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Gut-specific and developmental expression of a Caenorhabditis elegans cysteine protease gene
Molecular and Biochemical Parasitology, 51 (1992) 239 250 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
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MOLBIO 01695
Gut-specific and developmental expression of a Caenorhabditis elegans cysteine protease gene Celeste R a y and James H. M c K e r r o w UCSF School of Medicine, Department of Pathology, San Francisco, CA, U.S.A. (Received 20 August 1991; accepted 22 October 1991)
A Caenorhabditis elegans cysteine protease gene fragment, amplified by PCR using conserved eukaryotic protease gene sequences as primers, was used as a probe to isolate c D N A and genomic clones. The genomic clone, which had a coding sequence of 987 bp interrupted by 2 small introns, was physically mapped to the middle of linkage group V. The predicted amino acid sequence of the mature C. elegans cysteine protease was homologous to those of other eukaryotic cysteine proteases, particularly to that of the nematode parasite Haemonchus contortus (50%) and to the cathepsin B-like hemoglobinase of the trematode parasite Schistosoma mansoni (54%). The pro region of the C. elegans protease was homologous only to that of the H. contortus enzyme, implying a similar mechanism of protease activation. The C. elegans cysteine protease gene was temporally regulated: abundant 1.1-kb transcripts were detected in larvae and adults, but not in embryos. Transcription also was spatially regulated, occurring only in the intestine. Like the vitellogenin genes, which also are transcribed exclusively in the intestine, the 5' end of the C. elegans cysteine protease gene had at least one copy of each of 2 heptameric sequences which may be transcriptional regulatory elements governing gut-specific expression. Key words: Caenorhabditis elegans; Cysteine protease; Gut development; Parasitic helminth
Introduction
Eukaryotic lysosomal cysteine proteases are well characterized as enzymes involved in intracellular protein degradation. Recently, possible extracellular roles for this class of enzymes have been suggested from studies of tumor cell invasion [1,2], tissue degradation by macrophages [3] and parasite metabolism [4]. Structurally and biochemically similar cysteine proteases have been identified in many nematode and trematode parasites. The most extensively studied of the parasite cysteine Correspondence address: Celeste Ray, UCSF School of Medicine, Department of Pathology, P.O. Box 0506, San Francisco, CA 94143-0506, U.S.A. Fax: 415 476-9672. Abbreviations: PCR, polymerase chain reaction. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M74797.
proteases are the cathepsin B-like hemoglobinase of the trematode blood fluke Schistosoma mansoni [5] and the proteases of the nematode Haemonchus contortus [6,7]. Production of these enzymes is developmentally regulated, occurring at high levels only in actively feeding stages. Moreover, the cysteine proteases of S. mansoni [5], Angiostrongylus cantonensis [8], and Ascaris suum [8] are located primarily in the intestine, an observation which supports their role as digestive enzymes. Although these proteases are potential targets for serodiagnosis and anti-parasite chemotherapy, effective design and application of such reagents will depend on knowledge of basic parasite biology, gut development, and gene regulation. Two similar but distinct cysteine protease activities also have been identified in the freeliving nematode Caenorhabditis elegans [9,10]. Although these enzymes comigrate with lysosomal markers in isopycnic centrifugation, their functions were unknown. We have used
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a C. elegans cysteine protease gene fragment generated by the Polymerase Chain Reaction (PCR) [11] as a hybridization probe to isolate both genomic and cDNA clones encoding a single C. elegans cysteine protease. The results described here establish a basis for using C. elegans as a model system for studying parasite cysteine protease gene regulation.
Materials and Methods
Worm strains and media. General culture methods were those previously described by Brenner [12]. The C. elegans strain CB1490 (him-5[e1490]), which produces males at high frequency, was grown at room temperature on enriched NG agarose plates with Escherichia coli OP50 as a food source. The dauerconstitutive strain DR40 (daf-1), provided by the Caenorhabditis Genetics Center, was maintained at 15°C. Bacterial strains, plasmids, and restriction enzymes. E. coli strain DH5-~ was used as the host for plasmid pBluescript KS (Stratagene) and its derivatives. Strains containing these plasmids were grown in LB medium supplemented with 60 #g ml-~ ampicillin and plasmid DNA was isolated by the mini alkaline lysis procedure of Birnboim and Doly [13]. Restriction enzymes were obtained from BRL and New England Biolabs and were used as recommended by the suppliers. Isolation of the C. elegans cysteine protease gene. A PCR-generated, 160-bp cysteine protease gene fragment from C. elegans [11] was labeled with 32,3 p using random primers [14] and was used to screen C. elegans cDNA and genomic libraries by plaque hybridization [15]. The C. elegans cDNA library, constructed in 2uber2, was obtained from Chris Martin. Positive phage were converted to plasmids using helper phage R408 [16], and the 0.8-1.0kb ApaI/SstI inserts from 5 positive clones were subcloned into the ApaI/Sstl sites of pBluescript KS. Partial nucleotide sequencing revealed that the cDNA inserts all had the
same nucleotide sequence, and the plasmid harboring a l.l-kb insert with the furthest 5' extension was named pCR1. Genomic clones of the C. elegans cysteine protease gene were isolated from a 2EMBL3 library obtained from A. Kamb in Cynthia Kenyon's laboratory. Three positive phage were grown as plate lysates and purified through sucrose gradients [17]. DNA was obtained from the phage as described [17]. Restriction fragments which hybridized to the original probe fragment were identified by Southern blotting [18] and were subcloned into pBluescript KS. As determined by partial nucleic acid sequencing, all 3 of the plasmids contained all or part of the same cysteine protease gene. One of the plasmids, pCRI4, contained the complete structural gene on a 2.8-kb EcoRI fragment inserted into the EcoRI site of pBluescript KS.
DNA sequencing and primer extension. The nucleotide sequences of the inserts in pCR1 and pCR14 were determined by the dideoxy chain termination method [19] using the Sequenase kit (United States Biochemical) and double-stranded DNA templates isolated by alkaline lysis [13]. The nucleotide sequence of the coding region shown in Fig. 1 was read from both strands. Primer extension analysis was done by the methods of Geliebter [20] using poly(A) + RNA isolated from adult male and hermaphrodite worms (see below). The primer was a synthetic oligonucleotide having the nucleotide sequence 5'-CAAGCGTGACTGCGCAG-3'. Worm fractionation and RNA isolation. For separation of adult males and hermaphrodites, worms were washed off several enriched NGM agarose plates (150 mm) with M9 buffer [12] followed by flotation in 1 M sucrose. Nylon filters (Spectrum Medical Industries, Inc.) were used to remove larvae from adults and to fractionate the adults by sex as described by Klass and Hirsh [21]. Different larval stages were isolated from synchronous cultures established as described by Austin and Kimble [22]. Embryos were
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examined microscopically to insure that both older and earlier embryos were represented. Dauer larvae of strain CB1490 were isolated from starved plate cultures by treatment with 1°/0 SDS [23]. The dauer-constitutive strain DR40 was grown at 15°C, the permissive temperature, on enriched N G M agarose plates then shifted to room temperature for 78 h to allow dauer formation. Dauer larvae were isolated by 1% SDS treatment as described above. All worm fractions were examined microscopically to determine homogeneity then were frozen at - 8 0 ° C for RNA extraction. Total RNA was isolated from the frozen worms by methods derived from Austin and Kimble [22]. The nucleic acid pellets were resuspended in water and poly(A) ÷ RNA was isolated using Poly(A) Quick columns from Stratagene.
Northern blots. Approximately 0.4 #g poly(A) ÷ RNA from eggs, larvae and adults were fractionated by electrophoresis on 1.25% agarose/6.67% formaldehyde gels. The gels were blotted onto Nytran filters and hybridized to 32P-labeled probes [17]. Bound probe was removed from the blots by boiling the filters for 20 min in 0.01 x SSC/0.5% SDS. In situ hybridization. Cysteine protease m R N A was detected in whole worms using digoxigenin-labeled probes. The protocol was originally designed by Tautz and Pfeile for use in Drosophila [24] and was adapted for use in worms by C. Oh, B. Edgar, D. Greenstein, and G.Ruvkun (personal communication). The probes were the 1.1-kb ApaI/SstI cysteine protease fragment from pCR1 and the 2.1-kb HindlII C. elegans pharyngeal myosin fragment from pMyo-II, pMyo-II contains a fragment of the rod portion of the pharyngeal myosin heavy chain C [25]. These fragments, purified from low-melting agarose gels and digested with DdeI to reduce the average probe size to 200-300 bp, were then labeled with digoxigenin using random hexamers as described in the Boehringer Mannheim Genius Kit. Worms from enriched N G M agarose plates were washed in M9 buffer and resuspended in
5 ml fixing solution (2.2% paraformaldehyde/ 80 mM KCI/20 mM NaC1/2 mM EGTA/15 mM Pipes, pH 7.4/0.5 mM spermidine/0.2 mM spermine/0.5% fl-mercaptoethanol/20 mM vanadyl ribonucleoside complex). 3 ml nheptane and 1 ml 90% methanol/10% 0.25 M EGTA were added, the emulsion vortexed for 20 min at room temperature, then frozen in a dry ice/ethanol bath. The worms were thawed, vortexed another 20 rain at room temperature, pelleted in a clinical centrifuge, washed in 10 ml methanol, rinsed in 50% ethanol/50% xylene, and soaked in 100% xylene for 2 h at room temperature. Sequential washes were done in 50% ethanol/50% xylene, 100%o ethanol, 100°/0 methanol, and 50% methanol/50% PBTS containing 5% (w/w) paraformaldehyde. (PBTS is 1 x PBS/ 0.1% Tween 20/0.01% SDS.) Animals were fixed in PBTS containing 5% paraformaldehyde for 20 min at room temperature and rinsed 3 times in PBTS. The cuticles were permeabilized by incubating the worms 10 min at 37°C in PBTS containing 100 #g m1-1 Proteinase K, washing twice in PBTS containing 2 mg ml-1 glycine, and washing twice in PBTS. Post-fixing then was done for 2 min at room temperature in PBTS containing 5°/0 paraformaldehyde, followed by 5 washes in PBTS. For each hybridization reaction, 20 pl of fixed, permeabilized worms were put into microfuge tubes and rinsed sequentially in 50% PBTS/50% hybridization solution then in 100% hybridization solution (50% deionized formamide/5 x SSC/10% dextran sulfate (Sigma #D6001)/100 /~g m1-1 salmon sperm DNA/100 pg ml-1 E. coli tRNA/50 #g ml-1 heparin/0.1% Tween 20/10 mM Tris-HC1, pH 7.5/0.005% SDS). Prehybridization was at 50°C in 200 pl hybridization solution, and hybridization was done overnight at 50°C in 100 /A hybridization solution containing approximately 0.1 #g m l - 1 digoxigenin-labeled probe. Sequential washes each were done for 20 min at 50°C in a volume of 500 pl using the following solutions: hybridization solution, hybridization solution with 5% dextran sulfate, twice in hybridization solu-
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tion without dextran sulfate, once in 50% hybridization solution/50% PBTS, and 5 times in PBTS. Animals were then incubated in 500 /A PBT (1 x PBS/0.1% Tween 20)/1% BSA (Sigma #A7030)/0.02% sodium azide for 90 min at 4°C. In order to detect bound probe, worms were incubated overnight at 4°C with alkalinephosphatase conjugated anti-digoxigenin antibodies (Boehringer Mannheim Genus kit) diluted 1:100 in 1% BSA/0.02% sodium azide, followed by six 20 rain washes in PBTS at 4°C. After 2 rinses in 100 m M NaC1/50 mM MgC12/100 m M Tris, pH 8.8/1 m M levamisol/ 10% Tween 20, animals were resuspended in l ml of this buffer containing 336 #g m1-1 nitroblue tetrazolium salt (Boehringer Genius Kit) and 174 gg ml-1 X-Phosphate (Boehringer Genius Kit). Reactions were incubated in the dark for 3 h at room temperature, and worms were mounted for microscopy on 2% agarose pads made in 50 mM Tris-HC1 (pH 9.5)/5 mM MgC12.
from the ATG translation initiation codon. The gcp-1 transcripts were not trans-spliced. The 5' end ofgcp-1 contained at least one copy of each of 2 heptamers which may be regulatory elements governing gut-specific expression [27] (Fig. 1). At the 3' end of the gene, a potential poly(A) addition signal was found 40 bp from the translation termination codon and at least 22 adenosines were found 14 bp further downstream in the c D N A clone. Although we have identified only one gene, we cannot rule out the possibility that C. elegans has other cysteine protease genes. Two distinct cysteine proteases have been identified biochemically in C. elegans [9,10]. One of these activities is similar to cathepsin B and may be the one encoded by gcp-l. The cysteine proteases of the evolutionarily related parasitic nematode H. contortus are encoded by a multigene family [7]. Because of the molecular similarities between gcp-1 and the protease genes of H. contortus (see below), C. elegans may also contain more than one cysteine protease gene.
Results and Discussion
The C. elegans cysteine protease gene has structural similarities to those J?om parasites. Small introns (45-59 bp) are characteristic of many C. elegans genes and may also be common in genes from parasitic worms. The HGPRTase gene from S. mansoni, for example, contains 4 small introns of 31, 33, 42, and 32 bp [28]. Because only a c D N A clone has been isolated for the S. mansoni cathepsin Blike hemoglobinase, nothing is known yet about the intron structure of this gene. Of the 2 cysteine protease genes isolated from H. contortus, AC-1 is a c D N A clone [6] and AC-2 is a genomic clone [7]. AC-2 has 11 introns ranging in size from 57 bp to over 5.2-kb [7]. Intron 1 occurs immediately after the putative A T G translation start codon and is 80 bp long. Likewise, the first intron of gcp-1 was in this same position and was 82 bp long. The 5' splice donor sequences of these 2 introns were identical at twelve nucleotides centered at the splice junction, whereas the 3' acceptor sequences were identical at 10 of the 12 nucleotides centered at the 3' splice site.
Isolation of a C. elegans eysteine protease gene. A 160-bp PCR-generated cysteine protease gene fragment from C. elegans was used to isolate c D N A and genomic clones encoding a single cysteine protease gene. This gene was designated gcp-1 for Gut-specific Cysteine Protease. The original EMBL3 genomic clone was sent to Alan Coulson and John Sulston at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England for physical mapping [26]. According to their analysis, the cysteine protease gene was located near the middle of chromosome V, between the genes daf-ll and myo-3. The cysteine protease coding sequence was 987 bp long and was interrupted by 2 small introns of 82 and 46 bp (Fig. 1). As determined by primer extension, the c D N A clone of gcp-1 was missing 23 bp of 5' sequence. Primer extension was also used to show that transcription was initiated 25 bp downstream from the T A T A T A A sequence and 28 bp upstream
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Fig. 1. Nucleotide and predicted amino acid sequences of the C. elegans cysteine protease gene. Nucleotides are numbered from the transcription start site at position + 1. Introns are in lowercase letters and the ends of the c D N A clone are indicated by solid triangles. The ATG translation start site, TAA translation termination signal, TATA promoter element, and A A T A A A poly(A) addition signals are underlined. Enclosed regions labeled 'Box 1' and 'Box 2' are potential regulatory elements ~ r gut-specific transcription which were first identified in the vitellogenin genes [27]. Consensus sequences ~ r each of these elements is written below the g~-I sequence. Capitol letters represent invariant bases, underlined bases are almost invafiant, and lower case letters indicate variable bases. In the amino acid sequence, the putative pro region cleavage site is indicated by an asterisk and the active site amino acid residues are boxed.
Although more divergent in size than the first introns, AC-2 intron 9 and gcp-1 intron 2 also interrupted the coding sequences at exactly the same positions and had similar nucleotide sequences at the splice junctions. Thus, the positions, sizes, and boundary sequences of the 2 gcp-1 introns are similar to those of 2 of the introns in the H. contortus AC-2 gene. The predicted amino acid sequence of the C. elegans cysteine protease is similar to mammalian cathepsin B and is compared to that of the H. contortus cysteine proteases and the S. mansoni cathepsin B-like hemoglobinase in Fig. 2. The mature C. elegans protease is
50% homologous to those of H. contortus and, if conservative amino acid substitutions are included, this similarity increases to 83%. The mature C. elegans cysteine protease also was 54% homologous to the S. mansoni hemoglobinase, with an overall similarity of 84%. In addition, although there was no homology with that of the S. mansoni hemoglobinase, the 84 amino acid pro region of the C. elegans cysteine protease (Fig. 2) was 28% identical to the H. contortus cysteine protease AC-2 and 31% identical to AC-1 (73-74% chemical similarity). This result is surprising since pro regions are highly divergent and indicates that
244
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Fig. 2. Comparison of the predicted amino acid sequence of the C. elegans cysteine protease gcp-1 to those of the H. contortus AC-1 [7] and S. mansoni sm31 [33] cysteine proteases. The amino acid sequence of AC-1 is identical to that of AC-2 except for seven amino acids [6] that are written above the AC-2 sequence. The sm31 pro region is not given since it shares no homology with the C. elegans and H. contortus proteases. The predicted site of pro region cleavage is indicated by an arrowhead and the active site residues are boxed. Double dots designate identical amino acids, single dots indicate amino acids that are chemically similar. Dashes represent gaps that were introduced to facilitate sequence alignment.
the proteases from these 2 species may share similar activation mechanisms. Unlike the proteases from H. contortus and S. mansoni, that from C. elegans does not contain any potential N-linked glycosylation sequences.
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Expression of gcp-1 is temporally regulated. Northern blots with poly(A) + R N A isolated from different developmental stages of C. elegans were prepared and screened at high stringency with the e D N A insert of pCR1 as a probe (Fig. 3). Abundant 1.1-kb transcripts migrating as a single band were present in approximately the same amounts in L1-L4 larvae, dauer larvae, and adults, but were not detected in embryos. The presence of gep-1 m R N A in dauer larvae was confirmed using the dauer-constitutive strain DR40 (data not shown). Accumulation of gcp-1 m R N A in L1 larvae did not depend on the presence of food,
Fig. 3. Northern blot analysis of gcp-1 transcription. Poly(A) + R N A isolated from different developmental stages of C. elegans CB1490 was fractionated on a 1.25% agarose/6.67% formaldehyde gel, blotted onto a Nytran filter, and hybridized with the 32P-labeled gcp-1 e D N A insert of pCR1. Following autoradiography, the blot was boiled and rehybridized with a C. elegans actin-1 fragment from pW-16 210 (M. Krause). Lanes (left to right): adult hermaphrodites; adult males; dauer larvae; embryos; L1 larvae; L2 larvae; L3 larvae; L4 larvae.
245 since the larvae were isolated after allowing eggs to hatch in M9 buffer. These results indicate that gcp-1 expression is temporally regulated, occurring at or near hatching when the embryos contain about 550 cells. When this same blot was probed with a C. elegans actin-1 gene fragment (pW-16-210 obtained from M. Krause), all of the lanes contained a 1.3-kb band (Fig. 3), indicating that comparable amounts of RNA were in each lane. If C. elegans has more than one cysteine protease gene, the bands seen in the Northern blot could be the result of hybridization to transcripts from these genes in addition to those from gcp-1. Because of the stringency at which this blot was hybridized the other genes would have to be highly homologous to gcp-1 and produce transcripts of similar size. Moreover, these genes would be temporally regulated since no transcripts were detected in embryos. These results are similar to those found with the H. contortus AC-2 gene which is also developmentally regulated: it is transcribed at high levels in blood-feeding adults and at 30fold reduced levels in non-feeding larvae [7]. Likewise, high levels of the S. mansoni cathepsin B-like hemoglobinase activity are associated with feeding adult-stage worms. That parasites delay activation of their cysteine protease genes until t h e adult stage could be the result of adaptation to a parasitic lifestyle. Parasite-derived proteases are highly antigenic, and parasitic worms like schistosomes usually have a tissue-migratory phase before reaching their ultimate residence in the bloodstream. It may, therefore, be in the parasites' best interests to delay protease expression until reaching their main food source. In contrast, free-living worms, like C. elegans, could begin producing digestive enzymes as soon the worms hatch and begin searching for food. An unexpected observation was that nonfeeding dauer larvae contained as much cysteine protease m R N A as did L1-L4 larvae and adults. Dauer larvae may remain in this state of arrested development for several months, and production of high levels of a
digestive protease would be metabolically wasteful and could potentially damage the intestine. Reape and Burnell [29] have shown that inhibition of RNA synthesis does not inhibit dauer recovery, implying that dauer larvae store m R N A made during earlier larval stages. Because pharyngeal pumping (feeding) is one of the first signs of dauer recovery, production of digestive enzymes should be one of the initial events in dauer recovery. The gcp1 m R N A we detected in dauer larvae, therefore, may have been made earlier and was being stored in a translationally inactive state. If this is the case, gcp-1 expression may be subject to translational as well as transcriptional control.
Expression of gep-1 is spatially regulated. The spatial regulation of gcp-1 expression was analyzed using in situ hybridization (Fig. 4). Whole worms were probed with the cDNA insert from pCR1 which had been labeled with digoxigenin. A digoxigenin-labeled pharyngeal myosin-2 gene fragment [25] also was used as a positive control. Bound probe was detected colorimetrically using anti-digoxigenin antibodies conjugated to alkaline phosphatase. Cysteine protease m R N A was located exclusively in the intestinal cells of C. elegans, indicating that expression was spatially regulated (Fig. 4). In general, we observed that staining was more intense in larvae than in adults (Fig. 4c-d), probably as a result of cuticle permeability differences. In addition, the anterior gut stained darker than did the posterior (Fig. 4d), a result which could also be due to differential permeability or to differential expression in different parts of the intestine. Because staining was observed only when probe was added and the pattern of staining was specific for each probe used, we conclude that gcp-1 expression is tissuespecific. Other C. elegans genes are known to be expressed only in the intestine. These include the vitellogenin [30] and esterase genes [31]. Zucker-Aprison and Blumenthal [27] have identified 2 repeated heptameric sequences in the promoters of the C. elegans and Caeno-
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Fig. 4. In situ hybridization. Samples containing mixed stages of whole worms were fixed and permeabilized, then incubated in hybridization buffer containing digoxigenin-labeled probes (see Materials and Methods). Bound probe was detected colorimetrically (blue color; dark areas in these photographs) using anti-digoxigenin antibodies (1:100) that were conjugated to alkaline phosphatase (Boehringer Mannheim). (a) Adults and larvae incubated without probe. (b) Larva incubated with the pharyngeal myosin-2 fragment from pMyo-II [25]. (c) and (d) Adults and larvae, respectively, incubated with the gep-1 cDNA insert of pCR1. The scale bar represents 70 microns.
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rhabdit& briggsae vitellogenin genes that may be binding sites for transcriptional regulators. Preliminary DNA sequence data of the C. elegans cysteine protease gene promoter showed that this gene had at least one copy of each of the heptamers (Fig. 1). Interestingly, the 5' untranslated region of the H. contortus cysteine protease gene AC-2 also has copies of these 2 heptamers. In addition, regions of the esterase promoter that are important for gutspecific expression have recently been identified using sequential deletions of this region [32]. The pattern of esterase expression from these deletion mutants suggests that both activators and repressors regulate gut-specific expression. A similar analysis can be done using gcp-1 to determine if these same regions are present and if the heptamers do indeed function as regulator binding sites. Although the vitellogenin and esterase genes are like gcp-1 in their gut-specificity, all 3 genes differ in temporal regulation: the vitellogenin genes are only expressed in adult hermaphrodites; the esterase gene is expressed early in embryogenesis (100-150 cell stage); and gcp-1, the cysteine protease structural gene, was expressed late in development, near the time the eggs hatched (550 cell stage). By determining the molecular basis of gcp-1 spatial and temporal regulation and comparing these results to those already obtained for the vitellogenin and esterase genes, new insights can be obtained about the mechanisms of nematode gut development and gut-specific gene regulation. The molecular similarities described here between the cysteine proteases of C. elegans and those of parasites, indicate that C. elegans can be used as a model system to study parasite protease gene regulation at a level not yet possible using parasitic worms. We can, for example, use gene fusions and site-directed mutagenesis to determine the specific domains involved in temporal and spatial regulation of gcp-1 transcription. This information should provide further clues to understanding nematode gut development and regulation of digestive protease genes in both free-living and parasitic nematodes.
Acknowledgements We thank C. Kenyon and J. Austin for their comments on this manuscript, and C. Kenyon for providing C. elegans strain CB1490 and the EMBL3 genomic library, and for many helpful suggestions. We also thank D. Cowing for providing the in situ hybridization protocol, and S. Shivakumar for advice regarding total RNA isolation from synchronous cultures. C. elegans strain DR40 was obtained from the Caenorhabditis Genetics Center which is supported by the National Institutes of Health Division of Research Resources. This work was supported by grants from the Edna McConnell Clark Foundation and W.H.O. Tropical Disease Research on Filariasis to J.H.McK.
References 1 Sloane, B.F., Rozhin, J., Hatfield, J.S., Crissman, J.D.and Honn, K.V. (1987) Plasma membrane-associated cysteine proteases in human and animal tumors. Exp. Cell Biol. 55, 209~24. 2 Troen, B.R., Ascherman, D., Atlas, D~ and Gottesman, M.M. (1988) Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. J. Biol. Chem. 63, 254-261. 3 Schorlemmer, H-U., Davies, P. and Allison, A.C. (1976) Ability of activated complement components to induce lysosomal enzyme release from macrophages. Nature 261, 48M9. 4 McKerrow, J.H. (1989) Parasite proteases. Exp. Parasitol. 68, 111 115. 5 McKerrow, J.H. and Doenhoff, M.J. (1988) Schistosome proteases. Parasitol. Today 4, 334-340. 6 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. 7 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, 181-192. 8 Maki, J. and Yanagisawa, T. (1986) Demonstration of carboxyl and thiol protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis, and Ascaris suum. J. Helminthol. 60, 31-37.
9 Sarkis, G.J., Ashcom, J.D., Hawdon, J.M. and Jacobson, L.A. (1988) Decline in protease activities with age in the nematode Caenorhabditis elegans. Mech. Ageing Dev. 45, 191-201. 10 Sarkis, G.J., Kurpiewski, M.R., Ashcom, J.D., JenJacobson, L. and Jacobson, L.A. (1988) Proteases of the nematode Caenorhabditis elegans. Arch. Biochem. Biophys. 261, 80-90. 11 Sakanari, J., Staunton, C.E., Eakin, A.E., Craik, C.S.
249 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. 12 Brenner, S. (1974) The genetics of the nematode Caenorhabditis elegans. Genetics 77, 71-94. 13 Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant DNA. Nucleic Acids Res. 7, 1513-1523. 14 Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 613. 15 Benton, W.D. and Davis, R.W. (1977) Screening Lambda-gt recombinant clones by hybridization to single plaques in situ. Science 196, 180-182. 16 Russel, M., Kidd, S. and Kelley, M.R. (1986) An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene 45, 333 338. 17 Sambrook,J. Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2rid edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 18 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. 19 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 20 Geliebter, J. (1987). Dideoxy sequencing of RNA and uncloned cDNA. Focus 9, 5-8. 21 Klass, M. and Hirsh, D. (1981) Sperm isolation and biochemical analysis of the major sperm protein from Caenorhabditis elegans. Dev. Biol. 84, 299-312. 22 Austin, J. and Kimble, J. (1989) Transcript analysis of glp-I and lin-12, homologous genes required for cell interactions during development of C. elegans. Cell 58, 565-571. 23 Cassada, R.C. and Russell, R.L. (1975) The dauer larva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev. Biol. 46, 326342.
24 Tautz, D. and Pfeifle, C. (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene huchback. Chromosoma 98, 81-85. 25 Meyer, B.J. and Casson, L.P. (1986) Caenorhabditis elegans compensates for the difference in X chromosome dosage between the sexes by regulating transcript levels. Cell 47, 871-881. 26 Coulson, A., Sulston, J., Brenner, S. and J. Karn. (1986) Toward a physical map of the genome of the nematode Caenorhabditis elegans. Proc. Nat. Acad. Sci. USA 83, 7821-7825. 27 Zucker-Aprison, E. and Blumenthal, T. (1989) Potential regulatory elements of nematode vitellogenin genes revealed by interspecies sequence comparison. J. Mol. Evol. 28,487-496. 28 Craig, S.P., III, Muralidhar, M.G., McKerrow, J.H. and Wang, C.C. (1989) Evidence for a class of very small introns in the gene for hypoxanthine-guanine phosphoribosyltransferase in Schistosoma mansoni. Nucleic Acids Res. 17, 1635 1647. 29 Reape, T.J. and Burnell, A.M. (1991) Dauer larvae recovery in the nematode Caenorhabditis elegans, I. The effect of mRNA synthesis inhibitors on recovery, growth, and pharyngeal pumping. Comp. Biochem. Physiol. 98B, 239-243. 30 Blumenthal, T., Squire, M., Kirtland, S., Cane, J., Donegan, M., Spieth, J. and Sharrock, W. (1984) Cloning a yolk protein gene family from Caenorhabditis elegans. J. Mol. Biol. 174, 1 18. 31 Edgar, L. and McGhee, J.D. (1986) Embryonic expression of a gut-specific esterase in Caenorhabditis elegans. Dev. Biol. 114, 109 118. 32 Aamodt, E.J., Chung, M.A. and McGhee, J.D. (1991) Spatial control of gut-specific gene expression during Caenorhabditis elegans development. Science 252, 579582. 33 Klinkert, M-Q., Felleisen, R., Link, G., Ruppel, A. and Beck, E. (1989) Primary structures of Sm31/32 diagnostic proteins of Schistosoma rnansoni and their identification as proteases. Mol. Biochem. Parasitol. 33, 113-122.
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MOLBIO 01695
Gut-specific and developmental expression of a Caenorhabditis elegans cysteine protease gene Celeste R a y and James H. M c K e r r o w UCSF School of Medicine, Department of Pathology, San Francisco, CA, U.S.A. (Received 20 August 1991; accepted 22 October 1991)
A Caenorhabditis elegans cysteine protease gene fragment, amplified by PCR using conserved eukaryotic protease gene sequences as primers, was used as a probe to isolate c D N A and genomic clones. The genomic clone, which had a coding sequence of 987 bp interrupted by 2 small introns, was physically mapped to the middle of linkage group V. The predicted amino acid sequence of the mature C. elegans cysteine protease was homologous to those of other eukaryotic cysteine proteases, particularly to that of the nematode parasite Haemonchus contortus (50%) and to the cathepsin B-like hemoglobinase of the trematode parasite Schistosoma mansoni (54%). The pro region of the C. elegans protease was homologous only to that of the H. contortus enzyme, implying a similar mechanism of protease activation. The C. elegans cysteine protease gene was temporally regulated: abundant 1.1-kb transcripts were detected in larvae and adults, but not in embryos. Transcription also was spatially regulated, occurring only in the intestine. Like the vitellogenin genes, which also are transcribed exclusively in the intestine, the 5' end of the C. elegans cysteine protease gene had at least one copy of each of 2 heptameric sequences which may be transcriptional regulatory elements governing gut-specific expression. Key words: Caenorhabditis elegans; Cysteine protease; Gut development; Parasitic helminth
Introduction
Eukaryotic lysosomal cysteine proteases are well characterized as enzymes involved in intracellular protein degradation. Recently, possible extracellular roles for this class of enzymes have been suggested from studies of tumor cell invasion [1,2], tissue degradation by macrophages [3] and parasite metabolism [4]. Structurally and biochemically similar cysteine proteases have been identified in many nematode and trematode parasites. The most extensively studied of the parasite cysteine Correspondence address: Celeste Ray, UCSF School of Medicine, Department of Pathology, P.O. Box 0506, San Francisco, CA 94143-0506, U.S.A. Fax: 415 476-9672. Abbreviations: PCR, polymerase chain reaction. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M74797.
proteases are the cathepsin B-like hemoglobinase of the trematode blood fluke Schistosoma mansoni [5] and the proteases of the nematode Haemonchus contortus [6,7]. Production of these enzymes is developmentally regulated, occurring at high levels only in actively feeding stages. Moreover, the cysteine proteases of S. mansoni [5], Angiostrongylus cantonensis [8], and Ascaris suum [8] are located primarily in the intestine, an observation which supports their role as digestive enzymes. Although these proteases are potential targets for serodiagnosis and anti-parasite chemotherapy, effective design and application of such reagents will depend on knowledge of basic parasite biology, gut development, and gene regulation. Two similar but distinct cysteine protease activities also have been identified in the freeliving nematode Caenorhabditis elegans [9,10]. Although these enzymes comigrate with lysosomal markers in isopycnic centrifugation, their functions were unknown. We have used
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a C. elegans cysteine protease gene fragment generated by the Polymerase Chain Reaction (PCR) [11] as a hybridization probe to isolate both genomic and cDNA clones encoding a single C. elegans cysteine protease. The results described here establish a basis for using C. elegans as a model system for studying parasite cysteine protease gene regulation.
Materials and Methods
Worm strains and media. General culture methods were those previously described by Brenner [12]. The C. elegans strain CB1490 (him-5[e1490]), which produces males at high frequency, was grown at room temperature on enriched NG agarose plates with Escherichia coli OP50 as a food source. The dauerconstitutive strain DR40 (daf-1), provided by the Caenorhabditis Genetics Center, was maintained at 15°C. Bacterial strains, plasmids, and restriction enzymes. E. coli strain DH5-~ was used as the host for plasmid pBluescript KS (Stratagene) and its derivatives. Strains containing these plasmids were grown in LB medium supplemented with 60 #g ml-~ ampicillin and plasmid DNA was isolated by the mini alkaline lysis procedure of Birnboim and Doly [13]. Restriction enzymes were obtained from BRL and New England Biolabs and were used as recommended by the suppliers. Isolation of the C. elegans cysteine protease gene. A PCR-generated, 160-bp cysteine protease gene fragment from C. elegans [11] was labeled with 32,3 p using random primers [14] and was used to screen C. elegans cDNA and genomic libraries by plaque hybridization [15]. The C. elegans cDNA library, constructed in 2uber2, was obtained from Chris Martin. Positive phage were converted to plasmids using helper phage R408 [16], and the 0.8-1.0kb ApaI/SstI inserts from 5 positive clones were subcloned into the ApaI/Sstl sites of pBluescript KS. Partial nucleotide sequencing revealed that the cDNA inserts all had the
same nucleotide sequence, and the plasmid harboring a l.l-kb insert with the furthest 5' extension was named pCR1. Genomic clones of the C. elegans cysteine protease gene were isolated from a 2EMBL3 library obtained from A. Kamb in Cynthia Kenyon's laboratory. Three positive phage were grown as plate lysates and purified through sucrose gradients [17]. DNA was obtained from the phage as described [17]. Restriction fragments which hybridized to the original probe fragment were identified by Southern blotting [18] and were subcloned into pBluescript KS. As determined by partial nucleic acid sequencing, all 3 of the plasmids contained all or part of the same cysteine protease gene. One of the plasmids, pCRI4, contained the complete structural gene on a 2.8-kb EcoRI fragment inserted into the EcoRI site of pBluescript KS.
DNA sequencing and primer extension. The nucleotide sequences of the inserts in pCR1 and pCR14 were determined by the dideoxy chain termination method [19] using the Sequenase kit (United States Biochemical) and double-stranded DNA templates isolated by alkaline lysis [13]. The nucleotide sequence of the coding region shown in Fig. 1 was read from both strands. Primer extension analysis was done by the methods of Geliebter [20] using poly(A) + RNA isolated from adult male and hermaphrodite worms (see below). The primer was a synthetic oligonucleotide having the nucleotide sequence 5'-CAAGCGTGACTGCGCAG-3'. Worm fractionation and RNA isolation. For separation of adult males and hermaphrodites, worms were washed off several enriched NGM agarose plates (150 mm) with M9 buffer [12] followed by flotation in 1 M sucrose. Nylon filters (Spectrum Medical Industries, Inc.) were used to remove larvae from adults and to fractionate the adults by sex as described by Klass and Hirsh [21]. Different larval stages were isolated from synchronous cultures established as described by Austin and Kimble [22]. Embryos were
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examined microscopically to insure that both older and earlier embryos were represented. Dauer larvae of strain CB1490 were isolated from starved plate cultures by treatment with 1°/0 SDS [23]. The dauer-constitutive strain DR40 was grown at 15°C, the permissive temperature, on enriched N G M agarose plates then shifted to room temperature for 78 h to allow dauer formation. Dauer larvae were isolated by 1% SDS treatment as described above. All worm fractions were examined microscopically to determine homogeneity then were frozen at - 8 0 ° C for RNA extraction. Total RNA was isolated from the frozen worms by methods derived from Austin and Kimble [22]. The nucleic acid pellets were resuspended in water and poly(A) ÷ RNA was isolated using Poly(A) Quick columns from Stratagene.
Northern blots. Approximately 0.4 #g poly(A) ÷ RNA from eggs, larvae and adults were fractionated by electrophoresis on 1.25% agarose/6.67% formaldehyde gels. The gels were blotted onto Nytran filters and hybridized to 32P-labeled probes [17]. Bound probe was removed from the blots by boiling the filters for 20 min in 0.01 x SSC/0.5% SDS. In situ hybridization. Cysteine protease m R N A was detected in whole worms using digoxigenin-labeled probes. The protocol was originally designed by Tautz and Pfeile for use in Drosophila [24] and was adapted for use in worms by C. Oh, B. Edgar, D. Greenstein, and G.Ruvkun (personal communication). The probes were the 1.1-kb ApaI/SstI cysteine protease fragment from pCR1 and the 2.1-kb HindlII C. elegans pharyngeal myosin fragment from pMyo-II, pMyo-II contains a fragment of the rod portion of the pharyngeal myosin heavy chain C [25]. These fragments, purified from low-melting agarose gels and digested with DdeI to reduce the average probe size to 200-300 bp, were then labeled with digoxigenin using random hexamers as described in the Boehringer Mannheim Genius Kit. Worms from enriched N G M agarose plates were washed in M9 buffer and resuspended in
5 ml fixing solution (2.2% paraformaldehyde/ 80 mM KCI/20 mM NaC1/2 mM EGTA/15 mM Pipes, pH 7.4/0.5 mM spermidine/0.2 mM spermine/0.5% fl-mercaptoethanol/20 mM vanadyl ribonucleoside complex). 3 ml nheptane and 1 ml 90% methanol/10% 0.25 M EGTA were added, the emulsion vortexed for 20 min at room temperature, then frozen in a dry ice/ethanol bath. The worms were thawed, vortexed another 20 rain at room temperature, pelleted in a clinical centrifuge, washed in 10 ml methanol, rinsed in 50% ethanol/50% xylene, and soaked in 100% xylene for 2 h at room temperature. Sequential washes were done in 50% ethanol/50% xylene, 100%o ethanol, 100°/0 methanol, and 50% methanol/50% PBTS containing 5% (w/w) paraformaldehyde. (PBTS is 1 x PBS/ 0.1% Tween 20/0.01% SDS.) Animals were fixed in PBTS containing 5% paraformaldehyde for 20 min at room temperature and rinsed 3 times in PBTS. The cuticles were permeabilized by incubating the worms 10 min at 37°C in PBTS containing 100 #g m1-1 Proteinase K, washing twice in PBTS containing 2 mg ml-1 glycine, and washing twice in PBTS. Post-fixing then was done for 2 min at room temperature in PBTS containing 5°/0 paraformaldehyde, followed by 5 washes in PBTS. For each hybridization reaction, 20 pl of fixed, permeabilized worms were put into microfuge tubes and rinsed sequentially in 50% PBTS/50% hybridization solution then in 100% hybridization solution (50% deionized formamide/5 x SSC/10% dextran sulfate (Sigma #D6001)/100 /~g m1-1 salmon sperm DNA/100 pg ml-1 E. coli tRNA/50 #g ml-1 heparin/0.1% Tween 20/10 mM Tris-HC1, pH 7.5/0.005% SDS). Prehybridization was at 50°C in 200 pl hybridization solution, and hybridization was done overnight at 50°C in 100 /A hybridization solution containing approximately 0.1 #g m l - 1 digoxigenin-labeled probe. Sequential washes each were done for 20 min at 50°C in a volume of 500 pl using the following solutions: hybridization solution, hybridization solution with 5% dextran sulfate, twice in hybridization solu-
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tion without dextran sulfate, once in 50% hybridization solution/50% PBTS, and 5 times in PBTS. Animals were then incubated in 500 /A PBT (1 x PBS/0.1% Tween 20)/1% BSA (Sigma #A7030)/0.02% sodium azide for 90 min at 4°C. In order to detect bound probe, worms were incubated overnight at 4°C with alkalinephosphatase conjugated anti-digoxigenin antibodies (Boehringer Mannheim Genus kit) diluted 1:100 in 1% BSA/0.02% sodium azide, followed by six 20 rain washes in PBTS at 4°C. After 2 rinses in 100 m M NaC1/50 mM MgC12/100 m M Tris, pH 8.8/1 m M levamisol/ 10% Tween 20, animals were resuspended in l ml of this buffer containing 336 #g m1-1 nitroblue tetrazolium salt (Boehringer Genius Kit) and 174 gg ml-1 X-Phosphate (Boehringer Genius Kit). Reactions were incubated in the dark for 3 h at room temperature, and worms were mounted for microscopy on 2% agarose pads made in 50 mM Tris-HC1 (pH 9.5)/5 mM MgC12.
from the ATG translation initiation codon. The gcp-1 transcripts were not trans-spliced. The 5' end ofgcp-1 contained at least one copy of each of 2 heptamers which may be regulatory elements governing gut-specific expression [27] (Fig. 1). At the 3' end of the gene, a potential poly(A) addition signal was found 40 bp from the translation termination codon and at least 22 adenosines were found 14 bp further downstream in the c D N A clone. Although we have identified only one gene, we cannot rule out the possibility that C. elegans has other cysteine protease genes. Two distinct cysteine proteases have been identified biochemically in C. elegans [9,10]. One of these activities is similar to cathepsin B and may be the one encoded by gcp-l. The cysteine proteases of the evolutionarily related parasitic nematode H. contortus are encoded by a multigene family [7]. Because of the molecular similarities between gcp-1 and the protease genes of H. contortus (see below), C. elegans may also contain more than one cysteine protease gene.
Results and Discussion
The C. elegans cysteine protease gene has structural similarities to those J?om parasites. Small introns (45-59 bp) are characteristic of many C. elegans genes and may also be common in genes from parasitic worms. The HGPRTase gene from S. mansoni, for example, contains 4 small introns of 31, 33, 42, and 32 bp [28]. Because only a c D N A clone has been isolated for the S. mansoni cathepsin Blike hemoglobinase, nothing is known yet about the intron structure of this gene. Of the 2 cysteine protease genes isolated from H. contortus, AC-1 is a c D N A clone [6] and AC-2 is a genomic clone [7]. AC-2 has 11 introns ranging in size from 57 bp to over 5.2-kb [7]. Intron 1 occurs immediately after the putative A T G translation start codon and is 80 bp long. Likewise, the first intron of gcp-1 was in this same position and was 82 bp long. The 5' splice donor sequences of these 2 introns were identical at twelve nucleotides centered at the splice junction, whereas the 3' acceptor sequences were identical at 10 of the 12 nucleotides centered at the 3' splice site.
Isolation of a C. elegans eysteine protease gene. A 160-bp PCR-generated cysteine protease gene fragment from C. elegans was used to isolate c D N A and genomic clones encoding a single cysteine protease gene. This gene was designated gcp-1 for Gut-specific Cysteine Protease. The original EMBL3 genomic clone was sent to Alan Coulson and John Sulston at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England for physical mapping [26]. According to their analysis, the cysteine protease gene was located near the middle of chromosome V, between the genes daf-ll and myo-3. The cysteine protease coding sequence was 987 bp long and was interrupted by 2 small introns of 82 and 46 bp (Fig. 1). As determined by primer extension, the c D N A clone of gcp-1 was missing 23 bp of 5' sequence. Primer extension was also used to show that transcription was initiated 25 bp downstream from the T A T A T A A sequence and 28 bp upstream
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I I G W G T E S G S P Y W L V A [ ] S W G V N 1057 T G G G G A G A G A G C G G T T T C T T C ~ G A T C T A C C G T G G A G A T G A T C ~ T G C G G ~ T T G ~ T C A G C A G T T W G E S G F F K I Y R G D D Q C G I E S A V 1123 G T C G C C G G A A A G G C C ~ G G T C T A A A T G C A T T A T G T A C A C ~ T T T T T G T ~ T T A C C A T C T G C C A A A T V A G K A K~ 1189 L ~ T G T T C A T ~ T T T A A A T T T G T T C T T A A A T C T T C T G A A A T A T A T A T A T C T T T A T T A A A A T T T A 1255 C G ~ G A G C ~ T T A C A A A A G A G C A T G A A A T C A ~ A T T ~ T C T A G T C A C A T T A T G ~ C ~
Fig. 1. Nucleotide and predicted amino acid sequences of the C. elegans cysteine protease gene. Nucleotides are numbered from the transcription start site at position + 1. Introns are in lowercase letters and the ends of the c D N A clone are indicated by solid triangles. The ATG translation start site, TAA translation termination signal, TATA promoter element, and A A T A A A poly(A) addition signals are underlined. Enclosed regions labeled 'Box 1' and 'Box 2' are potential regulatory elements ~ r gut-specific transcription which were first identified in the vitellogenin genes [27]. Consensus sequences ~ r each of these elements is written below the g~-I sequence. Capitol letters represent invariant bases, underlined bases are almost invafiant, and lower case letters indicate variable bases. In the amino acid sequence, the putative pro region cleavage site is indicated by an asterisk and the active site amino acid residues are boxed.
Although more divergent in size than the first introns, AC-2 intron 9 and gcp-1 intron 2 also interrupted the coding sequences at exactly the same positions and had similar nucleotide sequences at the splice junctions. Thus, the positions, sizes, and boundary sequences of the 2 gcp-1 introns are similar to those of 2 of the introns in the H. contortus AC-2 gene. The predicted amino acid sequence of the C. elegans cysteine protease is similar to mammalian cathepsin B and is compared to that of the H. contortus cysteine proteases and the S. mansoni cathepsin B-like hemoglobinase in Fig. 2. The mature C. elegans protease is
50% homologous to those of H. contortus and, if conservative amino acid substitutions are included, this similarity increases to 83%. The mature C. elegans cysteine protease also was 54% homologous to the S. mansoni hemoglobinase, with an overall similarity of 84%. In addition, although there was no homology with that of the S. mansoni hemoglobinase, the 84 amino acid pro region of the C. elegans cysteine protease (Fig. 2) was 28% identical to the H. contortus cysteine protease AC-2 and 31% identical to AC-1 (73-74% chemical similarity). This result is surprising since pro regions are highly divergent and indicates that
244
AC-2 _q~-i AC-2
(T) (A) MKYLVLALCTYLCSQSGADENAAQGIPLEAQRLTGEPLVAYLRRSQNLFEVNSDPTPDFEQK-::.:.:. ::. . : .... :. :::..:1.: .... :.:: ...... MKFLILT---ALCAVTLAFVPINHQSAVET--LTGQALVDYVNSAQSLFKTEHVEITEEEMKFK (D) • IMSIKY-KHQKLNLMVKEDPDPEVDIPPSYDPRDVW.
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SDRICIASKAEKQVNISATDIMTCCRPQCGDGCEGGWPIEAWKYFIYDGVVSGGEYLT-KDVCR : : : . : : . . : . . . :
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PYPI HPCGHHGN DTYYGE---CRGTAP--TPPCKRKCRPGVRKMYRI DKRYGKDAY IVKQSVKA :::: :: :. :. .. ::.:...:..: .. : ::..: .:: : . . . . PYPIAPC ....... TSGN---CP-ESK--TPS CSMS CQSGYSTAYAKDKHFGVSAYAVPKNAAS :::.. : :.:. : .. ::.:. .::. :.:.:..::: : :.:.: .. . PYPFPKCEGG .... TKGKY PPCG-S KI YNTPRCKQTCQRKYKTPYTQDKHRGKSS Y N V K N DI K A
(R)
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NDWGEKGY FRIVRGSNDCG I EGTIAAGIVDTES .:::.:.:.: : : . . . : : : : . . . . :: VNWGESGFFKI
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EDWGINGYFRIVRGRDECSIESEVIAGRIN
Fig. 2. Comparison of the predicted amino acid sequence of the C. elegans cysteine protease gcp-1 to those of the H. contortus AC-1 [7] and S. mansoni sm31 [33] cysteine proteases. The amino acid sequence of AC-1 is identical to that of AC-2 except for seven amino acids [6] that are written above the AC-2 sequence. The sm31 pro region is not given since it shares no homology with the C. elegans and H. contortus proteases. The predicted site of pro region cleavage is indicated by an arrowhead and the active site residues are boxed. Double dots designate identical amino acids, single dots indicate amino acids that are chemically similar. Dashes represent gaps that were introduced to facilitate sequence alignment.
the proteases from these 2 species may share similar activation mechanisms. Unlike the proteases from H. contortus and S. mansoni, that from C. elegans does not contain any potential N-linked glycosylation sequences.
oE
gcp-1
--
act-1
--
1 2 3 4
- - 1 . 1 Kb
Expression of gcp-1 is temporally regulated. Northern blots with poly(A) + R N A isolated from different developmental stages of C. elegans were prepared and screened at high stringency with the e D N A insert of pCR1 as a probe (Fig. 3). Abundant 1.1-kb transcripts migrating as a single band were present in approximately the same amounts in L1-L4 larvae, dauer larvae, and adults, but were not detected in embryos. The presence of gep-1 m R N A in dauer larvae was confirmed using the dauer-constitutive strain DR40 (data not shown). Accumulation of gcp-1 m R N A in L1 larvae did not depend on the presence of food,
Fig. 3. Northern blot analysis of gcp-1 transcription. Poly(A) + R N A isolated from different developmental stages of C. elegans CB1490 was fractionated on a 1.25% agarose/6.67% formaldehyde gel, blotted onto a Nytran filter, and hybridized with the 32P-labeled gcp-1 e D N A insert of pCR1. Following autoradiography, the blot was boiled and rehybridized with a C. elegans actin-1 fragment from pW-16 210 (M. Krause). Lanes (left to right): adult hermaphrodites; adult males; dauer larvae; embryos; L1 larvae; L2 larvae; L3 larvae; L4 larvae.
245 since the larvae were isolated after allowing eggs to hatch in M9 buffer. These results indicate that gcp-1 expression is temporally regulated, occurring at or near hatching when the embryos contain about 550 cells. When this same blot was probed with a C. elegans actin-1 gene fragment (pW-16-210 obtained from M. Krause), all of the lanes contained a 1.3-kb band (Fig. 3), indicating that comparable amounts of RNA were in each lane. If C. elegans has more than one cysteine protease gene, the bands seen in the Northern blot could be the result of hybridization to transcripts from these genes in addition to those from gcp-1. Because of the stringency at which this blot was hybridized the other genes would have to be highly homologous to gcp-1 and produce transcripts of similar size. Moreover, these genes would be temporally regulated since no transcripts were detected in embryos. These results are similar to those found with the H. contortus AC-2 gene which is also developmentally regulated: it is transcribed at high levels in blood-feeding adults and at 30fold reduced levels in non-feeding larvae [7]. Likewise, high levels of the S. mansoni cathepsin B-like hemoglobinase activity are associated with feeding adult-stage worms. That parasites delay activation of their cysteine protease genes until t h e adult stage could be the result of adaptation to a parasitic lifestyle. Parasite-derived proteases are highly antigenic, and parasitic worms like schistosomes usually have a tissue-migratory phase before reaching their ultimate residence in the bloodstream. It may, therefore, be in the parasites' best interests to delay protease expression until reaching their main food source. In contrast, free-living worms, like C. elegans, could begin producing digestive enzymes as soon the worms hatch and begin searching for food. An unexpected observation was that nonfeeding dauer larvae contained as much cysteine protease m R N A as did L1-L4 larvae and adults. Dauer larvae may remain in this state of arrested development for several months, and production of high levels of a
digestive protease would be metabolically wasteful and could potentially damage the intestine. Reape and Burnell [29] have shown that inhibition of RNA synthesis does not inhibit dauer recovery, implying that dauer larvae store m R N A made during earlier larval stages. Because pharyngeal pumping (feeding) is one of the first signs of dauer recovery, production of digestive enzymes should be one of the initial events in dauer recovery. The gcp1 m R N A we detected in dauer larvae, therefore, may have been made earlier and was being stored in a translationally inactive state. If this is the case, gcp-1 expression may be subject to translational as well as transcriptional control.
Expression of gep-1 is spatially regulated. The spatial regulation of gcp-1 expression was analyzed using in situ hybridization (Fig. 4). Whole worms were probed with the cDNA insert from pCR1 which had been labeled with digoxigenin. A digoxigenin-labeled pharyngeal myosin-2 gene fragment [25] also was used as a positive control. Bound probe was detected colorimetrically using anti-digoxigenin antibodies conjugated to alkaline phosphatase. Cysteine protease m R N A was located exclusively in the intestinal cells of C. elegans, indicating that expression was spatially regulated (Fig. 4). In general, we observed that staining was more intense in larvae than in adults (Fig. 4c-d), probably as a result of cuticle permeability differences. In addition, the anterior gut stained darker than did the posterior (Fig. 4d), a result which could also be due to differential permeability or to differential expression in different parts of the intestine. Because staining was observed only when probe was added and the pattern of staining was specific for each probe used, we conclude that gcp-1 expression is tissuespecific. Other C. elegans genes are known to be expressed only in the intestine. These include the vitellogenin [30] and esterase genes [31]. Zucker-Aprison and Blumenthal [27] have identified 2 repeated heptameric sequences in the promoters of the C. elegans and Caeno-
247
Fig. 4. In situ hybridization. Samples containing mixed stages of whole worms were fixed and permeabilized, then incubated in hybridization buffer containing digoxigenin-labeled probes (see Materials and Methods). Bound probe was detected colorimetrically (blue color; dark areas in these photographs) using anti-digoxigenin antibodies (1:100) that were conjugated to alkaline phosphatase (Boehringer Mannheim). (a) Adults and larvae incubated without probe. (b) Larva incubated with the pharyngeal myosin-2 fragment from pMyo-II [25]. (c) and (d) Adults and larvae, respectively, incubated with the gep-1 cDNA insert of pCR1. The scale bar represents 70 microns.
248
rhabdit& briggsae vitellogenin genes that may be binding sites for transcriptional regulators. Preliminary DNA sequence data of the C. elegans cysteine protease gene promoter showed that this gene had at least one copy of each of the heptamers (Fig. 1). Interestingly, the 5' untranslated region of the H. contortus cysteine protease gene AC-2 also has copies of these 2 heptamers. In addition, regions of the esterase promoter that are important for gutspecific expression have recently been identified using sequential deletions of this region [32]. The pattern of esterase expression from these deletion mutants suggests that both activators and repressors regulate gut-specific expression. A similar analysis can be done using gcp-1 to determine if these same regions are present and if the heptamers do indeed function as regulator binding sites. Although the vitellogenin and esterase genes are like gcp-1 in their gut-specificity, all 3 genes differ in temporal regulation: the vitellogenin genes are only expressed in adult hermaphrodites; the esterase gene is expressed early in embryogenesis (100-150 cell stage); and gcp-1, the cysteine protease structural gene, was expressed late in development, near the time the eggs hatched (550 cell stage). By determining the molecular basis of gcp-1 spatial and temporal regulation and comparing these results to those already obtained for the vitellogenin and esterase genes, new insights can be obtained about the mechanisms of nematode gut development and gut-specific gene regulation. The molecular similarities described here between the cysteine proteases of C. elegans and those of parasites, indicate that C. elegans can be used as a model system to study parasite protease gene regulation at a level not yet possible using parasitic worms. We can, for example, use gene fusions and site-directed mutagenesis to determine the specific domains involved in temporal and spatial regulation of gcp-1 transcription. This information should provide further clues to understanding nematode gut development and regulation of digestive protease genes in both free-living and parasitic nematodes.
Acknowledgements We thank C. Kenyon and J. Austin for their comments on this manuscript, and C. Kenyon for providing C. elegans strain CB1490 and the EMBL3 genomic library, and for many helpful suggestions. We also thank D. Cowing for providing the in situ hybridization protocol, and S. Shivakumar for advice regarding total RNA isolation from synchronous cultures. C. elegans strain DR40 was obtained from the Caenorhabditis Genetics Center which is supported by the National Institutes of Health Division of Research Resources. This work was supported by grants from the Edna McConnell Clark Foundation and W.H.O. Tropical Disease Research on Filariasis to J.H.McK.
References 1 Sloane, B.F., Rozhin, J., Hatfield, J.S., Crissman, J.D.and Honn, K.V. (1987) Plasma membrane-associated cysteine proteases in human and animal tumors. Exp. Cell Biol. 55, 209~24. 2 Troen, B.R., Ascherman, D., Atlas, D~ and Gottesman, M.M. (1988) Cloning and expression of the gene for the major excreted protein of transformed mouse fibroblasts. J. Biol. Chem. 63, 254-261. 3 Schorlemmer, H-U., Davies, P. and Allison, A.C. (1976) Ability of activated complement components to induce lysosomal enzyme release from macrophages. Nature 261, 48M9. 4 McKerrow, J.H. (1989) Parasite proteases. Exp. Parasitol. 68, 111 115. 5 McKerrow, J.H. and Doenhoff, M.J. (1988) Schistosome proteases. Parasitol. Today 4, 334-340. 6 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. 7 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, 181-192. 8 Maki, J. and Yanagisawa, T. (1986) Demonstration of carboxyl and thiol protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis, and Ascaris suum. J. Helminthol. 60, 31-37.
9 Sarkis, G.J., Ashcom, J.D., Hawdon, J.M. and Jacobson, L.A. (1988) Decline in protease activities with age in the nematode Caenorhabditis elegans. Mech. Ageing Dev. 45, 191-201. 10 Sarkis, G.J., Kurpiewski, M.R., Ashcom, J.D., JenJacobson, L. and Jacobson, L.A. (1988) Proteases of the nematode Caenorhabditis elegans. Arch. Biochem. Biophys. 261, 80-90. 11 Sakanari, J., Staunton, C.E., Eakin, A.E., Craik, C.S.
249 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. 12 Brenner, S. (1974) The genetics of the nematode Caenorhabditis elegans. Genetics 77, 71-94. 13 Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant DNA. Nucleic Acids Res. 7, 1513-1523. 14 Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 613. 15 Benton, W.D. and Davis, R.W. (1977) Screening Lambda-gt recombinant clones by hybridization to single plaques in situ. Science 196, 180-182. 16 Russel, M., Kidd, S. and Kelley, M.R. (1986) An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene 45, 333 338. 17 Sambrook,J. Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2rid edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 18 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. 19 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 20 Geliebter, J. (1987). Dideoxy sequencing of RNA and uncloned cDNA. Focus 9, 5-8. 21 Klass, M. and Hirsh, D. (1981) Sperm isolation and biochemical analysis of the major sperm protein from Caenorhabditis elegans. Dev. Biol. 84, 299-312. 22 Austin, J. and Kimble, J. (1989) Transcript analysis of glp-I and lin-12, homologous genes required for cell interactions during development of C. elegans. Cell 58, 565-571. 23 Cassada, R.C. and Russell, R.L. (1975) The dauer larva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Dev. Biol. 46, 326342.
24 Tautz, D. and Pfeifle, C. (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene huchback. Chromosoma 98, 81-85. 25 Meyer, B.J. and Casson, L.P. (1986) Caenorhabditis elegans compensates for the difference in X chromosome dosage between the sexes by regulating transcript levels. Cell 47, 871-881. 26 Coulson, A., Sulston, J., Brenner, S. and J. Karn. (1986) Toward a physical map of the genome of the nematode Caenorhabditis elegans. Proc. Nat. Acad. Sci. USA 83, 7821-7825. 27 Zucker-Aprison, E. and Blumenthal, T. (1989) Potential regulatory elements of nematode vitellogenin genes revealed by interspecies sequence comparison. J. Mol. Evol. 28,487-496. 28 Craig, S.P., III, Muralidhar, M.G., McKerrow, J.H. and Wang, C.C. (1989) Evidence for a class of very small introns in the gene for hypoxanthine-guanine phosphoribosyltransferase in Schistosoma mansoni. Nucleic Acids Res. 17, 1635 1647. 29 Reape, T.J. and Burnell, A.M. (1991) Dauer larvae recovery in the nematode Caenorhabditis elegans, I. The effect of mRNA synthesis inhibitors on recovery, growth, and pharyngeal pumping. Comp. Biochem. Physiol. 98B, 239-243. 30 Blumenthal, T., Squire, M., Kirtland, S., Cane, J., Donegan, M., Spieth, J. and Sharrock, W. (1984) Cloning a yolk protein gene family from Caenorhabditis elegans. J. Mol. Biol. 174, 1 18. 31 Edgar, L. and McGhee, J.D. (1986) Embryonic expression of a gut-specific esterase in Caenorhabditis elegans. Dev. Biol. 114, 109 118. 32 Aamodt, E.J., Chung, M.A. and McGhee, J.D. (1991) Spatial control of gut-specific gene expression during Caenorhabditis elegans development. Science 252, 579582. 33 Klinkert, M-Q., Felleisen, R., Link, G., Ruppel, A. and Beck, E. (1989) Primary structures of Sm31/32 diagnostic proteins of Schistosoma rnansoni and their identification as proteases. Mol. Biochem. Parasitol. 33, 113-122.