Cathepsin L Is Essential for Embryogenesis and Development ofCaenorhabditis elegans
2002; Elsevier BV; Volume: 277; Issue: 5 Linguagem: Inglês
10.1074/jbc.m106117200
ISSN1083-351X
AutoresSarwar Hashmi, Collette Britton, Jing Liu, David B. Guiliano, Yelena Oksov, Sara Lustigman,
Tópico(s)Selenium in Biological Systems
ResumoCysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterizedCe-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role forcpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematodeOnchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes. Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterizedCe-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role forcpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematodeOnchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes. cysteine protease cathepsin L gene green fluorescence protein expressed sequence tag RNA interference basic local alignment search tool reverse transcriptase α-amanitin-resistant gene first larval stage second larval stage third larval stage fourth larval stage polyethylene glycol β-galactosidase phosphate-buffered saline double-stranded RNA Cysteine proteases of the papain superfamily have long been recognized for their role in intracellular and extracellular protein degradation in a range of cellular processes (1Bond J.S. Butler P.E. Annu. Rev. Biochem. 1987; 56: 333-364Crossref PubMed Scopus (455) Google Scholar). Within the papain family, the cathepsins can be subdivided into more than 10 subfamilies on the basis of their primary sequence and enzymatic activity (2Santamaria I. Velasco G. Pendas A.M. Fueyo A. Lopez-Otin C. J. Biol. Chem. 1998; 273: 16816-16823Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The family includes cathepsin B, C, L, and Z, all of which contain an essential cysteine residue in their active site but differ in tissue distribution and in some enzymatic properties, such as substrate specificity and pH stability. Cathepsin B-like cysteine protease genes occur as a large multigene family in a wide range of parasitic and free-living nematodes. Several cathepsin B genes were reported to be expressed in Caenorhabditis elegans, some of which were restricted to the intestines of larval and adult transgenic worms (3Britton C. McKerrow J.H. Johnstone I.L. J. Mol. Biol. 1998; 283: 315-327Crossref Scopus (47) Google Scholar, 4Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 5Ray C. McKerrow J.H. Mol. Biochem. Parasitol. 1992; 51: 239-250Crossref PubMed Scopus (59) Google Scholar). Interestingly, the structurally similar Hemonchus contortus (3Britton C. McKerrow J.H. Johnstone I.L. J. Mol. Biol. 1998; 283: 315-327Crossref Scopus (47) Google Scholar, 4Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 5Ray C. McKerrow J.H. Mol. Biochem. Parasitol. 1992; 51: 239-250Crossref PubMed Scopus (59) Google Scholar) and Schistosoma mansoni (6Tort J. Brindley P.J. Knox D. Wolfe K.H. Dalton J.P. Adv. Parasitol. 1999; 43: 161-266Crossref PubMed Google Scholar) cathepsin B homologues were also expressed in the gut and were suggested to be potentially involved in feeding (5Ray C. McKerrow J.H. Mol. Biochem. Parasitol. 1992; 51: 239-250Crossref PubMed Scopus (59) Google Scholar, 7McKerrow J.H. Drug Disc. Desi. 1994; 2: 437-444Google Scholar), such as nutrient digestion. Heterologous transformation of C. elegans with an H. contortus cathepsin B gene promoter has demonstrated also conservation of the mechanisms controlling its spatial expression in free-living and parasitic nematodes (8Britton C. Redmond D.L. Knox D.P. McKerrow J.H. Barry J.D. Mol. Biochem. Parasitol. 1999; 103: 171-181Crossref PubMed Scopus (47) Google Scholar), and therefore both enzymes were hypothesized to be not only structurally similar, but also functionally homologous and important for proper feeding (4Pratt D. Armes L.G. Hageman R. Reynolds V. Boisvenue R.J. Cox G.N. Mol. Biochem. Parasitol. 1992; 51: 209-218Crossref PubMed Scopus (65) Google Scholar, 7McKerrow J.H. Drug Disc. Desi. 1994; 2: 437-444Google Scholar). Cathepsin L and Z-like proteases were shown to be present in many parasitic nematodes, where they are speculated to have diverse biological functions including invasion, feeding, molting, and immune evasion (reviewed in Refs. 6Tort J. Brindley P.J. Knox D. Wolfe K.H. Dalton J.P. Adv. Parasitol. 1999; 43: 161-266Crossref PubMed Google Scholar, 9Koiwa H. Shade R.E. Zhu-Salzman K. D'Urzo M.P. Murdock L.L. Bressan R.A. Hasegawa P.M. FEBS Lett. 2000; 471: 67-70Crossref PubMed Scopus (95) Google Scholar, and 10Shompole P.S. Jasmer D.P. J. Biol. Chem. 2000; 276: 2928-2934Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Many of these cathepsins have homologues in C. elegans suggesting that they may be involved in functions conserved across different nematode species. Yet not much is known about their precise function. In Dirofilaria immitis (11Richter J.K. Sakanari J.A. Frank G.R. Grieve R.B. Exp. Parasitol. 1992; 75: 303-307Crossref PubMed Scopus (38) Google Scholar) and Brugia pahangi 1X. Hang, C. Britton, and J. McKerrow, unpublished data.1X. Hang, C. Britton, and J. McKerrow, unpublished data. cysteine proteases were shown to be associated with molting as well as activities that might facilitate larval migration. The potential role of cysteine proteases during molting was indirectly established in Onchocerca volvulus by showing that the peptidyl monofluoromethyl ketones, low molecular weight irreversible cysteine protease inhibitors, inhibit the molting of third stage larvae (L3) in a time- and dose-dependent manner (12Lustigman S. McKerrow J.H. Shah K. Lui J. Huima T. Hough M. Brotman B. J. Biol. Chem. 1996; 271: 30181-30189Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). These irreversible inhibitors can block cathepsin Z and L-like, but not the B-like enzyme activities, suggesting that a cathepsin L, as well as a cathepsin Z, might be involved in the molting process. The target cysteine proteases were indirectly localized in the granules of the glandular esophagus ofO. volvulus L3 using the biotin-Phe-Ala-CHN2inhibitor (12Lustigman S. McKerrow J.H. Shah K. Lui J. Huima T. Hough M. Brotman B. J. Biol. Chem. 1996; 271: 30181-30189Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In further studies, a larval O. volvuluscysteine protease named LOVCP was cloned (12Lustigman S. McKerrow J.H. Shah K. Lui J. Huima T. Hough M. Brotman B. J. Biol. Chem. 1996; 271: 30181-30189Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). LOVCP as well as its homologue in Toxocara canis and C. eleganswere recently classified as a novel monophylectic group in the papain family of cysteine proteases, named cathepsin Z (13Falcone F.H. Tetteh K.K. Hunt P. Blaxter M.L. Loukas A. Maizels R.M. Exp. Parasitol. 2000; 94: 201-207Crossref PubMed Scopus (20) Google Scholar). The O. volvulus cathepsin Z was shown to be required for molting and the development of fourth-stage larvae (L4) based on its localization using monospecific anti-LOVCP antibodies. The native enzyme was localized in molting L3 in the region where the separation between the cuticles of L3 and L4 takes place. The general role of the O. volvulus cathepsin Z-like enzyme as well as its C. elegans homologue (14Hashmi S. Tawe W. Lustigman S. Trends Parasitol. 2001; 17: 387-392Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) during molting was therefore hypothesized to be as a proteolytic enzyme involved in cuticle degradation, similar to that observed in the entomopathogenic fungiMetarhizium anisopliae with its endogenous cysteine protease Pr4 (15Cole Jr., S.C. Charnleg A.K. Cooper R.M. FEMS Microbiol. Lett. 1993; 113: 189-196Crossref Google Scholar). This function as well as other yet unknown functions of cathepsin Z and L cysteine proteases were previously hypothesized to be present in nematodes based on the immunolocalization of onchocystatin, an endogenous cysteine protease inhibitor, in thin sections of O. volvulus. Onchocystatin was expressed in L3, molting L3, adult worms, and eggshells around developing microfilaria (16Lustigman S. Brotman B. Huima T. Prince A.M. Mol. Biochem. Parasitol. 1991; 45: 65-75Crossref PubMed Scopus (71) Google Scholar, 17Lustigman S. Brotman B. Huima T. Prince A.M. McKerrow J.H. J. Biol. Chem. 1992; 267: 17339-17346Abstract Full Text PDF PubMed Google Scholar), suggesting that its target cysteine protease(s) is possibly involved in regulating activities such as molting, cuticle remodeling, and embryogenesis during the development of the parasite in the host. Importantly, C. elegans has two homologues of onchocystatin (14Hashmi S. Tawe W. Lustigman S. Trends Parasitol. 2001; 17: 387-392Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), suggesting the presence of their target enzymes in this worm as well. The indirect approaches as described above, however, do not provide conclusive information on the precise function of these proteases. Recently, a new family of cathepsin L-like sequences with similarity to previously characterized mammalian cathepsin L-like enzymes was identified in the O. volvulus as well as in other filarial L3 EST data bases, 2D. Guiliano, unpublished data.2D. Guiliano, unpublished data. and in other parasitic nematodes. 3C. Britton and L. Murray, unpublished data.3C. Britton and L. Murray, unpublished data.Their function is not as yet known. We have identified a related cathepsin L-like protease sequence (T03E6.7) within the C. elegans complete genome data base (ACeDB), and used the C.elegans powerful system for investigating its functionsin vivo during development. To determine the function of the cathepsin L protease we took advantage of methods available in C. elegans that enable analysis of gene promoter activity and gene function in individual cells. Based on the obtained information we could predict its potential physiological role in filarial parasites. This study is the first to directly demonstrate the functional importance of cathepsin L in nematode development. A BLAST search (18Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69707) Google Scholar) of the C. elegans genome data base (ACeDB, www.sanger.ac.uk/projects/C_elegans/wormpep/1) and the nr data base using the Ov-CPL (accession number AF331036) and other filarial nematodes amino acid sequences, Di-CPL (accession number AF001101), Bp-CPL (accession number AF031819.1) identified a predicted C. elegans cathepsin L-like gene (T03E6.7), which was namedCe-cpl-1.4 ACe-cpl-1 cDNA clone containing the full-length sequence was also identified in the EST data base (accession number C12099) and the corresponding λ ZAP II phage was obtained from Yuji Kohara (clone yk146d10, C. elegans consortium, National Institute of Genetics, Mishima, Japan). The pBluescript phagemid was excised and the DNA sequenced in both directions to confirm the predicted amino acid sequence. Signal sequences and putative cleavage sites were identified using the SignalP server (www.cbs.dtu.dk/services/SignalP). Prediction of the pro-region cleavage site as well as the active sites were based on alignment of the CPL protein sequences was made using Cluster W multiple sequence alignment. Analysis of the promoter region ofCe-cpl-1 gene was performed using Genefinder provided by BCM server (www.hgsc.bcm.tmc.edu/searchlauncher). C. elegans strains used in this study were the wild-type Bristol N2 strain and unc-76 mutant strain DR96 (unc-76(e911)V) (19Brenner M. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar), both provided by theCaenorhabditis Genetics Center. Strains were maintained on NGM agar plates as previously described (19Brenner M. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). Semi-quantitative RT-PCR (sqRT-PCR) was carried out using first strand cDNA generated from total RNA collected from synchronous L1, L2, L3, L4, and young adultC. elegans cultures at 2-h intervals, as previously described (20Johnstone I.L. Barry J.D. EMBO J. 1996; 15: 3633-3639Crossref PubMed Scopus (133) Google Scholar). The stage-specific cDNA samples were kindly provided by Iain Johnstone (Wellcome Center for Molecular Parasitology, University of Glasgow, UK) and used according to the established protocols. Gene-specific cDNA fragments were amplified using two sets of PCR primers, one set was specific forCe-cpl-1 (CPF1, sense, 5′-GTCTCCGTGCTCTGGGTCGGTTCCGTATC-3′ and CPR1, antisense, 5′-CCATGGTGTCGACACCGAGGAGTCATAC-3′) and the other set was specific for an internal control, the ama-1transcript (20Johnstone I.L. Barry J.D. EMBO J. 1996; 15: 3633-3639Crossref PubMed Scopus (133) Google Scholar). The primers were designed to span an intron to distinguish cDNA from contaminating gDNA products. The following PCR conditions, which allowed reactants to remain in excess, were used: 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 3 min. Amplified products were separated on 2% agarose gels, Southern blotted, and probed with the appropriate end-labeled oligonucleotides. After autoradiography, specific bands corresponding to amplified cpl-1 and control ama-1 products were excised from the blot for each time point and counted in scintillant. The relative content of the transcript corresponding to the Ce-cpl-1 gene is expressed as the ratio of the signal for cpl-1 for each developmental stage to that ofama-1. The sqRT-PCR was carried out on two occasions, with very similar results, one of which is presented. The double-stranded RNA interference (RNAi) procedure was carried out as described by Fireet al. (21Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11651) Google Scholar) and Tabara et al. (22Tabara H. Grishok A. Mello C.C. Science. 1998; 282: 430-431Crossref PubMed Scopus (518) Google Scholar). Three different regions of the Ce-cpl-1 gene were PCR amplified from cosmid T03E6 DNA using Vent DNA polymerase (New England Biolabs, Beverly, MA) and the following PCR primers (underline indicates artificial restriction enzyme sites introduced for cloning): CPexon1F (XhoI), sense, 5′-AATCTCGAGATTCATTCTTCTGGCACTG-3′, and CPexon1R (XbaI), antisense, 5′-AAATCTAGATTCTTACCAAGTCAGC-3′ (PCR product 275 bp); CPexon3F (PstI), sense, 5′-TTTCTGCAGCCAGATGAGGTTGAC-3′ and CPexon3R (PstI), antisense 5′-ACCCTGCAGGTAAAGTTGGAAGCTGC-3′ (PCR product 453 bp); CPexons3+4F (XbaI), sense, 5′-AAATCTAGACGACTGCTCTACCAAGTAC-3′, and CPexons3+4R (XhoI), antisense, 5′-GACCTCGAGATAACTGGCCTTGGTGGC-3′ (PCR product 1.07 kb). Positions and sizes of the resulting PCR products are indicated in Fig. 1B. The three DNA fragments and the full-lengthCe-cpl-1 cDNA present in clone yk146d10 were cloned separately into pBluescript (Stratagene, La Jolla, CA) and used as templates for RNA synthesis. The double-stranded RNA (dsRNA) used for RNAi experiments was prepared following the Fire et al. (21Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11651) Google Scholar) protocol. Briefly, forin vitro transcription, each of the pBluescript plasmid DNA constructs was linearized with the appropriate restriction enzyme and single-stranded sense or antisense RNA was synthesized using the RiboMAX RNA Large-Scale Production System (Promega, Madison, WI) according to the manufacturers instructions. Equal amounts of each set of sense and antisense RNA strand were then annealed by incubation in injection buffer (20 mm KPO4, 3 mmK citrate, 2% PEG 6000, pH 7.5) for 10 min at 68 °C and 30 min at 37 °C. Double-stranded RNA (concentration 0.5 mg/ml) was then injected into the gonad of 25–30 young adult C. eleganshermaphrodites. Injected worms were left for 16–24 h at 20 °C to recover and to lay any eggs present in utero prior to microinjection, and then transferred to individual plates at 24-h intervals. The F1 progeny was quantified and examined for embryonic lethality or abnormal development. RNAi using the soaking protocol was done on L3 and L4 as described by Tabara et al. (22Tabara H. Grishok A. Mello C.C. Science. 1998; 282: 430-431Crossref PubMed Scopus (518) Google Scholar). Briefly, 15 C. elegans worms were incubated in 15 μl of 0.2 m sucrose in 0.1 × PBS containing 1 mg/ml dsRNA pre-mixed with 1 μl of Lipofectin (Invitrogen, Carlsbad, CA). After 24 h, soaked larvae were transferred to individual plates and their development examined for 4–5 days. Larvae soaked in 0.2 m sucrose in 0.1 × PBS without dsRNA served as the control. The length and width of each treated worm were measured under microscope using ocular and stage micrometer. Data were analyzed by One-way Analysis of Variance (ANOVA) and Student-Newman-Keuls Comparison Test (GraphPad InStat Software Inc.). Probability values of less than 0.05 were considered significant. Loss ofcpl-1 transcript following dsRNA injection was examined by RT-PCR using the same cpl-1 and ama-1 internal primers described above. Approximately 20 RNAi injected adult hermaphrodites or 300 RNAi mutant embryos were collected and washed twice in 1 ml of PBS. Wild-type adults and embryos were used as controls for normal gene expression levels. Adult and embryo pellets were then resuspended in 200 μl of lysis buffer (0.5% SDS, 5% β-mercaptoethanol, 10 mm EDTA, 10 mmTris-HCl, pH 7.5, and 0.5 mg/ml proteinase K), quick-frozen at −80 °C for 10 min, followed by incubation at 55 °C for 1 h. The RNA was extracted using Total RNA Isolation Reagent (Advanced Biotechnologies Ltd.) and the RT-PCR was carried out using SuperScript One-Step RT-PCR System (Invitrogen) according to the manufacturers instructions. Each 50-μl reaction mixture was split into two tubes into which either cpl-1 primers or ama-1 control primers were added. After 35 cycles of amplification, the RT-PCR products were separated on 2% agarose gels. Both transcriptional and translationalCe-cpl-1 reporter gene fusion constructs were generated and their expression patterns examined. For the transcriptional fusion construct, a promoter region of 1.76-kb ofcpl-1 upstream sequence, was generated by PCR on T03E6 cosmid DNA using Vent DNA Polymerase (New England Biolabs) and the following PCR primers: CPpromF1 (SphI), sense, 5′-ACAGCATGCTCCCGAAAAAAACTTCAATATTCTG-3′, corresponding to positions −1761 to −1736 relative to the ATG start codon, and CPpromR1 (XbaI), antisense, 5′-CGGTCTAGACTGGAATTTTATAACATTTAAAAT-3′, complementary to positions −2 to −25 relative to the ATG start codon (Fig. 1A). Following restriction enzyme digestion, the PCR fragment was cloned into lacZ reporter vector pPD96.04 containing a nuclear localization signal (kindly supplied by A. Fire). The translational fusion construct (pSL104) contained a 3.6-kb genomic fragment including 1.03 kb of the potential promoter region and all the four exons and three introns. This fragment was amplified by PCR on T03E6 cosmid DNA using the following primers: F1 (SphI), sense, 5′-CATGCATGCATCTCACCGTCTTCACCAGG-3′ corresponding to position −1030 to −1010 relative to the ATG start codon and R1 (AgeI), antisense, 5′-GCTACCGGTGCGACTCCGCAGTGATTGTT-3′, complementary to position 2550 to 2570 within the cpl-1 gene relative to the ATG start codon. The PCR amplified gene fragment was first cloned into the PCR2.1 cloning vector using the TOPO cloning kit (Invitrogen) and then subcloned into the gfp reporter vector pPD95.75 (kindly supplied by A. Fire). Plasmid DNA of the construct was prepared using the ConcertTM Rapid Plasmid Miniprep System (Invitrogen) and sequenced to confirm that the last exon of cpl-1 is in-frame with gfp. Transformation of C. elegans was performed by microinjection of plasmid DNA into the distal arm of the hermaphrodite gonad as described previously (23Fire A. EMBO J. 1986; 5: 2673-2680Crossref PubMed Google Scholar, 24Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2426) Google Scholar). Reporter plasmid DNA corresponding to the transcriptional construct (25 μg/ml) or translational construct (60 μg/ml) was co-injected with a marker plasmid DNA (pRF4 at 100 μg/ml) containing a dominant mutant allele of the rol-6gene (su1006) (24Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2426) Google Scholar). Transformants were identified by their right roller phenotype (25Kramer J.M. French R.P. Park E.C. Johnson J.J. Mol. Cell. Biol. 1990; 10: 2081-2089Crossref PubMed Scopus (262) Google Scholar). To allow examination of expression from transcriptional fusion constructs in the absence of any enhancers from therol-6 gene, transformation rescue of the unc-76mutant strain DR96 was carried out with plasmid p76–16B (a gift of Laird Bloom, MIT Center for Cancer Research), which contains the wild-type neuronally expressed unc-76 gene. Lines in which F2 and subsequent generations showed the roller orunc-rescue phenotype were stained for β-galactosidase expression, as previously described (3Britton C. McKerrow J.H. Johnstone I.L. J. Mol. Biol. 1998; 283: 315-327Crossref Scopus (47) Google Scholar) using 4′6-diamidino-2-phenylindole (final concentration 0.1%) as a co-stain to aid in the identification of cell types. GFP was visualized by mounting live transgenic worms on a 2% agarose pad in 0.01% sodium azide, that inhibits their movement, and viewing under a fluorescence filter. At least three independent lines were examined for each construct. A fragment of Ce-cpl-1 cDNA encoding the mature enzyme (Fig. 1A, amino acids 121–337) was amplified and cloned into the BamHI and XhoI sites of the pGEX4T-3 expression vector (Amersham Bioscience Inc., Piscataway, NJ). The recombinant GST-Ce-mCPL-1 protein was expressed in the form of inclusion bodies and was therefore purified from the 50 mm Tris-HCl, pH 8.0, insoluble pellet. The pellet was solubilized in 6 m urea in 50 mmTris-HCl buffer, pH 8.0, followed by preparative separation using the Prep Cell (Bio-Rad) according to the manufacturers instructions. Fractions containing the purified recombinant GST-Ce-mCPL-1 polypeptide were identified using antibodies against GST. A mouse antiserum was raised using the repetitive multiple site immunization strategy (26Kilpatrick K.E. Wring S.A. Walker D.H. Macklin M.D. Payne J.A. Su J.L. Champion B.R. Caterson B. McIntyre G.D. Hybridoma. 1997; 16: 381-389Crossref PubMed Scopus (73) Google Scholar). Each mouse received a total of 10 μg of GST-mCPL-1 in RIBIs adjuvant on days 0, 3, 6, 8, and 10. For detection of the nativeCe-CPL-1 enzyme in C. elegans embryos, gravid hermaphrodites were washed off culture plates in PBS and cut open to release the embryos. The embryos were then collected and fixed in methanol/acetone using the freeze-cracking protocol (27Kemphues K.J. Wolf N. Wood W.B. Hirsh D. Dev. Biol. 1986; 113: 449-460Crossref PubMed Scopus (62) Google Scholar). For whole mounted immunostaining, mixed-stage population of larvae and adults were collected and washed in PBS and Ruvkun Fixation buffer before being fixed and permeabilized using 1% paraformaldehyde in Ruvkun Fixation buffer for 30 min and two freeze-thaw cycles in a dry ice/ethanol bath (28Finney M. Ruvkun G.B. Cell. 1990; 63: 895-905Abstract Full Text PDF PubMed Scopus (490) Google Scholar). The fixed and permeabilized embryos or the whole worms were treated with a blocking solution (60 mg/ml normal goat serum in PBS, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h before reaction with antibodies. Primary anti-GST-mCPL-1 antibodies were used at 1:20 and 1:40 dilution. Fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibodies were used at a 1:50 dilution. Immunostained specimens were viewed under fluorescence microscope using appropriate filter sets in the presence of mounting medium, Vectashield containing 4′6-diamidino-2-phenylindole (Vector Laboratories, Inc., Burlingame, CA). C. elegans mixed stage larvae, hermaphrodites, and embryos collected from 5% NaOCl-treated hermaphrodites were fixed for 60 min in 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 m phosphate buffer, pH 7.4, containing 1% sucrose. The fixed worms were then processed for immunoelectron microscopy as previously described (17Lustigman S. Brotman B. Huima T. Prince A.M. McKerrow J.H. J. Biol. Chem. 1992; 267: 17339-17346Abstract Full Text PDF PubMed Google Scholar). Thin sections of C. elegans embedded worms were probed with mouse antisera raised against the recombinant C. elegansGST-Ce-mCPL-1 fusion polypeptide before incubation with 15-nm gold particles coated with anti-mouse IgG (Amersham Bioscience, Inc.). Mouse preimmune serum or antibodies to GST were used as controls. The localization of the native CPL in O. volvuluswas done as previously described (17Lustigman S. Brotman B. Huima T. Prince A.M. McKerrow J.H. J. Biol. Chem. 1992; 267: 17339-17346Abstract Full Text PDF PubMed Google Scholar) using thin sections of O. volvulus L3 and adult-embedded worms and rabbit antisera raised against the recombinant O. volvulus mature region or the pro-region. Rabbit preimmune serum or antibodies to GST were used as controls. The antisera raised against the CPL enzymes recognized by Western blot their corresponding recombinant proteins and did not cross-react with recombinant proteins expressing the cathepsin Z-like proteins of C. elegans or O. volvulus (data not shown). The potential promoter region of Ov-cpl was amplified by sequential PCR reactions using O. volvulus genomic library, λ Fix II (kindly provided by Dr. R. D. Walter), the vector primer and gene-specific primers designed based on known sequences. A 1.3-kb fragment containing the ATG start codon was amplified first followed by another 1.8-kb fragment after using the vector primer and an antisense primer corresponding to a region within the 1.3-kb fragment. The final region identified as the putative promoter of the gene was ∼2.5 kb upstream of the ATG of Ov-cpl encoding region (accession number AF442768). For the transcriptional fusion construct, this fragment was amplified from O. volvulus genomic DNA using the following pair of PCR primers: OVcplp5′ (XbaI), sense, 5′- CGTCTAGACTTAGATTTCCATCCCGACGAG-3′, corresponding to positions −2525 to −2504 relative to the ATG start codon, and OVcplp3′ (XmaI), antisense, 5′-GACCCGGGTGAGTTTTTTTCTGTTTCTGTTTTCC-3′, complementary to positions +1 to −25 relative to the ATG start codon. Following restriction enzyme digestion, the PCR fragment was cloned into lacZ reporter vector pPD90.23 containing a nuclear localization signal (kindly supplied by A. Fire). The fusion gene containing the cpl putative promoter was designatedOv-cpl:lacZ (pSL117A). Using a heterologous transformation, we created C. elegans transgenic lines expressing theO. volvulus promoter as described in generation and expression of C. elegans reporter gene constructs. Based on BLASTP search of ACeDB using the mature region of theOv-CPL amino acid and the other filarial nematode sequences, a related sequence was identified in cosmid T03E6.7 (accession nu
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