Cloning of a cysteine protease required for the molting of Onchocerca volvulus third stage larvae.

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 μM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.

Nematodes characteristically grow and develop through four molts; in parasitic nematodes, the last two are always in the final host. The process of molting involves the separation of the cuticle from the underlying epidermis (apolysis), the formation of a new cuticle arising from the outermost surface of the epidermis, and the shedding of the old cuticle (ecdysis) (1). The actual mechanics of molting can be very different among the various nematode genera. Some nematodes reabsorb the old cuticle during molting (2), some shed the old cuticle intact (3), and some do not undergo ecdysis until entering the mammalian host (4). Proteases are thought to play an essential role in molting by digesting the old cuticle, degrading cuticular anchoring proteins, or activating peptide molting hormones or other molting enzymes by processing of proenzymes (5)(6)(7). A 44-kDa zinc metalloprotease in Hemonchus contortus was shown to be responsible for the digestion of the ring region of the second stage larvae cuticle before molting (8,9). Proteases active during molting have been also observed in other species, including Phocanema decipiens (10), Ancylostoma (11), and Filariae (5,12). In Dirofilaria immitis and Brugia pahangi, metalloproteases were shown to be intimately associated with molting as well as activities that might facilitate larval migration (5,12).
Onchocerciasis, or river blindness, is one of the leading causes of infectious blindness and severe chronic dermatitis, afflicting about 18 million people in West Africa and Latin America (13). The Onchocerca volvulus parasite is transmitted by bites of Simulium black flies. Our ability to study the molting process of O. volvulus third stage larvae (L3) has been enhanced after the conditions for in vitro cultivation of larval stages were developed (14). Ultrastructural examination of larval stages during molting have shown that the formation of the new cuticle of fourth stage larvae (L4) 1 is already under way on days 1 and 2 of culture. The new epicuticle was evident in some areas beneath the basal layer of the old cuticle and irregularly shaped "lakes" (separated areas between the cuticles) appeared below the old basal layer. On day 2 and 3 in culture, these lakes fused into a continuous layer of separation, and by day 4 or 5, most of the larvae have completed the molt (14). The appearance of the lakes and their fusion into a continuous separation before ecdysis suggested the possible activation of enzymatic processes that promote the separation of the old L3 cuticle from the new one (15).
In our previous studies we had identified and cloned a functional O. volvulus cysteine protease inhibitor, onchocystatin (16). Interestingly, onchocystatin was localized by immunogold electron microscopy in the hypodermis and the cuticles of L3 and L4 during the molting of L3 to L4, especially around the region of cuticle separation. As the postulated role for cystatin has been the regulation of cysteine protease activities (17), these studies indirectly suggested that a cysteine protease may be involved in the molting process of O. volvulus larvae. We now show that a cysteine protease is indeed one of the enzymes involved in the molting of O. volvulus larvae and is an essential enzyme for the successful development of L4.

MATERIALS AND METHODS
In Vitro Culturing of L3 in the Presence of Cysteine Protease Inhibitors-One approach to study the role of cysteine proteases in the molt-ing process was to culture the larvae in the presence of specific irreversible inhibitors of cysteine proteases and examine their effect during the molting of L3 to L4. We have used the peptidyl monofluoromethylketone (FMK) compounds containing a peptide sequence targeting the active sites of the cysteine proteases, Cathepsin B and L (18).
Simulium yahense black flies were infected with O. volvulus microfilariae, and after 7-8 days L3 were harvested as described previously (14,19). L3 were cultured in groups of 10 larvae in 96-well plates containing 1 ϫ 10 5 bovine peripheral blood lymphocytes/0.15 ml of culture medium (1:1 NCTC 135 and Iscove's modified Dulbecco's medium plus 20% heat-inactivated fetal calf serum, 100 units of penicillin/ ml, 100 g of streptomycin/ml, and 5 g of amphotericin B (Fungizone)/ ml). Using these conditions, we routinely achieved 40 -60% successful L3 to L4 molting by day 5 in culture. L3 were cultured in vitro for 6 days at 37°C in a humidified 5% CO 2 incubator in the presence of increasing concentrations of cysteine protease inhibitors: benzyloxycarbonyl-Phe-Ala-fluoromethylketone (Z-Phe-Ala-FMK), Z-D-Phe-Ala-FMK, Z-Phe-Arg-FMK, and Z-Phe-Phe-FMK, and the number of molting larvae was determined on day 6. Molting was manifested by shedding of the thick L3 cuticle and a marked increase in the motility of the larvae. Larval viability was also assessed visually after the uptake of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) by the larvae and its reduction into the blue formazan derivative as described previously (20,21). Larvae were scored live when they stained blue uniformly along their entire length and dead when the larvae remained unstained or partially blue. The results shown for each inhibitor concentration are the average number of larvae that molted or the average number of larvae that were scored alive in 5-10 wells containing a total of 50 -100 L3. Each experiment was repeated at least twice. The standard error between experiments never exceeded 10% and did not vary with the concentration of the inhibitors.
Ultrastructure of Larvae Cultured in the Presence of Cysteine Protease Inhibitors-Larvae that did not molt in the presence of cysteine protease inhibitors were collected and fixed overnight at 4°C with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), washed in the same buffer, and processed for electron microscopy examination as described previously (14). For examination of the ultrastructure of larvae undergoing normal molting, L3 were cultured in vitro for 5 days and larvae from day 1 to 5 in culture were collected. The larvae were fixed as described above.
Molecular Biology Methods-General procedures for preparation of plasmid DNA, subcloning, Southern blotting, hybridization, and preparation of probes were followed as described by Sambrook et al. (22). For subcloning PCR products, the DNA fragments were made blunt-ended with Klenow DNA polymerase for 5 min at 37°C, or cut with the appropriate restriction enzyme. The products were fractionated on 1.2% agarose gels and gel-purified by electroelution followed by ethanol precipitation. The products were ligated into pBluescript SKϪ (Stratagene, La Jolla, CA) previously digested with SmaI for blunt end ligation, or other restriction enzymes for ligation of cohesive ends.
Automated DNA sequencing was done by the Microchemistry Facility at the New York Blood Center using the fluorescent dye termination method and Taq DNA polymerase on an ABI model 373A sequencer (Applied Biosystems Inc., Foster City, CA). In order to minimize sequencing errors due to PCR artifacts (misreading by Taq polymerase during amplification), for each target cDNA sequence, at least three independent clones were isolated and sequenced in both orientations and their sequences were compared to derive the final consensus sequence representative of the cDNA fragments. All the oligonucleotide primers used during the course of these studies are listed in Table I volvulus can only be obtained in limited quantity from clinical samples, a PCR-based approach was used to clone the larval cysteine protease, and later we confirmed its identity by immunolocalization and Western blot analysis with antibodies against the recombinant polypeptide. A specific DNA fragment was initially isolated from genomic DNA prepared from O. cervicalis adult worms by PCR using degenerate primers designed for the cysteine and the asparagine active site motifs of cysteine proteases as described by Sakanari et al. (23) and Eakin et al. (24). The reaction was conducted in a volume of 100 l for 60 cycles, with the following profile: 1 min at 94°C, 2 min at 25°C, and 3 min at 72°C, followed by a final 7 min extension at 72°C. A DNA fragment of about 700 bp was purified and named OC700. This fragment would provide codon usage for onchocerca at conserved active site residues. OC700 contained two exons and one intron. In its 5Ј region, an exon of 130 bp encoding 43 amino acids was identified, which contained at its 5Ј end the sequence CGSCW originating from the forward primer. In its 3Ј region an exon of 153 bp was identified with an open reading frame encoding 51 amino acids, which had a significant homology to other cysteine proteases and which contained the conserved amino acid of the histidine active site of cysteine proteases in addition to the asparagine active site which was derived from the reverse primer (data not shown). A TaqI/HindIII 135-bp fragment from the 3Ј exon region coding for 45 amino acids was used to screen an EcoRI O. volvulus genomic library (a gift from Dr. Perler, New England Biolabs). 9 ϫ 10 5 plaque-forming units were screened by DNA hybridization at 37°C following the protocols of Sambrook et al. (22). Four genomic clones containing DNA inserts of 5-6.5 kb were isolated and purified. The largest clone (clone 10 ϭ 6.5 kb) was chosen to be analyzed in greater detail. The EcoRI fragment was subcloned into pBluescript SKϪ, and the plasmid DNA of the transformant was purified and sequenced from both directions using the T3 and T7 primers and specifically designed internal primers. Sequence analysis of the 5Ј region (2230 bp) of the genomic clone indicated that this region contained a specific but partial genomic sequence coding for the onchocerca cysteine protease, OVCP. The remainder of the 5Ј coding region of the OVCP gene was purified and sequenced after a specific fragment was generated by PCR from O. volvulus genomic DNA using a specific reverse primer to the genomic sequence (OVCP10) and a forward primer specific to the 5Ј coding region of the cDNA sequence (see below) including the first methionine (OVCP-Met).
In order to confirm this clone was expressed in larvae and to obtain a full-length cDNA of the O. volvulus larval cysteine protease, we first cloned the 3Ј region of the cDNA by a modification of the 3Ј rapid amplification of cDNA ends (RACE) protocol (25). A first-strand cDNA using oligo(dT) adaptor primer (SWE17) and L3 O. volvulus poly(A) ϩ RNA was synthesized and used as a template for PCR using a specific primer corresponding to the gene sequence in the exon coding for a region around the conserved motif of the histidine active site, DIDHIIS (CPF6) and the adaptor primer (SWE18) ( Table I). The first-strand cDNA was denatured for 5 min at 95°C in buffer containing 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 2.5 mM MgCl 2 , 0.1% Triton X-100, 0.01% gelatin, 200 M of each dNTP, and 50 pmol of each primer. Following denaturation, 2.5 units of Taq polymerase (Promega Corp., Madison, WI) was added to the reaction. Amplification was carried out by cycling for 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C. Following amplification the PCR products were fractionated on a 1.2% agarose gel, and bands were excised and purified by phenol/chloroform extraction, treated with Klenow and T4 Kinase, subcloned into the SmaI site of pBluescript KSϪ, and sequenced. One PCR product of about 350 bp was found to be specific, containing an open reading frame encoding 67 amino acids and 176 bp of the 3Ј noncoding region. The 67 amino acids contained in addition to the histidine active site motif the asparagine active site motif and all the intervening amino acids encoded by the last two exons of the genomic clone, confirming that the cDNA was specific for a mRNA encoded by the OVCP genomic clone. The rest of the larval cDNA of OVCP was then obtained by PCR using the SL1 primer, a 22-nucleotide splice leader sequence of Caenorhabditis elegans that is also present in many transcripts of filarial parasites (26,27), and a specific primer to the noncoding region (CPR3, Table I) at the same conditions described above.
Expression of Recombinant OVCP and Antibody Production-The region containing the propeptide and the mature enzyme of onchocerca ( Fig. 4, underlined arrows) was amplified by PCR and subcloned into the pGEX-4T-3 expression vector (Pharmacia Biotech, Inc.) which was successfully used for the expression of an active onchocystatin (16). The ligation mixture was used to transform E. coli subcloning efficiency DH5␣™-competent cells (Life Technologies, Inc.). The ligation junction of the pGEXLOVCP plasmid was sequenced to verify the correct read-ing frame. Transformants expressing the GST-LOVCP fusion polypeptides were identified after SDS-gel electrophoresis (SDS-PAGE) by staining with Coomassie Blue and by Western blot analysis using rabbit anti-GST antibodies, followed by goat anti-rabbit antibodies conjugated to horseradish peroxidase and detection by 3,3Ј,5,5Ј-tetramethylbenzidine (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). Large scale purification of GST-LOVCP fusion polypeptides was carried out according to the procedure described before (16). 250 ml of culture yielded about 0.5 mg of pure GST-LOVCP fusion polypeptide. The GST-LOVCP fusion polypeptide was not active as a cysteine protease when tested in a continuous fluorometric assay with Z-Phe-Arg-AMC (Z-Phe-Arg-4-amino-7-methylcoumarin) or Z-Arg-Arg-AMC (data not shown). Antiserum to the GST-LOVCP fusion polypeptide was prepared by subcutaneous injection of a rabbit with 100 g of protein mixed in complete Freund's adjuvant (Difco), followed by repeated injection 3 and 4 weeks later in incomplete Freund's adjuvant (Difco). The rabbit was bled 2 weeks after the third injection.
Identification of a Cysteine Protease in O. volvulus Molting Larvae-Initially, we used an indirect approach in order to identify the endogenous cysteine protease in molting larvae. We took advantage of the irreversible binding of labeled cysteine protease inhibitors to the enzyme during the molting process (28,29). Larvae were cultured in the presence of 50 M iodinated Mu-Tyr-O-methyl-homoPhe-FMK (28), and then we collected the worms after 2 days, extracted the proteins in SDS-PAGE sample buffer (final concentration of 2% SDS, 5% 2-␤mercaptoethanol in 62.5 mM Tris-HCl, pH 6.8) and separated them on a 12% SDS-PAGE gel. The iodinated proteins were then identified by autoradiography. In addition, stage-specific soluble extracts were prepared from about 100 L3 or 100 larvae collected on day 1, 2, or 3 in culture by homogenization on ice in a prechilled 100 mM sodium acetate buffer, pH 5.0. These extracts and culture supernatant collected from wells containing larvae cultured for 2 days were then incubated with 1 mM biotinylated inhibitor, biotinyl-Phe-Ala-diazomethane (biotin-Phe-Ala-CHN 2 , Biosyn Diagnostics, Belfast, Northern Ireland), for 60 min at 37°C in buffer conditions that allow cysteine protease activity: 2 mM cysteine, 1 mM EDTA and 0.1% Brij in 100 mM sodium acetate buffer, pH 5.0 (29). The reactions were stopped by the addition of SDS-PAGE sample buffer and boiling. The binding of the biotinylated inhibitor to the endogenous cysteine protease was determined by running the extracts on a 12% SDS-PAGE gel, transferring to nitrocellulose filters by electroblotting, and detection with streptavidin-alkaline phosphatase and nitro blue tetrazolium (Biosyn Diagnostics). Each lane contained the equivalent of 40 larvae or the supernatant from 50 cultured larvae.
Once we raised antibodies against the recombinant LOVCP protein, we used them to identify the endogenous cysteine protease by immunoprecipitation of [ 35 S]methionine metabolically labeled larval extracts (30), and by immunoblot analysis of larval extract prepared in SDS-PAGE sample buffer (31).
Localization of the Cysteine Protease in Larval Stages of the Parasite-O. volvulus L3 were cultured in vitro for 4 days, and larvae from days 1, 2, 3, and 4 in culture were collected. The larvae were fixed for 30 min in 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, containing 1% sucrose, and were then processed for immunoelectron microscopy as described previously (16). Thin sections of embedded worms were probed with rabbit antisera raised against the recombinant cysteine protease, GST-LOVCP, before interaction with 15 nm gold particles coated with protein A (Amersham Life Sciences). Preimmune serum from the rabbit or antibodies to GST were used as controls.
In addition, larvae were cultured for 1 day in the presence of 100 mM biotin-Phe-Ala-CHN 2 , after adding the inhibitor on day 0, 1, 2, or 3. After 24 h the larvae were collected and fixed as described above. The biotin-Phe-Ala-CHN 2 was detected in thin sections of the embedded worms by 15 nm gold particles coated with streptavidin (Amersham Life Sciences). The cysteine protease in these worms was detected with antibodies to the GST-LOVCP fusion polypeptide

The Effect of Specific Cysteine Protease Inhibitors on L3
Molting-The presence and location of the cysteine protease inhibitor, onchocystatin, suggested the possible involvement of a cysteine protease in the development of O. volvulus L4. To test this possibility, we studied the effect of four specific irreversible inhibitors (peptidyl monofluoromethylketone compounds) of the cysteine proteases, cathepsin B and cathepsin L, on the viability of molting L3 and the ability of L3 to molt to L4 in vitro. Parasite viability was assessed visually after the uptake of MTT by the larvae. L3 were cultured in the presence of increasing concentrations of the inhibitors, and the number of molting larvae was determined on day 6. All inhibitors reduced, in a dose-dependent manner, the molting ability of L3 in vitro (Fig. 1). In comparison with 50% molting under normal culture conditions, 50 -80% of molting was inhibited by 100 M Z-Phe-Ala-FMK, Z-D-Phe-Ala-FMK, Z-Phe-Arg-FMK, and Z-Phe-Phe-FMK. Z-Phe-Ala-FMK, Z-D-Phe-Ala-FMK, and Z-Phe-Arg-FMK at 250 M completely inhibited molting. Z-Phe-Phe-FMK at 250 M inhibited only 70% of the molting. The inhibition was not due to a lethal effect of the inhibitors on L3. In parallel experiments larvae were treated with MTT on different days during culture in the presence of various concentrations of the inhibitors, and their viability were assessed after 24 h. The viability curves in Fig. 1 represent the data from larvae cultured for 3 days in the presence of the compounds. L3 that were cultured in the highest concentration (250 M) of Z-Phe-Ala-FMK, Z-Phe-Arg-FMK, and Z-Phe-Phe-FMK were still viable. With Z-D-Phe-Ala-FMK, only concentrations greater than 100 M had any significant effect on L3 viability. For the viability curves in Fig. 1, we used the data from day 3 in culture because this has been shown to be the optimal time to determine if larvae are both viable and ready for molting. Larvae usually complete their L3 to L4 molt by day 5 (14). Similar viability results were also obtained on successive days; the larvae were motile and viable during all days in culture (data not shown). Since the cysteine protease inhibitors were not lethal to L3 or L4 larvae at all concentrations, we could assay their effects on the molting process.
To identify the point during the molting process that is most sensitive to protease inhibitors, culture medium containing 100 M Z-Phe-Ala-FMK or Z-Phe-Arg-FMK was added to larvae on day 0, or on days 1, 2, and 3 by replacing the normal culture The nucleotides in lowercase are an extended sequence 5Ј to the specific primer and contain a restriction site for subcloning purposes. b (Ϫ) and (ϩ) indicates negative or antisense and positive or sense strand, respectively.
medium. The larvae were then allowed to remain in culture until day 6, when the number of molting larvae was determined. When added on day 0 or day 1 both inhibitors were able to inhibit completely the molting (Fig. 2). However, when Z-Phe-Arg-FMK was added on day 2 or day 3, only a partial inhibition was observed; 16 and 14% of the larvae molted, respectively, in comparison with 50% that molted under normal culture conditions. Interestingly, when Z-Phe-Ala-FMK was added on day 2, the inhibition was complete, but became partial when added on day 3 (72% inhibition). Similar results were obtained with the other inhibitors (data not shown). We then tested a second generation of inhibitors (kindly provided by Drs. Robert Smith and Mary Zimmerman of Prototek, Inc.) in which the amino acid at position P1 is not natural. This modification enhances half-life of the compounds both in vitro and in vivo. 2 Indeed, these compounds were effective at even lower concentrations. The molting of O. volvulus L3 to L4 was inhibited by 48 -88% and 81-100% at 10 and 50 M, respectively (Table II).  L3s were cultured in the presence of increasing concentrations of 4 different FMK inhibitors and the molting rate was determined on day 6. 50% L3 larvae molt by day 5 under normal control culture conditions. Larval viability after 3 days in culture in the presence of the inhibitors was assessed visually after the uptake of MTT by the larvae, and its reduction into the blue formazan derivative. Each experiment was repeated at least twice; the standard error between experiments never exceeded 10%. lake separation phase (Fig. 3, a-c; Ref. 14); therefore, the larvae never completed the molting. These larvae had a visible L4 epicuticle and cuticle in addition to the outer L3 epicuticle and cuticle, indicating that the new L4 cuticle had been synthesized (Fig. 3, e and f). In some larvae, only partial regions of the L4 epicuticle were seen (Fig. 3d). However, we could not find any larvae in which the separation between the cuticles was partial or complete as seen under normal culture conditions on days 1-4 ( Fig. 3, a-c). In addition we found that many larvae had unusual swollen L3 cuticles (Fig. 3, e and f).
Cloning of the O. volvulus Cysteine Protease, OVCP-A specific 642-bp fragment, designated OC700, was obtained by PCR amplification of O. cervicalis genomic DNA using degenerate primers based on the cysteine and the asparagine active site motifs that are well conserved among the papain-family cysteine proteases (32). Translation of all the possible open reading frames indicated that OC700 encoded portions of a cysteine protease including 51 amino acids with a high degree of similarity to the papain family, containing the conserved histidine active site motif of cysteine proteases plus the cysteine and asparagine active site motifs encoded by the primers. The fragment also contained a 359-bp intron. A TaqI/HindIII 135-bp fragment from the 3Ј exon was used as a hybridization probe to screen an O. volvulus genomic DNA library (a gift from Dr. F. Perler, New England Biolabs). Out of four genomic clones, the largest clone (clone 10 ϭ 6.5 kb) was chosen to be analyzed further. A 2230-bp region of this genomic clone contained a partial gene encoding a cysteine protease with six exons and five introns encoding all three active site motifs: cysteine, histidine, and asparagine. In a 642-bp overlap, the O. volvulus genomic clone was 90.4% identical to the OC700 genomic fragment from O. cervicalis (93.6% identity in the amino acids encoded by the two exons, 6/51 differences in the 3Ј exon; 0/43 in the 5Ј exon). Intron similarity was only 87.7%, with 315 out of 359 bp identical (data not shown). The 5Ј end of the O. volvulus gene was obtained by PCR using an internal primer (OVCP10) and a primer specific to the first 6 amino acids of the cDNA (OVCP-Met) using genomic DNA of O. volvulus as the template. The cDNA was obtained by PCR as described below. This additional fragment contained the remaining two exons and two introns. The composite sequence of the O. volvulus cysteine protease gene (gOVCP) contains eight exons and seven introns (see Fig. 4, arrowheads).
The composite full-length cDNA sequence of the O. volvulus L3 cysteine protease, LOVCP, was obtained from two separate PCR products using oligo(dT) adaptor-primed first strand cDNA of L3 as the template. The 3Ј region was obtained using a specific primer to the histidine active site motif (CPF6) and the 3Ј adaptor primer, and the 5Ј region was obtained using the C. elegans SL1 sequence and a specific primer to the 3Ј noncoding region of the first fragment (CPR3). The nucleotide sequence of LOVCP and the predicted amino acid sequence are shown in Fig. 4 (33). Sequence analysis from the postulated initiator methionine indicates a putative signal peptide of 20 amino acid residues, which is common in many cysteine proteases. Using the Ϫ1, Ϫ3 prediction method of von Heijne (34), a potential signal peptidase cleavage site is indicated at residue 21, lysine, which would result in a proform of the enzyme with 286 amino acids and a predicted mass of 32 kDa. Comparison with the other members of the papain superfamily (35) suggested that the LOVCP proenzyme is processed into the mature protease by cleavage at the lysine residue (amino acid 66, Fig. 4), perhaps by autohydrolysis, as has been demonstrated for cathepsin L (36,37). Cleavage of the propeptide region (45 amino acids) would result in a putative mature enzyme with 241 amino acids and a predicted mass of 27 kDa. The conserved cysteine, histidine, and asparagine active site residues are at position 31, 178, and 200, respectively, in the mature protein (Fig. 4, C, H, and N in bold at positions 96, 244, and 265 in the cDNA sequence, respectively). The mature enzyme contains two putative N-glycosylation sites (residues 187-189 and 286 -288). An additional putative N-glycosylation site is present in the signal peptide (residues 16 -18).
A homology search of LOVCP amino acid sequence against the PIR Protein Data Base showed a significant sequence similarity with other members of the cysteine protease family of the papain superfamily including papain, actinidain, cathepsin C, mammalian cathepsin L, and cathepsin B, and many of the protozoan, plant, and Schistosoma mansoni cathepsin-like enzymes, ranging from 22% to 35% identity in the amino acid sequences (data not shown).
An interesting feature of the onchocerca cysteine protease is a stretch of 5 amino acids, CGSC 112 W, 74 amino acids downstream from the cysteine active site motif that are an exact repeat of the 5 amino acids of the cysteine active site motif CGSC 31 WAF (Fig. 4, shaded areas). This is the first cysteine protease that has such a repeat in its structure. In addition, three amino acids contained in the cysteine active site motif, GSC, are repeated twice; the first one 37 amino acids downstream from Cys 31 , and the other one 3 amino acids downstream from Cys 112 (Fig. 4, shaded areas).
The Protease Gene Is Single-copy-In order to determine the copy number of the OVCP protease gene, genomic DNA of O. volvulus was digested with EcoRI or HindIII and probed by Southern blot analysis with a 900-bp fragment of LOVCP at 52°C. The 900-bp probe, which did not contain cleavage sites for any of the two restriction enzymes, hybridized with a single restriction fragment from each digest (Fig. 5):; 5 kb in EcoRI and 9.4 kb in the HindIII digest. Hybridization at lower temperatures (25°C, 37°C, and 42°C) did not reveal any additional bands. When other genomic DNA from parasitic and non-parasitic nematodes Strongyloides stercoralis (the worms were kindly provided by Dr. Shah, Pennsylvania), D. immitis (the worms were kindly provided by Dr. Carlow, New England Biolabs), and C. elegans (the worms were kindly provided by Dr. Radice) were tested, none of them hybridized with the LOVCP probe at 42°C (data not shown).
Identification of the Endogenous Larval Cysteine Protease-The endogenous cysteine protease in molting larvae was identified initially with labeled inhibitors. One of the peptidyl monofluoromethylketone that has a tyrosyl residue, Mu-Tyr-O-methyl-homoPhe-FMK, was labeled with 125 I and used in vitro to label the target cysteine protease during molting. This inhibitor reacts covalently and specifically with the cysteine residue in the active site and does not react with inactive or denatured enzymes or with other proteases or proteins (28). Larvae were cultured for 2 days in the presence of the radiolabeled inhibitor, collected, and washed four times in PBS. Lysates were prepared and subjected to electrophoresis in a 12% acrylamide gel. An autoradiogram of the gel clearly showed one radiolabeled band with a molecular mass of 72 kDa (Fig. 6a).
A major band of 72 kDa was also identified in the soluble and nondenatured extracts of larvae collected on day 2 and 3 and in the culture supernatant collected from larvae that were cultured for 2 days in vitro. To detect the endogenous active enzyme, the samples were reacted with biotin-Phe-Ala-CHN 2 (29) before running on a SDS-PAGE gel and electrophoresis onto nitrocellulose filter. The inhibitor did not detect an active enzyme in extracts from L3 or L3 collected after only 1 day in culture (Fig. 6b). The similarity between the radiolabeled band and biotinylated bands confirmed that the endogenous active cysteine protease in molting larvae that binds to the inhibitors is a 72-kDa protein species in SDS-PAGE gels.
As the cloned cysteine protease cDNA encoded a preproform of the enzyme with a molecular mass of 34.5 kDa, it was of interest to find out the relationship between the 72-kDa protein identified by the inhibitors and the native protein corresponding to the recombinant enzyme. Soluble extracts, prepared from L3 cultured for 1 day in normal medium before being metabolically labeled for 24 h with [ 35 S]methionine, were incubated with antibodies against the recombinant O. volvulus LOVCP protein, and the immune complexes were precipitated with protein A-Sepharose and evaluated by 4 -20% SDS-PAGE electrophoresis and fluorography. Three proteins of 72, 48, and 37 kDa were recognized (Fig. 6c). Crude extracts prepared from L3 collected on day 2 in culture were also analyzed by immunoblot analysis with the same antibodies. Three proteins of 72, 37, and 23 kDa were recognized (Fig. 6d).
The similarity between the 72-kDa protein band recognized by the antibodies and the 72-kDa band recognized by the inhibitors confirmed our assumption that we have cloned the larval cysteine protease targeted by the cysteine protease inhibitors in vitro.
Localization of the Onchocerca Cysteine Protease in Larval Stages of the Parasite-We previously reported the localization of the protein corresponding to onchocystatin in the basal layer of molting L3, around the fused lakes, where the separation between the cuticles would take place, and in the cuticle of the newly formed L4 (16). In this study, immunoelectron microscopy staining of O. volvulus thin sections of larvae during molting with antibodies raised against the recombinant O. volvulus LOVCP protein permitted the localization of the onchocerca cysteine protease in the intermediate stages of O. volvulus molting larvae. Examination of thin sections of molting larvae revealed the newly formed epicuticle and cuticle of L4 in areas beneath the basal layer of the old L3 cuticle, and the separation between the cuticles below the old basal layer of the L3 (Fig. 7, b-d), as seen during normal molting (Fig. 3, a  and b). The antibodies reacted with proteins in the areas around the region where the separation between the cuticles takes place. The protein is also seen in the cuticles after their complete separation; in some areas of the cuticle of the newly formed L4 (Fig. 7, d and e), and in the lower part of the old cuticle, where the separation occurred (Fig. 7, c and d). Interestingly, the density of staining is highest in larvae after 1 day in culture before the separation between the cuticles starts, and is absent from L3 (Fig. 7a). In addition, specific labeling by the antibodies was observed in the hypodermis of molting larvae inside secretory vesicles (Fig. 8a), in the basal lamina lining the pseudocoelom (Fig. 8b), and in cells of epidermal cord (Fig. 8c). A control normal rabbit serum or serum against GST alone did not cross-react with any proteins in the larvae (data not shown).
Labeling of thin sections of larvae that were cultured for 24 h in the presence of biotin-Phe-Ala-CHN 2 with streptavidin-gold particles detected the inhibitor in many granules in the glandular part of the esophagus (Fig. 8d). The larvae in Fig. 8d are from day 2 in culture. Similar labeling was also observed in larvae cultured for 24 h in the presence of the inhibitor on day 0 and 1, but not when they were cultured with the inhibitor on day 3 (data not shown). Thin sections of larvae cultured without the inhibitor did not react with the streptavidin-gold particles (data not shown). When thin sections from these larvae were probed with the anti-GST-LOVCP antibodies, the enzyme was co-localized to where the inhibitor was present, the granules in the glandular part of the esophagus. In addition, staining was also observed in the basal lamina lining the pseudocoelom (Fig. 8e). DISCUSSION We previously proposed that a cysteine protease has a critical role in the molting process of the O. volvulus infective larvae, L3 (15,16). The results from this study clearly support this hypothesis. We have shown the inhibitory effects of a panel of FMK compounds on the molting of L3 to L4. The FMK are known to be highly specific inhibitors of cysteine proteases of the cathepsin B, L, and C activities (18). The fact that increased concentrations of the Z-Phe-Phe-FMK were required to demonstrate biological effects is consistent with limitations in the transport of this inhibitor across the cuticle of the larvae. Since these inhibitors were not lethal to the larvae, their effects appeared to be specific to the molting process and, therefore, indirectly indicated that a specific cysteine protease is present and active in the larvae during molting. A putative target enzyme was identified in extracts of larvae collected on day 2 and 3 (Fig. 6b). The inhibitors, Z-Phe-Ala-FMK and Z-Phe-Arg-FMK had to be present during the first 24 -48 h, the beginning of the molting process, to exert a complete inhibitory effect on molting (Fig. 2). After the 3rd day in culture, only a partial inhibitory effect was observed. The partial effects on days 2 and 3 could be due to a subpopulation of larvae that were faster in their molting process and therefore were not affected anymore when the inhibitors were added. Many times during in vitro culturing, we have seen larvae that have already molted by day 3 (14). These findings implied that the effect of the inhibitors was more specific to the changes in the cuticle that occur during the molting process on days 1-3 than in earlier events. At this time the separation between the old and new cuticles is in progress to completion (Fig. 3, a and b). This conclusion is supported by the ultrastructural studies of the larvae that did not molt (Fig. 3, d-f). The FMK inhibitors induced morphological abnormalities in the cuticles of these larvae. It appeared that the separation between the old and new cuticles was inhibited, inducing in many larvae a very unusual swelling of the old cuticles, yet the synthesis of the cuticle of the L4 was not affected. In addition, in immunogold labeling experiments we were able to localize the cysteine protease with an antisera raised against the recombinant cysteine protease that was cloned from L3 of O. volvulus, LOVCP. Notably, these antibodies reacted in molting larvae with a protein present in the same areas where onchocystatin, the endogenous cysteine protease inhibitor of O. volvulus, was localized (16). Both proteins were localized in the areas around the region where the separation between the cuticles takes place, in some areas of the cuticle of the newly formed L4, and in the lower part of the old cuticle, where the separation had occurred (Fig. 7).
We propose that the biological effects of the FMK inhibitors were specifically due to the inhibition of a cysteine protease needed for the degradation of cuticular proteins in the region where the L3/L4 cuticle separation occurs. The inhibition of L4 development correlates with the timing of the appearance of the lakes in the cuticles, a process that precedes ecdysis, or larval exsheathment, the emergence of the L4 from the old cuticle of L3. A cathepsin L-like cysteine protease was recently shown to be also important for the molting of B. pahangi L3. 3 An endogenous cysteine protease that has a probable function in the excystment of Paragonimus westermani metacercariae was recently described by Chung et al. (38). In this parasite, the authors have suggested that the dormant metacercariae begins to secrete the cysteine proteases on its arrival in duodenum, probably in response to the host signal of pH change. Although the mRNA encoding the onchocerca larval cysteine protease was isolated from vector-derived L3, enzyme activity was detected predominantly after the L3 were cultured at 37°C for 24 h. This was confirmed by immunogold labeling of larvae at different stages in the molting process (Fig. 7), as well as by detection of an active enzyme with a biotinylated inhibitor (Fig. 6b). One possible explanation is that the transcription of the protease mRNA, and the translation of enzymatically inactive protease proform, may occur in the L3 and during the 1st day in culture. However, conversion of the protease to an active form occurs only after the parasite has started the molting process and the protease has been sequestered in the cuticle for 24 h. Another possibility is that the enzyme is complexed with the endogenous inhibitor, onchocystatin, and is dissociated from the inhibitor after 24 h when the separation between cuticles starts. The timing of the enzyme activation in both cases would be consistent with the detection of the active enzyme by the labeled inhibitor in larval extracts of larvae cultured for more than 24 h. Cuticle degrading proteases in an inactive complex were also found in the molting fluid of silkmoth larvae. Activation of the molting enzymes was thought to occur by dissociation of an inhibitor, the activation of proenzymes, or release of enzymes from a compartmentalized state (reviewed in Ref. 39). In O. volvulus larvae, cultured in the presence of a biotinylated derivative of a fluoromethylketone inhibitor, the enzyme-inhibitor complexes were co-localized in granules in the glandular part of the esophagus (Fig. 8,  d and e), indicating that the enzyme is probably compartmen- , and in cells of epidermal cord, C (c). In thin sections of larvae from day 2 in culture that were cultured in the presence of biotin-Phe-Ala-CHN 2 for 24 h, the biotin was detected with streptavidin-gold (d). Note the presence of the label in the granules, g, which surround the glandular esophagus, Eo. Thin sections of these larvae were also probed with rabbit anti-GST-LOVCP antibodies and then with protein A coupled to 15 nm gold particles for indirect enzyme localization (e, bar 0.25 m). The endogenous cysteine protease was co-localized in the granules, g, which surround the glandular esophagus. In addition, the label was detected in the basal lamina lining the pseudocoelom (arrows). talized in these organelles before being transported via the pseudocoelom fluid and the secretory vesicles to the cuticle (Fig. 8, a and b). In previous immunogold labeling studies, we found that onchocystatin was also localized to the same internal organelles as the enzyme (data not shown).
The PCR approach used to clone the cysteine protease of O. volvulus L3 (LOVCP) allowed us to circumvent the considerable difficulty in obtaining adequate quantities of the onchocerca enzyme for partial amino acid sequence determination. We believe that LOVCP encodes the cysteine protease involved in molting of onchocerca larvae for several reasons. First, we found only one copy of the gene (Fig. 5) by Southern blot analysis at low stringency; it is expressed in L3 and molting larvae, antibodies raised against the recombinant enzyme localized the protein in the cuticles of molting larvae (Figs. 7 and 8), and antibodies raised against the recombinant enzyme recognized the same 72-kDa protein band that was detected by the cysteine protease inhibitors (Fig. 6). The sequence of the onchocerca cysteine protease clearly places the enzyme as a member of the papain family of cysteine proteases. The amino acid sequence of the O. volvulus cysteine protease deduced from the cDNA sequence has significant identity to many members of the papain superfamily of cysteine proteases, sharing 22-35% identical amino acids (data not shown). However, the onchocerca enzyme is unique in having a 5-amino acid repeat, CGSCW, which is part of the cysteine active site motif, CGSC-WAF, and two additional repeats of GSC. The significance or the function of the 5-amino acid repeat is unknown. Expression of an active enzyme and comparative studies using deleted and site-directed mutants of the cDNA coding for LOVCP in E. coli or Pichia pastoris may provide clues to the possible function of these repeated amino acids.
By homology the predicted amino acid sequence of LOVCP is mostly similar to cathepsin C or cathepsin L (data not shown). The predicted molecular mass of the mature O. volvulus enzyme encoded by LOVCP is 27 kDa, whereas the molecular mass of the active enzyme detected by the cysteine protease inhibitors, and of one of the protein bands recognized by antibodies directed against the recombinant enzyme, is 72 kDa. The 37-kDa protein band recognized by the antibodies may correspond to the preproform of the enzyme (predicted molecular mass of 34.5 kDa), and the 23-kDa protein band may correspond to the mature protease (Fig. 6). The reasons for the size differences between the endogenous and recombinant proteases are not clear. It may be due to post-translational modification such as N-linked glycosylation, the endogenous enzyme forming dimmers resistant to the reducing agents in the SDS-PAGE buffers, or if the enzyme is bound to another protein to help export it to the hypodermis and the cuticle. Size differences between the endogenous and recombinant cysteine proteases were also observed in Trypanosoma cruzi (37).
The cloning of a cysteine protease required for the molting of O. volvulus L3 provides us with the opportunity to study the structure-function relations between the O. volvulus enzyme and its endogenous inhibitor, onchocystatin, and will also be useful for studying the regulation of cysteine protease activity during molting. Acknowledgments-We thank Dong-Hun Lee for DNA sequencing and preparation of synthetic oligonucleotides and Massah Abdullai and Johnette Brown for technical assistance. We are grateful to Dr. Robert Smith and Prototek Inc. for the FMK compounds. We are also grateful to Dr. Francine Perler and Dr. Colvin Redman for comments on the manuscript and to Dr. Xiquiang Hong for useful discussions.