Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni

MOLECULAR

ELSEVIER

ECHENKAL PARASITOLOGY Molecular

and Biochemical

Parasitology

73 (1995) 7-18

Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni Anja Michel a, Hossam Ghoneim b, Maristella Resto bpl, Mo-Quen Klinkert b, Werner Kunz a,* a Institut fir Genetik and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universitiit, Universitiitsstrape D-40225 Diisseldorf, Germany b Istituto di Biologia Cell&are, Consiglio Nazionale delle Ricerche, Viale Marx 43, 00137 Rome, Italy Received 29 November

I,

1994; accepted 31 May 1995

Abstract A cDNA encoding Schistosoma mansoni cathepsin L was isolated from a cDNA library and sequenced. Alignment of the proposed amino-acid sequence with known members of cathepsin L shows highest homologies with sequences from mouse and rat. An expression plasmid was constructed in Escherichia coli to produce recombinant schistosome cathepsin L with an extension of six histidines at its N terminus. Using antibodies raised against the purified fusion protein, two polypeptide bands with approx. molecular masses of 38 and 31 kDa were identified in a schistosome extract. By use of specific radioiodinated inhibitors, a radioactively labeled protein could be detected at 31 kDa, suggesting that this is the active mature enzyme. The larger protein of 38 kDa did not react with the inhibitor, indicating that it represents the inactive precursor molecule. Immunohistological experiments revealed that the proteinase is localized in structures associated with the reproductive system of females and with the subtegumental region of the gynecophoric canal of males. However, Northern blot hybridization demonstrates that more transcripts are present in female parasites than in males. Genomic Southern blotting suggests that schistosome cathepsin L is expressed from a single-copy gene. Keywords:

Schistosoma

mansoni; Cysteine proteinase;

Cathepsin

L; Cathepsin

B; Immunohistology

1. Introduction

Abbreviations: DTT, dithiothreitol; LB, Luria-Bertani broth medium. Note: The sequence data reported in this paper have been submitted to the GenBank TM data base with the accession number z-32529. * Corresponding author. Tel. and Fax: (49-211) 311-2333; e-

mail: Wemer.Kunz@uni-duesseldorfde ’ Present address: Department of Biology, University of Puerto Rico, San Juan, Puerto Rico 00931-3360 0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)00092-5

Schistosoma mansoni is one of the causative agents of schistosomiasis, a parasitic disease affecting 200 million people. Schistosomes are the only mammalian trematodes which are sexually dimorphic. In recent years, several groups have characterized female specific proteins, for instance egg-shell precursors [ll. In an effort to shed more light on the molecular mechanism underlying expression of female-specific gene products, we have been engaged

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in the isolation and cloning of cDNAs encoding products which are potentially specific or preferentially expressed in female schistosomes. The method of subtractive hybridization was utilized to enrich for cDNAs of a library, prepared from RNA of female parasites after incubation with excess male RNA. One of the cDNA clones thus isolated was chosen for further characterization. Its amino-acid sequence deduced from the nucleotide sequence shows extensive homology to the cathepsin L subfamily of cysteine proteinases [2]. Until recently, it was broadly believed that cathepsin L and cathepsin-L-like cysteine proteinases were present only in protozoan parasites [3-61, while cathepsin B and cathepsin-B-related enzymes were found in nematodes and trematodes [7-91. Contrary to previous observations, a cathepsin-B-like enzyme has now been reported in the protozoon Leishmania mexicana [lo], and furthermore cathepsin L has been characterized in at least two trematodes, Fasciola hepatica [11,12] and S. mansoni [13]. In this paper, we present a detailed characterization of a variant clone of cathepsin L from S. mansoni. Notably the deduced amino-acid sequence of our clone is only 40% homologous to that of the recently published S. mansoni cathepsin L clone, pSmCL1 [13]. 5. mansoni cathepsin L described here is not a femalespecific product, since cathepsin L transcripts have also been detected by Northern blot analysis in male parasites. However, cathepsin-L-related RNA in females is at least 5-times more abundant. This cysteine proteinase exhibits an unusual feature in that, unlike many of the known parasite cathepsins, S. mansoni cathepsin L is not localized in the gut or gut epithelial cells. It is, therefore, not expected to be a digestive enzyme, as postulated for cathepsin B [14,15], even though until now there is little direct evidence for such a function in hemoglobin degradation in vivo. In female schistosomes cathepsin L is localized predominantly in regions related to the reproductive system, whereas in males it is found mainly in the subtegumental region of the gynecophoric canal. Several highly conserved cathepsins are known to be expressed in a variety of tissues of numerous plants and animals, and they appear to carry out multiple housekeeping functions, such as protein turnover [2] or processing of proenzymes [16l, as

73 (1995) 7-18

well as specialized functions like tumour invasion [17]. With regards to cysteine proteinases related to the cathepsins from different parasites, a number of roles have been postulated; for example, in invasion into host tissues, in evasion of the host immune system or in nutrition (reviewed in Ref. 18). The isolation of the schistosome cathepsin L cDNA and its expression in Escherichia coli will provide us with a useful handle for a more detailed analysis of its biochemical properties and to further our understanding of the complex behaviour of cysteine proteinases in the parasite.

2. Materials and methods 2.1. Parasite stocks A Schistosoma mansoni stock from Liberia was maintained in Biomphalaria glabrata and in Syrian hamsters. Recovery of worms was done by perfusion according to Duvall and De Witt [19]. Male and female worms were separated with a fine brush and stored in liquid nitrogen. 2.2. Synthesis of S. mansoni cDNA libraries in AZAPII Construction of the cDNA libraries was carried out with the ZAP cDNA synthesis kit, according to the manufacturer’s instructions (Stratagene). 5 pg each of poly(A)+ RNA prepared from male and female schistosomes was used for the synthesis of double-stranded cDNA. cDNA of each sex was ligated separately with AZAPII-EcoRI + Xhol-treated vector and packaged using Gigapack Gold packaging extracts (Stratagene). 2.3. Preparation of DNA from the hZAPII-specific library Large-scale phagemid and plasmid preparations from the S. mansoni cDNA libraries were done as described [20]. Phagemids were isolated from the female AZAPII cDNA libraries in an in vivo excision step. Male DNA was recovered in the form of plasmids from the pellet of bacterial cells. In order to enrich for female-specific cDNAs, biotinylated sense

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A. Michel et al. /Molecular and BiochemicalParasitology73 (1995) 7-18

transcripts from the male library were hybridized with antisense female phagemids [20,21]. 2.4.

Expression

(His),-cathepsin

and

purification

of

recombinant

L fusion protein

To express schistosome cathepsin L in Escherichia coli, we chose the pDS56 expression vector [22], which uses six histidines at the N terminus as a carrier. (His), fusion proteins produced in this system have the advantage of needing only one affinity-chromatography step for purification. The 1057-bp BamHI-Sal1 fragment of clone pUR-5C6 was subcloned into pDS56, previously digested with BamHI-XhoI. Expression of recombinant cathepsin L was carried out as previously described [23]. The yield from a lOO-ml induced culture was between 150 and 200 pg fusion protein. A total of 500 pg of the purified recombinant product spread over 4 injections at 2-3-week intervals was used to immunize a rabbit. 2.5. Immunoblotting Total proteins prepared from S. mansoni (equivalent to one parasite per lane) or from bacterial cells (0.3 ml induced culture) expressing fusion proteins were separated on SDS-PAGE (15% polyacrylamide) using the minigel system (Bio-Rad), transferred to nitrocellulose membranes and probed for seroreactivity with antibodies raised in rabbits against recombinant cathepsin B or L. Antisera were preincubated with proteins from E. coli lysates to reduce background reactivities. Recombinant cathepsin B is represented by an MS2 polymerase-cathepsin B hybrid protein of 50 kDa (previously described as MS2-Sm3 1 [24]), comprising a polypeptide encoded by the complete S. mansoni cathepsin B cDNA sequence fused to the first 99 amino acids of the RNA polymerase of the bacteriophage MS2 [25]. 2.6. Active-site

labeling

For radiolabeling of active cysteine proteinases in cell extracts, we used lz51 derivatives of tyrosinecontaining diazomethane compounds. Benzyloxycarbonyl-tyrosinyl-alanyl-diazomethane (Z-Tyr-AlaCHN,) and Z-Leu-Leu-Tyr-CHN, [26] were iodi-

nated using Iodo-Gen iodinating reagent (Pierce) [27]. The labeling of proteins was carried out by preincubating lo-20 pg of soluble schistosome proteins in a total volume of 15 ~1 of reaction buffer at 37°C for 30 min, with or without the non-radioactive form of the inhibitors at 100 PM [28]. Inactivation reactions with Z-[1251]Tyr-AlaCHNz were performed in 50 mM Na . acetate (pH 5.51, containing 1 mM EDTA/l mM DTT, whereas those with Z-Leu-Leu-[‘251]TyrCHN, were done in 50 mM Tris . HCl (pH 7.5) containing 5 mM CaCl,/l mM DTT/O.l% Triton X-100. Radioiodinated inhibitors (2 PM) were then added to the reaction mixture and incubated for 1 h. Proteins were separated on SDS-PAGE (12.5% polyacrylamide) and samples were analyzed by autoradiography. 2.7. Immunohistology Adult parasites were fixed in Bouin’s solution (picric acid/acetic acid/formaldehyde 15 : 1: 5) and embedded in paraplast. Sections for immunohistology were treated as described [29]. Anti-(His),cathepsin L antibody (1 : 2500) was detected with alkaline phosphatase conjugated goat anti-rabbit IgG (Dianova) (1 : 2500) with naphthol-AS-MX phosphate and Fast Red TR (Sigma) as substrate [30].

3. Results 3.1. Isolation of Schistosoma subtractive hybridization

mansoni

clones

by

cDNA libraries were propagated in hZAPI1 vector starting from RNA prepared from male and female parasites. The male cDNA library consisted of 1.5 X lo6 independent clones with cDNA inserts with an average length of 950 bp, the female library had 1.4 X lo6 clones with 950-bp inserts. For the production of a subtractive library enriched for cDNAs encoding female specific proteins, we carried out the steps, as described by Schweinfest et al. [20] and Swaarop et al. 1211. A total of 2547 white colonies were obtained, each of which was propagated in a well of microtiter plates, altogether giving 27 Ijlates.

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A. Michel et al. /Molecular

and Biochemical Parasitology

TO test the subtractive library and to isolate clones that are potentially female specific, we analyzed more than 600 clones on triplicate filters. The first filter was hybridized to radioactively labelled cDNA prepared from male parasites, the second to cDNA from female parasites and the third to DNA prepared from three clones previously characterized as female specific. This third filter served as a control in order to avoid analyzing previously characterized clones. Eight clones, neither reactive with the male-specific probe nor with the three female-specific clones, were classified as being potentially related to or at least preferentially expressed in female parasites. One of these clones, termed 5C6, will be described here in detail. 3.2. Sequencing and characterization of clone X6 Since the clone was more than 1 kb in length, we constructed three subclones in the vector pBluescript HSK, from which sequences of both cDNA strands were determined. At the 3’ end of the nucleotide sequence is the polyadenylation signal with the consensus sequence AATAAA. The deduced translation product of 330 amino acids has a calculated molecular mass of 38077 Da. The deduced amino-acid sequence was examined for homology with that of other proteins and was found to predict a cysteine proteinase belonging to the papain family, cathepsin L [2] (Fig. 1). The highest homology was found with mouse [31] with 46% of the amino acids being identical and 13% similar. The deduced amino-acid sequence could also be aligned with cathepsins L from human (44%) [32], Fasciola hepatica (43%) [12] and the slime mold Dictyostelium discoideum (41%) [33]. In addition, homologies were demonstrated with cysteine proteinases that have been characterized as cathepsin-L-like, including those from Trypanosoma cruzi [5] and T. brucei [34]. In comparing only the amino acids corresponding to the mature protein of the recently published S. mansoni cathepsin L clone, pSmCL1 [13], it becomes clear that

73 (1995) 7-18

pSmCL1 has a distinctly lower similarity with all known cathepsins L listed above. For example, while 5C6 is 54% identical and 12.1% similar to mouse cathepsin L, pSmCL1 is only 45.4% identical and 11.6% similar. The former has 52.6% of its amino acids identical and 13% similar to human cathepsin L, the latter 42.6% identical and 11% similar. Indeed, pSmCL1 is only 40% identical and 13% similar to our 5C6 sequence. Therefore, pSmCL1 was excluded from our alignment in Fig. 1. It is obvious that the proteins encoded by 5C6 and pSmCL1 are two different gene products. Clearly, there are too many differences in their amino-acid sequences to explain them as strain differences. However, it is not understood why S. mansoni should possess two variants of cathepsin L. Given the weaker homology of pSmCL1 with known cathepsins L, we propose to refer to that protein as a cathepsin-L-related product. Conserved cathepsin L sequences are centered around the catalytic residues characteristic of cysteine proteinases, namely that of Cys, His and Asn, as indicated by the asterisks (Fig. 1) and the regions surrounding these residues. Among other invariant residues are six cysteines, which according to X-ray data on papain [35], are probably responsible, for disulfide connections. Thus, cysteines at positions 135 and 178 (numberings according to the human sequence) would form a disulfide bridge, as would those at positions 169 and 211 and positions 269 and 322. In the two trypanosome sequences, cysteines at positions 178 and 269 are missing, but nine other cysteine residues, some of which have also been implicated in disulfide links [5] are found elsewhere on the sequence. It is also obvious that the trypanosome sequences are longer at the C terminus, making the alignment more difficult in this region, in spite of rather liberal placement of gaps. Like most cathepsins, the schistosome cathepsin L appears to be synthesized as a precursor molecule (for a review, see Ref. 36). A 17-amino-acid signal peptide has been identified on mouse cathepsin L through alignment of a cDNA sequence with protein

Fig. 1. Alignment of the predicted SC6 polypeptide with other cathepsin L amino-acid sequences. Amino-acid residues common to five or more than five sequences are in bold. Asterisks indicate amino acids of the active center; arrows indicate potential cleavage sites for posttranslational processing; s denotes positions of cysteines likely to form disulphide bridges. Gaps are shown as dashes. Amino-acid sequences were analyzed using the PC/GENE computer program version 6.6, 1991.

11

A. Micheletal./MolecularandBiochemicalParasitology 73 (1995)7-18

* Mouse

Human D.discoideum s.mansoni F.hepatica T.cruzi T. brucei Mouse Human D.discoideum s.lMnsoni F.hepatica T. cruzi T. brucei

EINL-----LLLLAVL------CLGTALATPKFDQT-FS~Q~STHRRLY-GTNEEE~IWEKMIRMIQL K)IICAMHNRLY-GMNEEGWRRAVWEKNMKMIEL MNP-----TLIUAF------CLGIASATLTFDHS-LEAQWT ~L---LVPLILLIFVNFSFANVRPNGR--RFSESQYRTAFTEWTLlCFQY-SSSEFSNRYSIPKSNWDYVDN MK-----VPLLLFSIIISVAIAQ--------HLSLQYDDIWKQWlCLllYNK~-SDSNEIRllKAIFMRWEKIQQ MR------FFVLAVLTVGVFASN------------DDLWHQWKRIYNKEX-NGADDEHRRNIWGKNVKHIQE MS--GWARALL~~~-CLVPAATASLHAEETLTSQF~~Q~GRVYESAAEEAFL MPRTEMVRFVRLPVVLLAMAACLASVALGSLHVEESLEMRI * HNGEYSNoQHGFSMEMNAPGD~BFRQVVNGYRHQKK----KGR--LFQEPLMLKIPKSWWREKGCVTPV IMQEYREGKHSFTMAMN~GDIFTSEEFRQVMNGFQNRKPR----KGK--VFQEPLFYEAPRSM~EK(PV WN---SKGDSQTVLGLNNFADITNEEYRKTYLGTRVNAHSYNGYDGRE-VLNVEDLQTNPKSIDURTI[NAVTPI HNLRHDLGLEGYTMGLNQFCD~~EIKTIMLSKVFGNSPL--WDDKK-EELELSNDPLPSKWDIQRDHOAVTPV HNLRHGLGLVmKLGLNQPTDLTFEEPKAKYLIEIPRSSEL--LS-RG-IPYKANKLAVPESIDWRDYYWTEV HAAANPHA----TFOVTPFSDLTREEFRSR---YHNGA?UIFAAAQERARVPVKVE%'GAPAAVDWRARGAVTAV QAAANPYA----TFGVTPFSDHTREEFRAR---YRNGASYFAAAQKRVRKTVNVTTG~AAVDWREKGAVTPV

61 61 68 60 53 71 74 129 129 138 131 123 138 141

Mouse Human D.discoideum S.mansoni F.hepatica T.cruzi T.brucei

201 201 210 202 194 210 213

Mouse Human D. discoideum S.marwoni F. hepatica T.cruzi T.brucei

271 271 281 272 264 279 282

Mouse

Human D.discoideum S.marlsoni F.hepatica T.cruzi T.brucei Mouse Human D. discoideum S.mansoni F.hepa tica T.cruzi T.brucei

Mouse

Human D.discoideum S.mansoni F.hepatica T. cruzi T. brucei

* * KNLDHOVLLVQYGYEGTDSN---------------------------------KNKYPnVRNSWGSElQOMEOIK ~D~H~W~GFESTESD-__---__--___-_____----__--___-__~--SWG~~~G~K TELDIiGVLWGYGVQGKDDEGPVLNRKQTIVIHKNEDNKVESSDDSSDSVRPKANNYW IVKNSWGTSWGIKGYIL FLL~-V~GR~N-___----------_---___-_--------__--__RKDYtQL~~SWGT~~OJGMNOYFK

313 313

EQLD~~LV-NS-__-----------_---__--_--------__--__NP~~~~~SWS-G~D~~R

356 310 302 317 320

I~________-________-_---___-____-_____---__--_______.______._____._--___-~_______--____-___-_---__--___________--__________________________-____-MSI[________--_______------_---_________----___-________________~____-____-L~________-____-___-__--___-__________---____~_____________________-___--F~_________-_______-___-________________________________--_____--_________

314 314 357 313 303

IAlCGSNQCLVKEEASSAWGGPGPTPEPTTTTTTSAPGPSPSYFVQMSCTDAACIVGCENVTLPTGQCLLTTSGV IEKGTNQCLMNQAVSSAWGGPTPPPPP--------PPPPSATFTQDFCEGKGCTKGCSHATFPTGECVQTTGE~QTTGVG

392 387

s DRD~CG~T~________-_SYP~___________________________--___________---__-D-CGIAS~________-_SYP~_-___________--________________________________DR~CGIASVS-_______-_SYPLV_--__________--_________________________________ N~CGIATNA________-_SFPLL---___-_____---___--____________________________ NRG~CGIAS~________-_S~~~FP_--______--__---____________________________

334 333 376 330 326

SAIVTCGAETLTENFLTSTHCSGPSVRSSVPLNKCNRLLRGSVEFFCGSSSSGRLADVDRQRRHQPYHSRHRRL SVIATCGASNLTQIIYPLSRSCSGPSVPITVPLDKCIPILIGSVEYHCSTNPPT~LVPHQ------------

467 450

D~T~~V~GSQD____-___--_______________--___-______GTD~~VIPJSWGT~G~D~~R ~QLD~~LV~DSA____-___--______________---__________AVP~~~IIlJS~TQWG~~~~R

12

A. Michel et al. /Molecular

A kDa

and Biochemical Parasitology

B 1

2

3

73 (1995) 7-I8

C

456

D

789

10

11

12

Fig. 2. Analysis of adult S. mansoni antigens and recombinant proteins. An extract of total S. mansoni proteins (lanes 1, 6, 9 and 12), E. coli extracts containing either recombinant cathepsin L (lanes 2, 5, 8 and 11) or recombinant cathepsin B (lanes 3, 4, 7 and 10) were loaded on SDS-polyacrylamide gels (A). Proteins were visualized by staining with Coomassie blue. Proteins were also transferred to nitroceilulose filters and immunoblotted with anti-(His),-cathepsin L antibodies (B), anti-MS2-cathepsin B antibodies (0, or preimmune serum (D).

sequences obtained for procathepsin L. Secondly, a 96-amino-acid propeptide has been demonstrated on mouse cathepsin L [31]. Moreover, the mature single chain proteinase is cleaved into a two-chain form. On the other hand, chicken liver cathepsin L is primarily a single chain enzyme [37]. It has not yet been determined whether schistosome cathepsin L exists as a single chain or as two chains. By analogy to proposed cleavage sites of known cathepsin L precursor molecules (arrows in Fig. l), the aminoacid sequence as deduced from 5C6 cDNA would appear to comprise a signal peptide of 18 amino acids and a propeptide of 97 amino acids. The mature enzyme would be 215 residues long and would have a calculated molecular mass of 24300 (see below).

masses of 38 and 31 kDa were observed to react with the antibody (Fig. 2B, lane 6). From this result, in analogy to other cysteine proteinases, we conclude that the schistosome cathepsin L has a transient proenzyme form of 38 kDa, which would subsequently be processed to a mature protein of 31 kDa. However, there is a difference between the sizes of the observed protein and the predicted protein (24.3

12

3

4

5

6

7

8

kDa 6645-

3.3 Specificity of anti-cathepsin L antibodies Total proteins from a schistosome extract and from cells expressing recombinant cathepsin L of approx. 38 kDa (arrowhead) and recombinant cathepsin B of 50 kDa (arrowhead) were separated by SDS-PAGE and stained with Coomassie blue (Fig. 2A, lanes 1, 2 and 3, respectively). In order to identify native cathepsin L in the schistosome extract, we carried out an immunoblot of total S. mansoni proteins using antibodies raised to recombinant cathepsin L. Two polypeptides with molecular

21Fig. 3. Affinity labeling of cysteine proteinases with radioactive inhibitors. S. mansoni extracts were incubated with radiolabeled Z-Tyr-[1251jAlaCHN, (lanes l-4) or Z-L.~U-L~U-T~~-[‘~~I]CHN~ (lanes 5-8). Proteins were incubated in duplicate without unlabelled inhibitor (lanes 1 and 4, and lanes 5 and 8) or pretreated with unlabelled Z-Tyr-AlaCHN* (lanes 2 and 6) or unlabelled Z-Leu-Leu-TyrCHN, (lanes 3 and 7).

A. Michel et al. /Molecular

and Biochemical

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72 (199.5) 7-18

13

sis, we examined whether antibody raised against (His),-cathepsin L fusion protein was immunoreactive with MS2-Sm31 fusion protein and vice versa. While anti-cathepsin L antibody did not react with the 50-kDa MS2-cathepsin B fusion protein, it recognized (His),-cathepsin L fusion protein of 38 kDa (Fig. 2B, lanes 4 and 5, respectively). On the other hand, anti-MS2 cathepsin B antibody was found to react with a 31-kDa polypeptide. as well as its precursor protein of 37 kDa in the schistosome extract (Fig. 2C, lane 91. In addition, the antibody gave a positive signal with the homologous fusion protein of 50 kDa, and not with the cathepsin L fusion protein (Fig. XT, lanes 7 and 8, respectively). We therefore conclude that immunologically cathepsins B and L are distinct from each other. Preimmune

Fig. 4. Analysis of total RNA isolated from adult male and female schistosomcs. Total RNA prepared from male (lane 1) and female worms (lane 2) were separated on a 6% agarose-formaldehyde gel and visualized by staining with ethidium bromide (A). prior to transfer on Hybond membrane. (B) Hybridization of male (lane 1) and female (lane 2) RNA was done using the antisense transcript from clone 5C6. Molecular mass marker (in bp) is I-kb DNA ladder (BRL).

kDa). Such a discrepancy could be explained if the protein has an anomalous behaviour on SDS-polyacrylamide gels. It is also likely that the schistosome procathepsin L sequence is cleaved, not at the postulated site, but upstream from it, so that a longer mature enzyme is produced. Alternatively, the endogenous proteinase could be glycosylated. Within the proposed amino-acid sequence are two potential N-linked glycosylation sites, but only one of the sites, at asparagine 325, is located on the sequence encoding the mature protein. In addition to the possibility of an N-linked glycosylation, the proteinase may also be modified by O-linked glycosylation moielies. In view of the significant similarity between cathepsin B and -L sequences, we determined whether the two proteins crossreact with each other at an immunological level. In an immunoblot analy-

Fig. 5. Southern blot analysis of S. mansoni genomic DNA. X yg of genomic DNA digested with various restriction enzymes were loaded on each lane. The filter was hybridized with a “P-labeled cDNA corresponding to the full-length SC6 clone and washed at high stringency. The positions of DNA size markers in bp are indicated.

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A. Michel et al. /Molecular

and Biochemical Parasitology 73 (1995) 7-18

serum taken from a rabbit immunized with (His),cathepsin L fusion protein showed no reactivity with any of the proteins from bacterial cells in duced for recombinants of cathepsin B or L (Fig. 2D, lanes 10 and 11, respectively) nor with schistosome proteins (lane 121. 3.4. Afsinity labeling of cathepsins B and L In order to confirm that the 31-kDa protein represents the active mature S. nzansoni cathepsin L molecule, we carried out affinity-labeling experiments using two different diazomethane inhibitors. Our choice of diazomethanes is such that they are capable of distinguishing the 31-kDa proteins of cathepsin B [8] from cathepsin L (this study), as well as from a 70-kDa calcium-dependent activity, that of calpain [38]. Furthermore, these compounds inacti-

vate cysteine proteinases via alkylation of cysteine residue at the active center and are, therefore, capable of reacting only with active mature enzyme forms. Thus, when radioiodinated Z-Tyr[izsI]AlaCHN,, which shows an affinity for cathepsins B and L [26] was incubated with a schistosome extract, it strongly labeled a 31-kDa protein band, shown in duplicate (Fig. 3, lanes 1 and 4). We deduce that this labeling represents both active cathepsins B and L. Pretreatment of the extract with unlabelled Z-TyrAlaCHN, blocked the subsequent labeling of the 31-kDa band (Fig. 3, lane 2). On the other hand, Z-Leu-LeuTyrCHN, is a specific inactivator of cathepsin L, as well as calpain [26] and can, therefore, compete with Z-Tyr-AlaCHN, for the labeling of cathepsin L. Thus, in the presence of cold Z-LeuLeu-TyrCHN, the labeling of the 31-kDa band was visibly decreased, verifying that the basis of the

Fig. 6. Immunohistological localization of cathepsin L in a section of a schistosome pair (A) and a section of negative control that has been incubated with the secondary antibody only (B). In the female ( Q 1, clear punctuate the tegument of the uterus (U). whereas the lining of the two shanks of the intestine (I) located on both sides of unlabelled (some dark material visible in the lower shank of the intestine results from haematin content of the gut). is restricted to distinct spots below the tegument (T) of the gynaecophoric canal. Scale = 200 pm.

the same worm pair as staining is visible below the uterus is completely In the male (6) staining

A. Michel et al. /Molecular and Biochemical Parasitology 73 (lY95) 7-18

response can be attributed in part to active cathepsin L (Fig. 3, lane 3). Weaker reacting bands visible at 23, 25, 28, 4.5 and 60 kDa are present-in all four lanes, indicating that they are non-specific. By incubating radioiodinated Z-Leu-Leu-Tyr[“‘I]CHN, with a schistosome extract, we were able to label active cathepsin L and calpain (Fig. 3, lanes 5 and 8, as duplicate samples). The specificity of this reaction was demonstrated by the preincubation of this extract with unlabelled Z-Tyr-AlaCHN,, which resulted in the disappearance of the 31-kDa band (Fig. 3, lane 6). Finally, pretreatment with unlabelled Z-Leu-Leu-TyrCHN, is seen to compete for the labeling of both cathepsin L as well as calpain (Fig. 3. lane 7). In conclusion, we were able to discriminate cathepsin L from B in an extract of schistosome proteins and on the basis of its different molecular mass, also calpain from the cathepsins. Moreover, we distinguished the active forms of cathepsins B and L from their inactive precursors. 3.5. Northern blot analysis of male and female schistosome RNA

In order to test clone 5C6 as being potentially female specific, we analyzed its hybridization to total RNA prepared from male and female parasites (Fig. 4A, lanes 1 and 2, respectively). Following transfer to Hybond membrane, the RNA was probed with [32 P]dUTP antisense transcripts from clone 5C6 directed by T7 polymerase. Based on the denatured DNA marker on the furthermost left lane, we estimated a transcript of 1300 bp in length. Despite the fact that identical amounts of RNA (20 pg) were loaded onto each lane, a hybridization signal five times weaker in intensity, as measured in the densitometer, was detected in the lane containing RNA from male worms (Fig. 4B, lane l), when compared to the one from female worms (Fig. 4B, lane 2). Therefore, 5C6 transcripts are not female-specific, but are expressed preferentially in female schistosomes. 3.6. Southern DNA

blot analysis of schistosome

genomic

Southern hybridization was conducted to determine the number of genes coding for cathepsin L in

IS

S. mansoni

using the full-length cDNA insert as a labeled probe. The probe produced only one hybridizing band with three different restriction enzymes used (Fig. 5). In addition, it hybridized to two PstI fragments of approx. 6 and 2 kb, as well as to two Sal1 fragments of 8 kb and 4 kb. as expected from the restriction map of the cDNA. Our results indicate that schistosome genomic DNA contains a single copy of the cathepsin L gene. 3.7. Immunohi.~tological

localization of cathepsin L

Antibodies raised to recombinant cathepsin L were used to localize the protein in tissue sections (Fig. 6). In the female. clear punctuate staining was observed below the epithelium surrounding the vitelloduct, the ovo-vitelloduct. the ootype, particularly at its posterior end, and the uterus along its entire length. Notably. however, the oviduct showed no reactivity. In the male parasite, staining was found below the tegument of the gynaecophoric canal and was strongest in the region below the testes.

4. Discussion In recent years, although the molecular cloning of several cathepsins L from a variety of parasites has been achieved and some of the corresponding gene products have been further characterized, the specific role of each of the enzymes has yet to be established. In Fasciola, both enzymes have been localized in the epithelial cells of the intestine [II]. The enzyme is concentrated within secretory vesicles from which it is released into the lumen of the gut, prior to discharge into the environment. Functions postulated for this enzyme include a role in evading the host’s immune system by cleavage of immunoglobulins and thus preventing antibody-mediated immune-effector cell attachment [39]. In this paper, we report localization of cathepsin L in S. mansoni in the cytons of the ventral tegument in the male as well as in the tegumental cytons of the female uterus. In addition, the enzyme has been localized in some less defined regions of the female reproductive tract at the posterior side of the ootype. Our immunhistological studies revealed

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staining in discrete granules, which is consistent with the schistosome cathepsin L being a lysosomal enzyme. However, further experiments are necessary to verify its subcellular location. Like in Fusciola, cathepsin L in schistosomes might also be secreted. Passage of oocytes. vitelline cells and eggs should be facilitated by a lubricating agent. Differences in the matrix lining the uterus and the ovovitelloduct led Spence and Silk [40] to conclude that the fluid contents are different. Besides. migration of reproductive cells through the ducts ahead of the eggshell-forming chamber is expected to put less of a mechanical stress on the duct wall than the transport of eggs encased in tough outer eggshells through the uterus. Consequently, a more resistant surface lining of the uterus is required to prevent any injury to it. Consistent with these cytological observations is a higher concentration of cathepsin L around the uterus compared with the region on the other side of the ootype. Cathepsin L may alter the viscosity of the lluid: such a function has been reported for an acidic protease in the seminal fluid of man [41]. A similar function may be attributed to cathepsin L located in the cytons of the tegument of the gynecophoric canal in the male. In S. mmsoni. sperms are continuously poured into the lumen of the gynecophoric canal [42] where they must find their way to the female genital porus. At the junction of the ovovitelloduct with the ootype, cathepsin L. as shown by immunohistology, is located in direct vicinity of the ootype. whereas the respective mRNA, visualized by in situ hybridization (not shown). is expressed more distal. This would be consistent with the assumption that synthesis and excretion of cathepsin L takes place in Mehlis glands, although until now. mainly neutral glycoproteins have been identified in the secretions of these glands [43]. The ootype is the site where eggshell precursor proteins are crosslinked, a process catalyzed by an enzyme identified as a phenoloxidase. The enzyme is synthesized in the vitellarium and stored together with its substrate in the eggshell granules. To prevent premature crosslinking, phenoloxidase must exist in an inactive state. Although nothing is known of the factors converting phenoloxidase to its active form, it is tempting to speculate that cathepsin L synthesized in the vicinity of the ootype is involved in its activa-

tion. At present, however, there are no data that directly support such a conjecture. Further biological and biochemical studies should assist us in understanding better the role of cathepsin L in the schistosome.

Acknowledgements We wish to thank Dr. Donato Cioli for helpful discussions, Dr. Elliott Shaw for the kind gift of diazomethane inhibitors, Lutz Gohr for computer work and Albert Kaltenberg for technical help. This investigation received financial support of the Deutsche Forschungsgemeinschaft (Ku 282,’ 13-2) and of the C.M.T.-Italian Ministry for Foreign Affairs-D.G.C.S. M.R. was partially supported by NIH grant No. T37TWO0046.

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