were selected after hybridising colony filter lifts [22] with a Fcpl probe, radioactively labelled using the random prime labelling kit of Amersham.

Screening of a F. hepatica cDNA library. A cDNA clone corresponding to the sequence Fcplc (Fig. 3), called Fcpl2, was isolated by screening an adult F. hepatica 2gtl0 library [23] by plaque hybridisation, with a 32p-labelled Fcpl probe. Hybridisation conditions were as described for Southern blotting. DNA sequencing and sequence analysis. The nucleotide sequence of the plasmid inserts was determined by double-stranded sequencing according to the dideoxy chain termination method [24], using a Pharmacia T7 sequencing kit. Nucleotide and amino acid sequence analysis and comparison were carried out using the G C G programs version 7.2 [25] and the GenEMBL database (release 34). The G C G programs 'peptide structure' and 'pepplot' were used to search for the presence of a the putative signal sequence. D NA preparation and Southern blot analysis. High molecular weight genomic DNA was isolated from adult flukes as described before [26]. DNA was digested using different restriction enzymes, separated in 0.8% agarose gels, transferred to Hybond y membranes (Amersham) and prehybridised for at least 4 h in hybridisation buffer (0.5% heparin/ 5x SSC/ 0.1% SDS/ 0.5% pyrophosphate/ 5× Denhardt's). Filters were then hybridised overnight at 65°C with 32p-labelled cDNA probes. Filters were washed at 65°C in 0.1 x SSC/0.1% SDS and exposed for 3 days to X-ray film, Northern blot analysis. Adult F. hepatica RNA was separated by electrophoresis [19] and blotted onto Hybond N filters, which were hybridised and washed as described for Southern blot analysis. Expression of recombinant poylypeptides. The inserts of all different cDNA clones were subcloned in the expression vector pGEX [27]. Expression of

14

V.T. Heussler, D.A.E. Dobbelaere/Molecular and BiochemicalParasitology64 (1994) 11-23

the GST fusion proteins was induced by isopropyl fl-o-thiogalactoside and purification of GST:fusion proteins was done as described [27]. GST:Fcpl was used for further analysis and antibody production. Production of anti-GST:Fcpl antibodies in rabbits. The immunisation protocol consisted of 3 subcutaneous injections at 3 weekly-intervals, using 25 #g of the purified GST:Fcpl fusion protein, emulsified with complete Freund's adjuvant for the first immunisation and with Freund's incomplete adjuvant for boosting. Test sera were collected after each booster injection and were kept at - 2 0 ° C until used. Fasciola antigen preparation and immunoblotting. Adult F. hepatica flukes were homogenised in 7.5 ml of PBS. The homogenate was centrifuged at 18 000 rev. min 1, for 1 h at 4°C, the supernatant collected and centrifuged at 40 000 rev. min 1, for 1 h at 4°C. This supernatant was used as crude somatic antigen (S-Ag). Some flukes were kept overnight in PBS (4 ml/fluke) and the resulting fluid was used as a source of excretory-secretory antigen (ES-Ag). Crude S-Ag was fractionated by gel filtration column chromatography (Ultragel AcA 44 g, IBF; column size 3 x 40 cm, Pharmacia; flow rate 10 ml h-~). Fractionation resulted in five peaks (A,B,C,D,E). The approximate molecular weight of the peaks are: peak A >90 kDa, peak B ~85 kDa, peak C ~66 kDa, peak D ~45 kDa, peak E ~21 kDa. The eluate of each peak was pooled and concentrated by ultrafiltration (Diaflo ':R ultrafilters YM10, Amicon). Antigen preparations were then loaded (5 #g/lane) onto 0.75-ram-thick 12% SDSPAGE gels [29], electrophoresed at constant voltage (200 V/200 mA) for 45 min, and antigens electrophoretically transferred to nitrocellulose filters (200 V/300 mA). Immunoblot analysis was carried out by incubating filters with hyperimmune serum (diluted 1:200). The filters were washed extensively with TBS-Tween, incubated with a horseradish peroxidase conjugate (HR recombinant Protein G, diluted 1:2000, Zymed), developed using the enhanced chemiluminescence kit (ECL R, Amersham) and exposed to Hyperfilm

MP ~ (Amersham). Gelatin substrate S D S - P A G E analysis. Gelatinsubstrate SDS-PAGE was carried out as described [29]. Gels were run with const. 100 rnA for 2.5 h using a BRL submarine minigel system which was cooled in an ice bath during electrophoresis. After electrophoresis the gels were washed for 2 h in 0.1 M sodium citrate containing 2.5% Triton X-100 with two changes. The gels were then immersed in 0.1 M sodium citrate, pH 4.5/ 2 mM dithiothreitol for 24 h at 37°C. Gels were stained in 45% methanol/10% acetic acid containing 0.25% Coomassie Brilliant Blue R 250 and destained in 20% methanol/8% acetic acid.

3. Results and discussion

Generation ofF. hepatica cDNA fragments by RTPCR. RT-PCR using degenerate primers specific for cysteine proteases, carried out on F. hepatica RNA, yielded a population of cDNA fragments of approx. 500 bp in length, corresponding to the size predicted from published cysteine protease cDNA sequences. The amplification products were cloned into the vector puC19 and 86 different clones were sequenced. By analysing the sequences and their open reading frames, 7 different cysteine proteases, termed Fcpl-7, could be identified. The majority of the clones were identical to Fcpl. Other abundant clones were represented by Fcp2 and Fcp3. Clones identical to Fcp4, Fcp5, Fcp6 and Fcp7, on the other hand, were only represented in small numbers. The cysteine or thiol protease family can be subdivided into different types and includes the lysosomal proteases cathepsin B, H and L as typical members. The various family members differ in their substrate specificity and perform a variety of functions [30]. The predicted amino acid sequences of the different F. hepatica protease clones are shown in Fig. 1A and B. Fcpl-4 and Fcp6 correspond to the cathepsin L group whereas Fcp5 and Fcp7 are more related to the cathepsin B group. Fig. I A illustrates a comparison of the predicted amino acid sequence of Fcpl-4 and Fcp6 with human cathepsin L [31]. The histidine residue at po-

15

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11-23

A Fcplc

HUmCL

MRFFV.LAVL MNPTLILAAF .

Fcp6 Fcp4 Fcp3 Fcp2 Fcpl Fcplc

HUmCL

Fcp6 FCp4 Fcp3 FCp2 Fcpl Fcplc

HumCL

**

Fcp4 Fcp3 Fcp2 Fcpl Fcplc HumQL

,

L ........ W HQWKRIYNKE TFDHSLEAQW TKWKAMHNRL

**

.

**

•

FTDLTFEEFK FGDMTSEEFR

YNGADDEHRR NIWGKNVKHI QEHNLRHGLG LVTYKLGLNQ YGMNEEGWRR AVWEKNMKMI ELHNQEYREG KHSFTMAMNA . ** . • ** . . . **

.

.......................................... .......................................... .......................................... .......................................... .......................................... SELLSRGIPY KANKLAVPES IDWRDYYYVT EVKDQGQCGS PRKGKVFQEP LFYEA..PRS VDWREKGYVT pVKNQGQCGS . • *** *** ** * * * * *

AFSTTGTM AFATTGW AFSTTGAT AFSTTGAL AFSTTGAV CWAFSTTGAV CWAFSATGAL ********

EGQYMKKQRT EGQYSRKYGS EGQYMKNQRT EGQYMKSQRI EGQFRKNERA EGQFRKNERA EGQMFRKTGR ***

SISFSDEQLV DC SRPWGNNG KTGFSEQQLV DCRRRHGNEG SISFSEQQLV DCSRDFGNYG NISFSEQQLV DCSGDFGNHG SASFSEQQLV DCTRDFGNYG SASFSEQQLV DCTRDFGNYG LISLSEQNLV DCSGPQGNEG ** . * * * * * * * * **

.ESSYPYTAV .EGDYPYEAM .ESSYPYRAV .ESSYSYRAD .ESYYPYQAV .ESYYPYQAV SEESYPYEAT

NLVGSEGPAR SWSGTRGPVA NLVGAGRPAA NLIGVEGPAA NLVGTEDLPA NLVGTEDLPA KAVATVGPIS

SPVDVESDFM VGIHADDGFQ VALDVESDFM VALDVNIDFM VALDADSDFM VALDADSDFM VAIDAGHESF

M.YRSGIYQS F.YSHGIYVS M.YRSGIYQS M.YRSGIYQD M.YQSGIYQS M.YQSGIYQS LFYKEGIYFE * ***

.

Fcp6

TVGVFASNDD CLGI.ASATL

***

.

EGQCRYNEQL DNRCRANRTK EGQCRYNEQL EGPCQYDRQL EGPCQYDGRL EGPCQYDGRL EESCKYNPKy .

.

GVAKVTGYYT GIVKVKSYTV GVAKVTGYYT GVAQVSGYFI AYAKVTGYYT AYAKVTGYYT SVANDTGFVD

.

.

.

VHSGSEVELK LKNESETHSR VHSGDEVELQ VHSQDEVALK VHSGDEIELK VHSGDEIELK IPKQ.EKALM

**

.

*

*

QTCLPFALNH STCSSWPANH QTCSPDRLNH EICSSRYLNH QTCLPDRLTH QTCLPDRLTH PDCSSEDMDH *

,

.

***

AKYLIEIPRS QVMNGFQNRK

91 99

•

CGGGLMENAY CNGGLMTSSY CNGGLMENAY CSGGLMEKAY CGGGYMENAY CGGGYMENAY CNGGLMDYAF • ** . **

QYLKQFGLET RYLMNNSLES EYLKRFGLET EYLRHFGLET EYLKHNGLET EYLKHNGLET QYVQDNGGLD ** ***

AVLAVGYGT. GVLWGYGA. GVLAVGYGT. AVLAVGYGT. AVLAVGYGS. AVLAVGYGS. GVLVVGYGFE ** ***

.QDGTD.. .EANSP.. .QDGTD.. .EDGTD.. .QDGTD.. . Q D G T D Y W 285 STESDNNKYW 296 * **

191 197

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

.......................................... .......................................... .......................................... .......................................... I V K N S W G T W W G E D G Y I R F A R N R G N M C G I A S L A S V P M V A R F P. LVKNSWGEEW GMGGYVKMAK DRRNHCGIAS AASYPTV ..... ******* * * ** . * * ***** ** * *

326 333

B FCD5 ................... F GA. S T M S D R I Fcp7 ................... F GAVGAMSDRV SchiCB GCKSIATIRD QSRCGSCWSF GAVEAMSDRS . *** * * * * *

CIASQGKHTP CIHSKGQMKP CIQSGGKQNV ** . **

VLSAENMVDC HLSARDLLSC ELSAVDLLTC *** *** .

CTSCGMGCNG GGFPLKLG...SIGKKTRSC HRWFVRIERW MPTILVPSLR CEFCGRGCRG GSPALAWDYW KSSGIVTGGS LEEPTGCAPY PFPKCAHHG. CESCGLGCEG GILGPAWDYW VKEGIVTASS KE~GCEPY PFPKCEHHT. ***** **** . ***** * * * * ** • * * * * * * * * * * * **

Fcp7

TSRDWTETPC ..SSGGYKPC

NQDVT.TPAC KHTCRPGYNM TYQKDKWYAR TVYKVPADEH RIMRELLTNG PMEVSFEVYG DFPSYKSGVY PEEYY..PA ..............................................................

SchiCB

...KGKYPPC

GSKIYNTPRC

Fcp5

.

,

**

.

**

KQTCQRKYKT .

.

**

.

PYTQDKHRGK .

**

SSYNVKNDEK •

.

**

AIQKEIMKYG •

.

.

PVEASFTVYE • . ** **

199

Q

DFLNYKSGIY KHITGEALGG ** **** .

HAIRIIGWGV

296

Fig, 1. (A) Alignment of the predicted amino acid sequences for Fcpl, 2, 3, 4,6, encoding F. hepatica cysteine proteases, with the sequence of human cathepsin L (HumCL). Also included in the alignment are the complete amino acid sequence of Fcplc (composite of Fcpla and Fcplb). Amino acid residues representing the catalytic triad are indicated in bold type. The amino acids which are conserved are marked with an asterisks. Dots were introduced to improve alignment. (B) Alignment of the predicted amino acid sequences for Fcp5 and Fcp7 with the sequence of S. mansoni cathepsin B (SchiCB). Asterisks mark amino acid residues which are conserved between at least one F. hepatica sequence and the S. mansoni sequence. Potential glycosylation sites are underlined. sition 276 o f the human cathepsin L, which forms part o f the catalytic triad and is required for ionpair formation with the active site cysteine (position 138) [32], is conserved in all sequences. The 6 cysteines o f human cathepsin L that can be found in the region o f comparison all aligned perfectly with the cysteine residues in the F. hepatica sequences, suggesting a similar tertiary structure. In addition, the glycine at position 181 o f the h u m a n cathepsin L, which is involved in substrate bind-

ing, is also present in all F. hepatica sequences. In Fig. 1B, the predicted amino acid sequences o f Fcp5 and Fcp7 are compared with S. mansoni cathepsin B [33]. In contrast to the cathepsin L group, cathepsin B-like proteases have potential N-glycosylation sites (underlined residues in Fig. 1B) and more conserved cysteine residues. Nine o f 11 cysteine residues present in the corresponding S. mansoni sequence are conserved in Fcp5. All cysteine residues are also conserved in the region

16

V.T. Heuss&r, D.A.E. Dobbe~ere/ Mo~cular and Biochemical Parasito~gy 64 (1994) 11 23 Protein

FCp1 Fcp2 o

~ 7 0 79

comparison

%identity

(78) 78 (86) 47 (61) 30 (49) 73 [83 ~

Fcp3

85

84

57

59

61

FCp5

<50

53

<50

FCp6

84

82

88

59

50

FCp7

56

57

55

<50

64

SchICB

30 (53 49 (62) 29 (53)

78 (84) 48 (61) 35 (51) 72 (81) 31 (56) 49 (64) 36 (54)

Fcp4

HIImCL

(%similarity)

~

51 (61) 32 (53) 84 (89) 37 (50) 50 (64) 30 (53 ~

36 (47) 49 (63 55

~

34 (43

47 (63) 32 (50

30 (50) 36 (55) 45 (51) 38 (54 ~

29 (52) 47 {65] 30 (52) 51

~

29 {53] 56 (63

57

57

57

56

<50

56

<50

<50

<50

<50

<50

55

<50

63

~ 2 9

(54)

<50

Fig. 2. Amino acid identities and similarities and nucleotide homology (expressed in %) obtained by comparison of cathepsin L- and B-like cysteine proteases of F. hepatica, S. mansoni (SchiCB) and homo sapiens (HumCL). The identity and similarity (in brackets) [25] between the different amino acid sequences are shown in the boxes above the diagonal; homology at the nucleotide level is shown below.

of overlap between Fcp7 and S. mansoni cathepsin B. The region of Fcp5 corresponding to amino acid 160 to 204 of the S. mansoni cathepsin B differs markedly from Fcp7 and S. mansoni cathepsin B. Homology resumes again from amino acid 205 to the end of the sequences. Such interspersed regions of low amino acid homology are not unusual and have also been described for other cathepsin B-like proteases [15]. Fig. 2 lists the homologies at the nucleotide and amino acid sequence level for the different F. hepatica proteases, human cathepsin L and S. mansoni cathepsin B. The overall amino acid identity between the F. hepatica proteases Fcpl, 2, 3, 4, 6 and human cathepsin L varied from 47% to 50%; the similarity ranged from 62% to 65%. Interestingly, Fcp5, based on amino acid identity, could also be classified in the cathepsin L group. The higher nucleotide homology and amino acid similarity compared to S. mansoni cathepsin B, and the conserved positions of 9 cysteine residues, however, justify its classification in the cathepsin B group. Cloning of the 3' and 5' ends of Fcpl by RACEPCR. The amplification strategy is summarised

in Fig. 3. Amplification by RACE-PCR of the 3' end of the cDNA corresponding to Fcpl resulted in cDNA fragments of approx. 750 bp which were

cloned into pUC 19. Sequence analysis confirmed that the 3' end of Fcpl had been cloned. The 3' E H S "Fcp$c"

M

K MMSS

'II

I

///

(E)

[

......

(L)

FWo I

~

dP5 ..~

AL

Fcplb

,

' ~ Re3

4

(E) dP5 ~

(E) ~

dP3

100 bp

Fig. 3. Strategy for cloning of a complete Fcpl cDNA. 'Fcplc' corresponds to the complete cDNA. The part of Fcplc shown in black corresponds to the region of overlap between clone Fcpla and clone Fcplb. The single letters indicate restriction sites which were used for cloning or Southern blot analysis (E, EcoRl; H, Hindlll; S, Sau3Al; M, Mspl, K, Kpnl; L, Sall). The restriction sites between brackets at the 5' and 3' ends of Fcpla and Fcpl indicate artificial restriction sites added to the primers. The A's at the ends of clone Fcplb indicate that the 'TA cloning system', was used. The arrows labelled Ro, Ri, PI, P2, dP5, Re3. dP3 represent the primers used for PCR. The white blocks at the 3' end of clone Fcpla and at the 5' end of clone F c p l b illustrate the two d(T),7 adapter primers which were used for RACE-PCR. Fcpl illustrates the cDNA which was amplified using the degenerate primers dP5 and dP3 and which was cloned into the vector pGEX for expression as a fusion protein in E. coil

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11-23

17

TGAGTAAA~AG~CAA~GATAACAATCAAA~GAT~GG~CTT~TATTAGCC~TC~TCACGGTCGGAGTGTTCGCCTCGAATGACGAT~GTGGCATC loo M R F F V L A V L T V G V F A A S N D D L W H Q 2 3 AATGGAAA~GAATATA~AATAAAGAATATAATGG~G~TGAC~TGAG~A~AGACGAAATAT~GGGG~AAAAA~G~GAAACATATCCAAGAAcACAACCT 2OO W K R I Y N K E Y N G A D D E H R R N I W G K N V K H I Q E H N L 56

ACGTcAC~GTCTCGG~CTC~TCACCTACAAG~GGGA~GAACCAA~CA~TGATTTGACA~GAGGAATTCAAGGCCAAATATCTAATAGAAATCCCA 3OO R H G L G L V T Y K L G L N Q F T D L T F E E F K A K Y L I E I P 89 CG~TcATCTGAG~ACTCTCAcGCG~TATcCCGTATAAGGCGAACAAGC~GCCGTAcCCGA~AGCA~GACTGG~GTGACTA~A~ATGTGACTGAGG ~00 R S S E L L S R G I P Y K A N K L A V P E S I D W R D Y Y Y V T E V I 2 ? dP5 T~AA~ATCAGGGAcAAT~TGG~CCTG~GGGCT1~CTCAACAACCGGTG~TGTGGAGGGACAG~TA~AAGAACGAAAGA~CTA~TGC~CA~CTC 5OO K D Q G Q C G S C W A F S T T G A V E G Q F R K N E R A S A S F S I 5 6 T~AGCAACAACTGG~CGA~GTACcC~TGATTT~GGcAA~ATGGTTGCGGTGGAGGATATATGGAAAACGCTTATGAATA~T~GAAACACAAC~ATTG 600 E Q Q L V D C T R D F G N Y G C G G G Y M E N A Y E Y L K H N G L 189 P2

Pl

GAAAcTGAGT~CTA~ATc~ATACCAGG~TGTG~AAGG~CC~TGTCAA~A~ATGGGcGG~GG~ATAT~CCAAAGTGACTG~CTACTATA~TGTGCA~700 E T E S Y Y P Y Q A V £ G P C Q Y D G R L A Y A K V T G Y Y T V H S 2 2 3 , cTG~c~ATGA~ATA~A~A~AAT~T~TCGGTACC~AAGACCT~cCG~CGGTCGC~ATGCGGA~CT~AC~CA~GATGTA~CAGA~TG~TAT 8oo G D E I E L K N L V G T E D L P A V A L D A D S D F M M Y Q S G I 2 5 6 ~ATCAGA~C~AAAC~GT~-~ACCG~ATCGC~GACTCAT~CAGTC~G~CTGTCGG~TAT~ATCACAAGA~GGTACTGACTA~G~A~T~AAAAAT 900 Y Q S Q T C L P D R L T H A V L A V G Y C S Q D G T D Y W I V K N 289 A~AACGT~GT~GT~A~AcG~ACA~GT]~CCAG~AAcCGAGGTAATAT~T~T~GAA~GC~TCTCTGGCCAGT~TCcC~ATG~TGG iooo S W G T W W G E D G Y I R F A R N R G N M C G I A S L A S V P M V A 3 2 3 CAC~ATTrCC~T~ATAATT~cT~TCA~AT~AAAACGCAATGAACAATAAATCTCACTCGG~CTT~CAAAAAAAAAAAAAAAAAAAAAAA1091 R F P * *

Fig. 4. Nucleotide and predicted amino acid sequence of Fcplc. The sequence shown is a composite of sequences obtained from Fcpla and Fcplb. The asterisks at the end of the amino acid sequence show stop codons. The arrow dP5 represents the degenerate sense primer used ~ r amplification of the 3' end. ( ~ r exact sequense of dP5, see Material and Methods). The arrows PI and P2 represents the gene-specific antisense primers used for amplification of the 5' end. The arrowhead between the amino acid A and S at position 15 and 16 illustrates the putative cleavage site of the signal peptide. The position of a potential polyadenylation signal, AATAAA is underlined (GenBank accession number Z22765).

clone, called Fcpla, was identical in the region of overlap with Fcpl and also contained a potential polyadenylation signal and part of a poly(A) tail (Fig. 4). In order to clone the missing 5' end, the nested RACE-PCR method described by Frohman was chosen [20]. This method strongly favours specific amplification and is based on the use of a gene-specific primer for cDNA synthesis (primer dP3) and two gene-specific primers for PCR (P1, P2). P1 and P2 (Fig. 4) were designed using the sequence information obtained from clone Fcpl and Fcpla. After two rounds of amplification of 30 cycles each, a single DNA fragment of approx. 750 bp was obtained. The amplification product was ligated in the TA cloning vector and sequenced. The resulting clone, Fcpl b, overlapped by 243 bp with the clones Fcpl and Fcpla and also contained the 5' end of the cDNA. Five mismatches were detected in the region corresponding to the degenerate primer dP5 which was used for the amplification of Fcpl and Fcpla. The combined sequence of the 5' clone Fcplb and the 3' clone Fcpla ic called Fcplc, and is 1.1 kb in length. It contains an open reading frame, start-

ing with an ATG at position 34 (Fig. 4) encoding a protein of 326 amino acids with a predicted molecular weight of 38 kDa. A stretch of 15 hydrophobic amino acids that could function as a signal sequence can be found at the N-terminus of the predicted peptide. Computer analysis predicts that this signal sequence [34] would be cleaved between the amino acids alanine and serine (position 15 and 16 indicated by an arrow). In analogy to other proteases, the six cysteine residues present in the Fcplc sequence, could form disulphide bridges between positions 129 and 172, 163 and 204 and between 262 and 311. A typical AATAAA polyadenylation signal is present at position 1047 1052. The total length of Fcplc is shorter than the size of the transcript (1.2-1.3 kb) detected by the probe Fcpl in northern blot analysis (Fig. 5). This could reflect either the fact that the 5' untranslated region is not complete, or could be due to differences in length of the poly(A) tail as has also been observed for the m R N A of the 'hemoglobinase' of S. mansoni [35]. In this case, it was shown that the poly(A) tail of the 'hemoglobinase' m R N A of adults was longer

18

V.T. Heussler, D.A.E. Dobbelaere/Molecularand BiochemicalParasitology64 (1994) 11-23

p ~4.4

kb

7.4 1.4

~0.2 Fig. 5. Northern blot analysis of total RNA isolated from adult flukes. A total of 65/~g RNA was first denatured in one tube and then subjected to 1.4% agarose gel electrophoresis(5/~g/ lane) m presence of ethidium bromide and transferred to a nylon membrane. After blotting the membrane was cut in strips for hybridisation with the different probes. The filter strips were probed with radiolabelled fragments of clones Fcpl, 2, 3, 4, 5, 6, 7. After hybridisation, the filter strips were exposed to X-ray film for 16 h. The migration of RNA marker is indicated (sizes in kb). than in juvenile flukes. Isolation of an Fcpl cDNA clone from a cDNA library. An adult F. hepatica c D N A library, generated in 2gtl0 [23] was screened with a radiolabelled Fcpl probe. A large number of plaques hybridised to the probe reflecting the abundance of the corresponding mRNA's. Sequencing of the ends of a number of these clones revealed the existence of several additional cysteine proteases, thereby confirming the existence of a large cysteine protease gene family in F. hepatica. The clone with the largest insert, t e r m e d Fcpl2 was sequenced completely. The sequence of F c p l 2 began at position 14 of the clone F c p l c and shared 99.7% identity with Fcplc. The minor differences in the nucleotide sequence did not disturb the reading frame. The mismatches could in part be caused by misincorporation by the Taq D N A polymerase during PCR, but, perhaps more likely, could also be due to population polymorphism between different strains of F. hepatica. That this could be the case is supported by the fact that the parasites used for generating the 2gtl0 library and those from which R N A was isolated for R T - P C R

originated from different continents [23] and could therefore belong to different strains or variants. In this regard, Yamasaki [36] has reported that different Fasciola species, such as F. hepatica and F. gigantica exist in Japan, but that intermediate forms of these two species could also be found. Northern blot analysis. Total RNA extracted from adult F. hepatica was examined by Northern blot analysis using Fcpl-7 as probes (Fig. 5). When probes Fcpl, 2, 3 and 6 were used, transcripts of 1.2-1.3 kb could be detected, which gave strong signals. In contrast, much weaker hybridisation signals were observed using Fcp4, 5 and 7 as probes. Transcripts hybridising to Fcp4 were approx. 1.5 kb in size. Fcp5 and Fcp7 hybridised to transcripts of 1.5 kb and 1.4 kb, respectively. Southern blot analysis. Southern blot analysis of genomic F. hepatica D N A using probes Fcpl-7 revealed two clearly different patterns of hybridisation (Fig. 6). Fcp4, 5 and 7 hybridised to a limited number of fragments, resulting in a simple pattern with relatively weak signals. Filters hybridised with Fcpl, 2, 3 and 6, on the other hand, showed a complex banding pattern with strong signals. The EcoRI patterns observed after hybridisation with Fcpl, 2, 3 and 6, were almost identical, but differences in the relative intensity of the hybridisation signals could be observed for individual bands. Hybridisation of the MspI-digested DNA, on the other hand, clearly showed restriction fragment length polymorphism, confirming that the probes detect different genes. The differences in intensity of the hybridisation signals obtained with the probes Fcp l, 2, 3 and Fcp6, compared to Fcp4, 5 and 7 strongly suggest that these genes are present in multiple copies. It is also worth noting that a strong correlation exists, between the signal intensities obtained by Southern blot analysis and the abundance of transcripts revealed by Northern blot analysis. Although filters were hybridised and washed under stringent conditions to reduce cross-hybridisation, the strong signals may, however, also partly result from cross-hybridisation due to the relatively high nucleotide homology which exists between Fcpl, 2,

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11 23

Mspl

EcoRI t',, (.0 tO ~,- o,) rV

gppp

e

kb 21

p

Sau3AI t,,, (.o t.q ~

pp p

¢'o

~

kb

i

kb m

I

5.1

5.1 42 315

t ~ ~ Z.01~.97.

t 'O

0,5

~

21

21

4.2 3.5

2.0 1.9 1.7 1.3 0.9 0.8

19

m

5.1

4.2

3.5

--

2.0 1,9 1.7

1.3

....

~ : #

0.9

0.9 0.8

0.5

0.5

i

Fig. 6. Southern blot analysis of F hepatica genomic DNA. For each gel, 52 #g of genomic F. hepatica DNA was digested with EcoRl, Mspl, or Sau3Al, respectively. 4 #g of digested D N A was loaded per lane. After gel electrophoresis, the DNA was transferred to a nylon membrane and cut in single strips. One filter strip of each digest was hybridised with a 32p-labelled cDNA probe corresponding to Fcpl-7. After hybridisation, filters were wasbed at high stringency and exposed to X-ray film for 2 days.

3 and 6 (Fig. 2). The organisation of protease genes in multiple copy gene families has also been found in other parasites such as S. mansoni and Haemonchus contortus [5,37,38]. Furthermore, a variety of different proteases have also been described at the protein level in several other parasites, such as Trypanosoma and Ascaris [39,40] to name only a few. Although these proteases may all have different functions in these organisms, the presence of multicopy protease gene families in different parasites may reflect common adaptation mechanisms which are required for parasite survival during their life cycles. Characterisation of the Fcpl protease by immunoblot analysis. The Fcpl fragment was subcloned into the pGEX vector and expressed as a GSTfusion protein in E. coll. The GST:Fcpl fusion protein was purified by affinity chromatography and used to generate antibodies in rabbits. Rabbit antiserum was used in immunoblot analysis of different F. hepatica preparations such as F. hepatica adult somatic antigen (S-Ag), size-fractionated F. hepatica antigen preparations (peak A-E) and also

secreted F. hepatica antigen (E/S-Ag) (Fig. 7 upper panel). The antiserum reacted specifically with a 30 kDa protein abundantly present in the S-Ag, in peak C, D and E antigen franctions and also in the E/S Ag fraction. An additional band of 38 kDa was also clearly recognised in S-Ag and peak C. Longer exposure also revealed bands in the range of 28 kDa, represening the different F. hepatica GST proteins (see below, Fig, 8). Proteases are proteolytically processed from a precursor form (proenzyme) to the mature enzyme. The band at 38 kDa possibly represents the proenzyme of the mature 30 kDa enzyme. The fact that only the 30 kDa protein is present in the E/SAg, and hence is secreted, is consistent with it being the mature form. Furthermore, proteolytic activity in substrate gels (peak C) is visible at 30 kDa, but not at 38 kDa (Fig. 7, lower panel). Stage-specific gene expression detected by immunoblot analysis. The antiserum raised against the GST:Fcpl fusion protein detected antigens with different molecular weigths in the different F. hepatica life cycle stages (Fig. 8 upper panel), A single protein of 48 kDa was recognised in the

20

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11-23

,.6 106

80

80--

49,5 49.5 --

32.5

32.5 --

27.5

27.5 --

18.5

18.5--

106 80 49.5 32.5

mI-

!

_

80-49.5 1

32.5 -27.5 --

I

27.5 18.5--

18.5 Fig. 7. Immunoblot analysis of adult F. hepatica antigens using anti-GST:Fcpl antiserum (upper panel). 5 ~g of crude somatic antigen (S-Ag), size fractionated protein extracts (peak A, B, C, D, E), and excretory/secretory antigen (E/S-Ag) were separated by gel electrophoresis, blotted and then probed with rabbit immune serum raised against GST:Fcpl. The predicted mature Fcpl protein (30 kDa) and the putative propeptide (38 kDa) are marked with arrows. Molecuar weight markers are indicated in kDa. (Lower panel): gelatine substrate SDSPAGE analysis of adult F. hepatica proteases. 0.5 #g protein of the protein fractions described above were loaded per lane and submitted to electrophoresis under non-reducing conditions. The protease activity at 30 kDa is marked by an arrow.

extracts of rediae and cercariae. In the lanes containing metacercarial extracts, a strong signal is visible at 32 k D a and additional bands could be seen at 52, 56 and 72 kDa. Proteins of 3l, 50 and 62 k D a are present in NEJ. Two double bands of 30/31 k D a and 37/38 k D a were predominant in antigen preparations of immature flukes, and 2 additional weak bands appeared at 27/28 kDa. A weak signal at 78 k D a could also be detected in adults, which became more obvious after longer exposure (not shown). F. hepatica is known to ex-

Fig. 8. Immunoblot analysis of extracts made from different life-cycle stages of F. hepatica. Filters with extracts of different stages of the parasite (5 /tg/lane) were probed with antiGST:Fcpl antiserum (upper panel). The same filter was then stripped and reprobed with anti-GST antibodies (lower panel). The 30 kDa protein and the putative propeptide (38 kDa) are marked with arrows. The GST doublet of 27/28 kDa is shown by an arowhead. Molecuar weight markers are in kDa.

press different forms of the detoxifying enzyme G S T [4l] and it can be expected that the antiG S T : F c p l serum also recognises Fasciola GST. The same filter was therefore stripped and reprobed with anti-GST antiserum. Reprobing confirmed that the 27/28 kDa proteins represent F. hepatica G S T (Fig. 8, lower panel). The 62 k D a band in N E J and the 78 k D a band in mature F. hepatica were also recognised by the anti-GST antibodies. This was unexpected, since all known G S T proteins described so far, have a molecular weight of less than 30 kDa. The exact nature of these proteins is not known. It is conceivable that they represent other molecules which have epitopes in common with GST. Alternatively, they could represent G S T in complex with other molecules. The fact that the mature parasite expresses

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11-23

GST is not surprising, since it lives in the bile ducts, an environment where large amounts of toxic substances are present. On the other hand, GST is also required for the detoxification of endogenous molecules and could therefore be expected to be present in all parasitic stages as is the case for S. mansoni [42]. It is therefore puzzling that GST could not be detected in the migrating immature F. hepatica flukes. Whichever way, the data presented in Fig. 8 strongly suggest that both the proteases detected by anti-GST:Fcpl antibodies and GST are expressed or processed in a stage-specific manner. This event is presently being studied in more detail. Proteases seem to play an important role for the survival of F. hepatica in the definite host and possible functions have already been suggested. Some investigators localised a 27-kDa protease of Fasciola sp. within the secretory granules [43]. Others described various F. hepatica proteases which are released by the parasite [3]. It has been proposed that F. hepatica proteases may play an important role in the extracellular degradation of host proteins, including hemoglobin [43]. Hemoglobinase activity has also been described by other authors and it is assumed that it might play a role in parasite nutrition [2,13]. In this regard, Dalton [3] found that some proteases secreted by immature and adult F. hepatica have an optimum of activity at physiological pH, in contrast to the gut-related proteases which function optimally at a pH of 3 4 . Released proteases could also help the parasite to counteract the immune system of the host. Chapman and Mitchell [2] reported on the cleavage of immunoglobulins by a F. hepatica protease. Other authors have described a cathepsin L-like protease, which prevents the adherence of eosinophils to NEJ [4] and is present in the E/S-Ag fraction of juvenile and adult flukes. Howell [1] demonstrated that immature F. hepatica release enzymes in vitro that are capable of cleaving collagen and postulated that these enzymes are involved in the penetration of the liver tissue. In our studies, antibodies directed against Fcpl also recognised secreted F. hepatica proteases. It is not yet clear, however, whether any of the activities listed above can be attributed to Fcpl or one of the other cysteine proteases.

21

Mammals express a battery of protease inhibitors which could be involved in blocking of the proteases of parasites and other infectious agents such as pathogenic fungi, bacteria and viruses [5]. Differences in the presence of such inhibitors could explain why animals, infected with F. hepatica, are divided into 'resistant' (cattle, rats) and 'susceptible' (sheep, mice) hosts. A heavy infestation of F. hepatica metacercaria can lead to the death of 'susceptible' hosts whereas 'resistant' hosts survive a comparable challenge. Perhaps, 'resistant' hosts express more effective protease inhibitors than 'susceptible' animals do and may thus inactivate the parasite proteases in a biochemical way rather than only by immunological mechanisms. This could result in a more limited damage in the 'resistant' host's tissues during the migration of the parasite, whereas the 'susceptible' host could suffer of extensive lesions. This theory of protease inhibition is supported by studies of Hill and Hastie [44] who determined the genetic drift of protease inhibitors in vertebrates; they found a remarkably high rate of mutation at the active site of these inhibitors and postulated that the selective pressure for this change was primarily due to exposure of the host to proteases derived from parasites and other infectious agents. In summary, the discovery of a large F. hepatica protease gene family, the abundance of steady state m R N A and the extensive protease activity in parasite extracts, all suggest an important role for these enzymes in F. hepatica. The localisation and the function of the different proteases of F. hepatica, described in this work, have yet to be elucidated. The availability of cloned protease cDNA fragments and specific antibodies, however, should facilitate further studies on the characterisation of proteases in F. hepatica. In addition, proteases could provide a model for studying gene regulation in a parasite which undergoes many morphological changes and could also be targets for biochemical intervention with enzyme inhibitors.

4. Acknowledgements This work is supported by a grant from the

22

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11 23

Swiss National Science Foundation (NF No. 3128712.90) and a generous donation for equipment by the Victor and Erna Hasselblad Foundation (Sweden). We thank Bruno Gottstein for continuous support and Richard Felleisen for critical reading of this manuscript. Jeremy Mottram (Wellcome Unit for Molecular Parasitology, Glasgow) is thanked for the PCR primers dP3 and dP5, Mario Zurita (Universidad Nacional Autonoma De Mexico) for providing the F. hepatica cDNA library and Cor Gaasenbeck (Centraal Diergeneeskundig Instituut, Lelystad) for the metacercariae.

5. References [1] Howell, R.M. (1966) Collagenase activity of immature Fasciola hepatica. Nature 209, 713~ 14. [2] Chapman, C.B. and Mitchell, G.F. (1982) Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica. Vet. Parasitol. 11, 165-178. [3] Dalton, J.P. and Heffernan, M. (1989) Thiol proteases released in vitro by Fasciola hepatica. Mol. Biochem. Parasitol. 35, 161~5. [4] Carmona, C., Smith, A., Dowd, A. and Dalton, J.P. (1992) A Fasciola hepatica cathepsin L proteinase prevents the adherence of eosinophils to newly excysted juveniles. Biochem. Soc. Trans. 20, 86S. [5] McKerrow, J.H. and Doenhoff, M.J. (1988) Schistosome proteases. Parasitol. Today 4, 33,1~340. [6] Mazingue, C., Camus, D., Dessaint, J.-P., Capron, M. and Capron, A. (1980) In vitro and in vivo inhibition of mast cell degranulation by a factor from Schistosoma mansoni. Int. Arch. Allergy appl. Immunol. 63, 178. [7l Marikovsky, M., Arnon, R. and Fishelson, Z. (1988) Proteases secreted by transforming schistosomula of Schistosoma mansoni promote resistance to killing by complement. J, lmmunol. 141,273-278. [81 Chabaudie, N. and Boulard, C. (1992) Effect of hypodermin A, an enzyme secreted by Hypoderma lineatum (Insect Oestridae), on the bovine immune system. Vet. Immunol. Immunopathol. 31, 167 177. [9] Tannicb, E., Scholze, H., Nickel, R. and Horstmann, R.D. (1991 ) Homologous cysteine proteinases of pathogenic and nonpathogenic Entamoeba histolytica. Differences in structure and expression. J. Biol. Chem. 266, 4798-4803. [10] Souza, A.E., Waugh, S., Coombs, G.H. and Mottram, J.C. (1992) Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana. FEBS Lett. 311, 124~127. [11] North, M.J. (1992) The characteristics of cysteine proteinases of parasitic protozoa. Biol. Chem. Hoppe Seyler 373, 401-406.

[12] Simpkin, K.G., Chapman, C.R. and Coles, G.C. (1980) Fasciola hepatica: a proteolytic digestive enzyme. Exp. Parasitol. 49, 281 287. [13] Rege, A.A., Herrera, P.R., Lopez, M. and Dresden, M.H. (1989) isolation and characterization of a cysteine proteinase from Faseiola hepatica adult worms. Mol. 8iochem. Parasitol. 35, 89 95. [14] Sakanari, J.A., Staunton, C.E., Eakin, A.E., Craik, C.S. and McKerrow, J.H. (1989) Serine proteases from nematode and protozoan parasites: isolation of sequence homologs using generic molecular probes. Proc. Natl. Acad. Sci. USA 86, 4863-4867. [15] Eakin, A.E., Bouvier, J., Sakanari, J.A., Craik, C.S. and McKerrow, J.H. (1990) Amplification and sequencing of genomic DNA fragments encoding cysteine proteases from protozoan parasites. Mol. Biochem. Parasitol. 39, 1-8. [16] Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-choroform extraction. Anal. Biochem. 162, 15(~ 159. [17] Heussler, V.T., Eichhorn, M., Reeves, R., Magnuson, N.S., Williams, R.O. and Dobbelaere, D.A. (1992) Constitutive IL-2 mRNA expression in lymphocytes, infected with the intracellular parasite Theileria parva. J. Immunol. 149, 562 567. [18] Errington, J. (1990) A rapid and reliable one-step method for isolating DNA fragments from agarose gels. Nucleic Acids Res. 18, 5324. [19] Heussler, V.T., Eichhorn, M. and Dobbelaere, D.A.E. (1992) Cloning of a full-length cDNA encoding bovine interleukin 4 by polymerase chain reaction. Gene 114, 273 278. [20] Frohman, M.A. (1990) Rapid amplification ofcDNA ends (RACE): user-friendly cDNA cloning. Amplifications 5, 11-15 [21] Schuster, D.M., Bucbman, G.W. and Rashtchian, A. (1992) A simple and efficient method for amplification of cDNA ends using 5' RACE. Focus 14, 4652. [22] Maniatis, T., Fritscb, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [23] Zurita, M., Bieber, D., Ringold, G. and Mansour, T.E. (1987) Cloning and characterization of a female genital complex cDNA from the liver fluke Fasciola hepatica. Proc. Natl. Acad. Sci. USA 84, 2340-2344. [24] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. [25] Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the Vax. Nucleic Acids Res. 12, 387-395. [26] Heussler, V., Kaufmann, H., Strahm, D., Liz, J. and Dobbelaere, D. (1993) DNA probes for the detection of Fasciola hepatica in snails. Molecular and Cellular Probes. Mol. Cell. Probes 7, 261-267 (1993). [27] Smith, D.B. and Johnson, K.S. (1988) Single-step purification of polypeptides expressed in Escherichia coil

V.T. Heussler, D.A.E. Dobbelaere/Molecular and Biochemical Parasitology 64 (1994) 11-23

as fusions with glutathione S-transferase. Gene 67, 314-40. [28] Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. [29] Fagbemi, B.O. and Hillyer, G.V. (1991) Partial purification and characterisation of the proteolytic enzymes of Fasciola gigantica adult worms. Vet. Parasitol. 40, 217-26. [30] Barrett, A.J. and Salvesen, G. (1986) Proteinase Inhibitors. Elsevier, Cambridge. [31] Joseph, L.J., Chang, L.C., Stamenkovich, D. and P., S.V. (1988) Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L: An abundant transcript induced by transformation of fibroblasts. J. Clin. Invest. 81, 1621-1629. [32] Lewis, S.D., Johnson, F.A. and Shafer, J.A. (1981) Effect of cysteine-25 on the ionization of histidine-159 in papain as determined by proton nuclear magnetic resonance spectroscopy. Evidenc for a his-159-cys-25 ion pair and its possible role in catalysis. Biochemistry 20, 48-51. [33] Klinkert, M.Q., Felleisen, R., Link, G., Ruppel, A. a 't Beck, E. (1989) Primary structures of Sm31/32 diagnostic proteins of Schistosoma rnansoni and their identification as proteases. Mol. Biochem. Parasitol. 33, 113-122. [34] von Heijne, G. (1983) Patterns of amino acids near signalsequence cleavage sites. Eur. J. Biochem. 133, 17-21. [35] El Maenawy, A.M., Aji, T., Phillips, N.F.B., Davis, R.E., Salata, R.A., Malhotra, I., McClain, D., Aikawa, M. and Davis, A.H. (1990) Definition of the complete Schistosoma mansoni hemoglobinase m R N A sequence and gene expression in developing parasites. Am. J. Trop. Med, Hyg. 43, 67 78. [36] Yamasaki, H., Aoki, T. and Oya, H. (1989) A cysteine proteinase from the liver fluke Fasciola spp.: purification,

[37]

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[43]

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characterization, localization and application to immunodiagnosis. Jpn. J. Parasitol. 38, 373-384. Pratt, D., Cox, G.N., Milhausen, M.J. and Boisvenue, R.J. (1990) A developmentally regulated cysteine protease gene family in Haemonchus contortus. Mol. Biochem. Parasitol. 43, 181-191. Cox, G.N., Pratt, D., Hageman, R. and Boisvenue, R.J. (1990) Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms. Mol. Biochem. Parasitol. 41, 25-34. Lonsdale Eccles, J.D. and Mpimbaza, G.W. (1986) Thioldependent proteases of African trypanosomes. Analysis by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels co-polymerized with fibrinogen. Eur. J. Biochem. 155, 469473. Knox, D.P. and Kennedy, M.W. (1988) Proteinases released by the parasitic larval stages of Ascaris suum, and their inhibition by antibody. Mol. Biochem. Parasitol. 28, 207-216. Hillyer, G.V., Soler de Galanes, M. and Battisti, G. (1992) Fasciola hepatica: host responders and nonresponders to parasite glutathione S-transferase. Exp. Parasitol. 75, 176186. Sher, A., James, S.L., Correa-Oliveira, R., Hieny, S. and Pearce, E. (1989) Schistosome vaccines: Current progress and future prospects. Parasitology 98, 61 68. Yamasaki, H., Kominami, E. and Aoki, T. (1992) Immunocytochemical localization of a cysteine protease in adult worms of the liver fluke Fasciola sp. Parasitol. Res. 78, 574~580. Hill, R.E. and Hastie, N.D. (1987) Accelerated evolution in the reactive centre regions of serine protease inhibitors. Nature 326, 12-13.