A cDNA encoding a pepsinogen-like, aspartic protease from the human roundworm parasite Strongyloides stercoralis1

Acta Tropica 71 (1998) 17 – 26

A cDNA encoding a pepsinogen-like, aspartic protease from the human roundworm parasite Strongyloides stercoralis 1 Sara G. Gallego a, Robert W. Slade b, Paul J. Brindley a,* a Molecular Parasitology Unit, Queensland Institute of Medical Research and Australian Centre for International and Tropical Health and Nutrition, P.O. Royal Brisbane Hospital, Herston, Queensland 4029, Australia b Malaria and Arbo6irus Unit, Queensland Institute of Medical Research and Australian Centre for International and Tropical Health and Nutrition, P.O. Royal Brisbane Hospital, Herston, Queensland 4029, Australia

Received 16 January 1998; received in revised form 10 May 1998; accepted 31 May 1998

Abstract Using degenerate oligonucleotide primers based on conserved active site residues, we have isolated a cDNA encoding an aspartic protease from the nematode parasite Strongyloides stercoralis, an important, enteric pathogen of humans. cDNAs encoding the aspartic protease were isolated from the infective, third stage larvae of the parasite as well as from free-living, rhabditiform larvae. Based on comparisons of other aspartic proteases, the cDNA encoded a short signal peptide, an enzyme pro-segment of 35 amino acid residues, and mature enzyme of 337 residues. Homology alignments using the proenzyme sequence showed that the novel S. stercoralis zymogen was 36% identical to human pepsinogen A and 36% identical to pepsinogen C (progastricin) from humans and macaques. Phylogenetic analyses using the Phylip program and analysis of Glx/Asx and Leu/Ile ratios indicated that the proenzyme was closely related to pepsinogen A-like enzymes from the free-living nematode Caenorhabditis elegans and Haemonchous contortus, a nematode parasite of the gastro-intestinal tract of sheep. We have termed this novel enzyme strongyloidespepsin. © 1998 Elsevier Science B.V. All rights reserved.

Abbre6iations: HuPgC, human pepsinogen C; SsPep, strongyloidespepsin. * Corresponding author. Tel.: + 61 7 33620413; fax: + 61 7 33620104; e-mail: [email protected] 1 Sequences described here have been lodged in GenBank with the accession number AF027166. 0001-706X/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0001-706X(98)00050-3

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Keywords: Protease; Aspartic protease; Pepsin; Pepsinogen; Strongyloides stercoralis

1. Introduction Strongyloides stercoralis is an important nematode pathogen of humans. It is transmitted by contact with soil contaminated with faeces, like hookworm, and is widely prevalent in populations living in tropical and sub-tropical latitudes. However, its unique ability to replicate in, and reinfect, the same person gives it a pathological significance quite distinct from other parasitic helminths. Infection is established by larvae penetrating the skin, after which the larvae migrate in the circulation to the lungs, cross into an alveolar sac, move up the bronchae, and are swallowed. Moulting follows and the adult nematodes—parthenogenetic females— develop in the mucosa of the duodenum and jejunum, shedding eggs from which larvae hatch and pass out with the faeces into the environment. Sequences from only a few protein-encoding genes and a small number of expressed sequence tags have been reported from this important helminth pathogen of humans (Harrop et al., 1995; Moore et al., 1996; Ramachandran et al., 1998). Expansion of the sequence information for genes and their associated proteins of S. stercocralis will enhance our understanding of this unusual nematode, particularly in view of the difficulty in maintaining the parasite in the laboratory (see Ramachandran et al., 1998). Here we report the transcript encoding a novel aspartic protease expressed by several larval stages of S. stercoralis, including the filariform, infective third stage larva of the parasite. Homology comparisons showed that the protease was a pepsinogen-like aspartic protease. This is one of the first gene sequences encoding aspartic proteases to be reported from the phylum Nematoda. We are aware of only four other aspartic proteases from nematodes; a cathepsin D-like protease from the hookworm Ancylostoma caninum (Harrop et al., 1996), a pepsinogen A-like enzyme from Haemonchus contortus (Longbottom et al., 1997), the barber’s pole worm of sheep and cattle, a cathepsin D-like enzyme from the free-living Caenorhabditis elegans (GenBank accession numbers include U34899 and U97000), and a partial cDNA from Onchocerca 6ol6ulus (Jolodar and Miller, 1997).

2. Materials and methods

2.1. cDNA libraries from the rhabditiform and the filariform stages of Strongyloides stercoralis The cDNA libraries have been described in detail, previously (Moore et al., 1996), and were kindly provided by Drs T. Nutman and T. Moore of the National Institute of Health, USA. In brief, three libraries were used here, including a library constructed from rhabditiform larvae, and two libraries constructed from infective,

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third stage, filariform larvae. Of the two libraries from the filariform larvae, one was constructed from parasites originally isolated from infected humans and subsequently maintained in Erythrocebus patas monkeys, while the second was constructed from parasites maintained in immunosuppressed dogs. All three libraries are directional cDNA libraries constructed in the phage vector M UNI ZAP XP (Stratagene, La Jolla, CA).

2.2. Homology PCR A degenerate oligonucleotide primer, 5%-TTGAYACNGGNTCATCAAYCTNTGG, designed to target the highly conserved regions surrounding the active site Asp32 residue of aspartic proteases (Becker et al., 1995; Harrop et al., 1996) was paired with a l-specific primer (5%-GTAAAACGACCGGCCAGT) and used in polymerase chain reaction (PCR) with the cDNA library(s) as the template. The PCR ‘touchdown’ conditions described by Don et al. (1991) and others (Harrop et al., 1995) involving denaturation at 94°C for 1 min, annealing at 59°C (four cycles), 54°C (four cycles), 49°C (four cycles) and 37°C (25 cycles), with the annealing cycles for 1 min each, extension at 72°C for 1 min, and a final extension at 72°C for 15 min were employed. PCR products were examined in ethidium bromide-stained agarose gels, and amplified sequences were purified from gels using the Wizard Cleanup System (Promega). The purified DNA was employed as the template for a second round of PCR in which the two original oligonucleotide primers were replaced with the primers 5’-GGCGAATTCTTGAYACNGGNTCATCAAAYCTNTGG (where Y is C or T and N is any of the four bases) and 5’-GGCCTGCAGGTTTTCCCAGTCACGAC, which were identical to the original pair except that clamp sequences and PstI and EcoRI restriction site sequences were included in order to facilitate cloning of the PCR products into the plasmid pUC19.

2.3. Sub-cloning, library screening Products obtained after the second round of PCR amplification were digested with EcoRI and PstI (New England Biolabs, Beverley, MA), purified as above, and ligated into linearized, dephosphorylated pUC19. Escherichia coli SURE strain cells (Stratagene) were transformed with the ligation products, after which the transformed cells were cultured on LB agar media supplemented with ampicillin, 5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside, and isopropyl-1-thio-b-D-galactopyranoside (Sambrook et al., 1989). White colonies isolated were isolated and then cultured in LB supplemented with ampicillin. Plasmid DNA was isolated from the cultures using the Wizard Miniprep Kit (Promega). The S. stercoralis cDNA libraries were screened by nucleic acid hybridization using inserts of recombinant plasmids as probes, and stringent hybridization and washing conditions as described by Church and Gilbert (1984). The probes were radiolabeled with a 32P.dCTP (DuPont, Wilmington, DE) by random oligomer priming (Oligo-Labelling Kit, AMRAD-Pharmacia, Melbourne, Australia). Positive plaques were isolated by secondary screening, after which phagemids were excised into pBlue-

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script SK (-) constructs using the ExAssist helper phage and E. coli (SOLR) cells according to the manufacturer’s instructions (Stratagene). Midipreps of recombinant pBluescript plasmids were prepared from bacterial cultures using Qiagen 100 columns (Qiagen).

2.4. Nucleotide sequencing, sequence analyses The nucleotide sequence of inserts of plasmids was determined using l- and gene-specific primers, the Taq DyeDeoxy Terminator Cycle Sequencing System (Applied Biosystems, Foster City, CA [ABI]) and an automated DNA sequencer (ABI, model 377). Analyses of nucleotide and deduced amino acid sequences were undertaken using the GCG package software (University of Wisconsin, Madison, WI). Gene and protein identifications were accomplished by comparisons using the BLAST algorithms (Altschul et al., 1997). Comparisons of related sequences were undertaken using the PileUp, Prettyplot, and PEPWINDOW programs (GCG). Hydropathicity indices were determined by the method of Kyte and Doolittle (1982). Functional sites and motifs on target proteins were located using ScanProsite (www.expasy.hcuge.ch/sprot/scnpsite.html).

2.5. Phylogenetic analysis Phylogenetic reconstruction of the inferred amino acid sequence was performed with the neighbor-joining method of Saitou and Nei (1987), as implemented in the ‘MEGA’ program of Kumar et al. (1993). Bootstrap confidence levels were based on 1000 resamplings.

3. Results and discussion

3.1. Protease gene isolated by homology PCR When the aspartic protease gene specific primer was paired with the universal M13 forward primer in a PCR using the S. stercoralis cDNA libraries as templates, products of : 900 nucleotides in length were amplified from the rhabditiform life stage library. After the product was cloned into a plasmid vector and sequenced, it was found to encode a open reading frame (ORF) with strong homology to genes of aspartic proteases from a number of organisms (not shown). Subsequently, the insert of the recombinant plasmid was isolated, radiolabeled, and employed to probe the S. stercoralis cDNA libraries. After screening 100000 phage plaques, several positive clones were identified in all three libraries after secondary screens, the longest of which (insert size : 1.2 kb) was isolated from the rhabditiform stage library. The phagemid was excised from this phage clone and the nucleotide sequence of its insert determined. The clone contained a recombinant insert of 1180 bp which included an ORF of 380 codons including the stop codon TTA, a 3%-untranslated region (UTR) of 20 bp which included the polyadenylation signal

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AATGAAA, and a poly A tract (GenBank accession no. AF027166). The sequence included all of the 900 bp fragment originally isolated by homology PCR using the aspartic protease-gene specific primer. A reverse-directed primer, 5%-GGCCTGCAGCTGTTCAAGTGTTATTAC, was designed from the region encoding the ORF and paired with the vector specific M13 reverse primer 5%-GGCCTGCAGCAGGAAACAGCTATGAC, but no fragments containing the extreme 5%-terminus of the cDNA or the 5%-UTR were found. In like fashion, repeated screens of the libraries using the radiolabeled insert of 1180 bp (above) failed to isolate a longer clone. Phage clones with insert sequences homologous with the 1180 bp length insert were located in libraries from both the rhabditiform and filariform larval stages of S. stercoralis, indicating that the protease was expressed in at least two life cycle stages of this roundworm parasite.

3.2. Homologies with pepsinogen-like, aspartic proteases Homology comparisons using the entire 380 deduced amino acid residues revealed that the S. stercoralis cDNA encoded was a novel, aspartic protease. Highest homologies were observed with pepsinogen C from monkey and human (both 36%) (Kanegawa and Takahashi, 1986; Hayano et al., 1988) human pepsinogen A (36%), (Kanegawa and Takahashi, 1986; Hayano et al., 1988) plasmepsin II prepropenzyme of Plasmodium falciparum (30%) (Hill et al., 1994), the pepsinogen-A like enzyme from H. contortus (Longbottom et al., 1997), and with the aspartic protease from C. elegans (GenBank accession no. U34889). Further, the S. stercoralis protease showed moderate identity to other groups of aspartic proteases including oryzasin from rice (Asakura et al., 1995) and chymosin from cattle (Harris et al., 1982). The homology to pepsinogen-like proteases indicated that the novel S. stercoralis aspartic protease was pepsinogen-like.

3.3. Structural and functional motifs An alignment of this novel S. stercoralis protease (SsPep) with several other aspartic proteases (S. japonicum cathepsin D-like protease, human pepsinogen C, and Pep-1, the pepsinogen A-like enzyme from H. contortus) is presented in Fig. 1. Using the SigCleave program, and by comparison with human pepsinogen C, cleavage of the signal peptide from the pro-segment likely occurs between alanine and alanine (Ala-P1) (open arrow, Fig. 1) (propeptide residues numbered Ala-1P, Ser-2P, etc. Tang and Wong, 1987). Similarly, cleavage of the pro-segment from the mature protease likely occurs between alanine (Ala-35P) and valine (Val-1) (solid arrow, Fig. 1). Thus Val-1 (SsPep numbering) was predicted to represent the NH2-terminal residue of the mature protease. The pro-segment was predicted to include 35 amino acid residues, and the mature enzyme was predicted to include 337 amino acid residues, of similar lengths to many other aspartic proteases (Tang and Wong, 1987). The mature enzyme has a predicted mass of 36560 Da and an isoelectric point of 4.62. Hydropathy analysis of the deduced amino acid sequence showed that the NH2-terminus of the S. stercoralis aspartic protease was highly

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Fig. 1. Multiple sequence alignment of the deduced amino acid sequences of SsPep with three other aspartic proteases, the cathepsin D-like aspartic protease from Schistosoma japonicum (SjAsp) (Becker et al., 1995), human pepsinogen C (HuPgC) (Hayano et al., 1988), and the pepsinogen A-like protease from Haemonchus contortus (HcPep1) (Longbottom et al., 1997). Amino acid numbering for HuPgC is at the right. Similar residues are shown in the boxes. Gaps have been introduced to maximize the alignment. The catalytic dyad of aspartic acid residues is indicated by stars. The open arrow indicates the position of cleavage of signal sequences from the proenzymes and the solid arrow indicates the position of cleavage of the pro-segment from the mature aspartic proteases. Lys-29P in the prosegemnt of SsPep is indicated with a the gray box, as is the COOH-terminal extension of the mature SjAsp enzyme.

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hydrophobic, which supported the prediction that the signal peptide sequence spanned the residues NH2-terminal to Ala-P1 (not shown). Aspartic proteases form a catalytic site by folding two separate domains together to produce active enzyme (Tang and Wong, 1987). The active site aspartic acid residues (Asp-31 and Asp-215, porcine pepsinogen numbering Tang and Wong, 1987) and vicinal residues are highly conserved in the aspartic class of proteases. The active site residues were Asp-32 and Asp-225 in the Strongyloides enzyme SsPep and these and the neighboring amino acids exhibited very high conservation with respect to other aspartic proteases (Fig. 1). Two forms of pepsinogens — the inactive precursors of pepsin—are known from vertebrates, pepsinogen A and pepsinogen C. In humans, pepsinogen A is more abundant than pepsinogen C. Pepsinogen A is found in the fundus and the body of the stomach whereas pepsinogen C is found throughout the stomach, in the proximal duodenum, and in the testes (Hayano et al., 1988). The deduced amino acid sequences of pepsinogen A and pepsinogen C diverge significantly. Quantitative differences in the Asx/Glx ratio and the Leu/Ile ratio have been used as a basis for the classification of mammalian pepsins A and C (Baudys and Kostka, 1983; Kanegawa and Takahashi, 1986; Hayano et al., 1988). Values for human pepsinogens for the Glx/Asx and for the Leu/Ile ratios are 0.81 and 0.84, respectively, for pepsinogen A and 1.56 and 2.00, respectively, for pepsinogen C (Kanegawa and

Fig. 2. Neighbor-joining tree of the sequences of fifteen proteases representative of several families of aspartic proteases. Percent bootstrap confidence levels are shown at the nodes. Used in the alignment were 226 orthologous residues from each protease, which is available upon request from the authors. The clade termed S. stercoralis represents the novel protease strongyloidespepsin.

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Takahashi, 1986; Hayano et al., 1988). The Glx/Asx and Leu/Ile values were 0.81 and 0.86, respectively, for the novel Strongyloides pepsinogen-like enzyme, determined using the prosegment and mature enzyme sequences. These results indicated that SsPep was more like pepsinogen A than pepsinogen C. Interestingly, a comparison of the pro-enzymes of the pepsinogen-like enzymes from the nematodes H. contortus (Longbottom et al., 1997) and C. elegans (GenBank U34889) showed that the Glx/Asn and Lue/Ile ratios were more pepsinogen A-like than pepsinogen C-like in both cases (not shown). Lys-36P (porcine pepsinogen numbering) of the prosegment is a conserved residue in most if not all aspartic proteases from eukaryotic cells (Foltmann, 1988). In the inactive zymogen, the Lys-36P residue is located between the two active site aspartic acid residues, and it appears to be essential for the correct folding of the zymogen. The homologue of Lys-36P in the Strongyloides protease appeared to be Lys-32P (SsPep numbering) (Fig. 1). Gln-14, as well as its adjacent residues at the S3 subsite, is considered an important determinant in substrate enzyme interactions (Tang and Wong, 1987). This residue was replaced with Phe-13 in the SsPep sequence. Tyr-78 in the (hairpin loop 2) which partly occludes the active site is also crucial in determination of substrate specificity (Tang and Wong, 1987), and was conserved in SsPep as Tyr-82. However, Thr-80 in loop 2 is replaced by a serine in human pepsinogen C (Hayano et al., 1988) and by Ser-84 in the Strongyloides protease. Lys-203 is conserved in many lysosomal aspartic proteases including cathepsin D from humans and chickens and the cathepsin D-like protease from Aedes aegypti (Cho and Raikhel, 1992; Metcalf and Fusek, 1993). By contrast, this is replaced by Glx-203 in non-lysosomal, aspartic proteases including porcine pepsinogen C and cathepsin E (Hayashi et al., 1988; Azuma et al., 1989), and in SsPep as Glu-199. In similar fashion to human pepsinogen A, the Strongyloides protease has only a single proline in place of the polyproline loop of renins and cathepsin D (Metcalf and Fusek, 1993). The hemoglobinase plasmepsin II from P. falciparum and the cathepsins D of Schistosoma mansoni and S. japonicum also lack these prolines found in the mammalian cathepsin D-like, aspartic proteases within loop 8 (Francis et al., 1994; Wong et al., 1997).

3.4. Phylogenetic analysis We selected 14 other sequences encoding aspartic proteases in order to construct a phylogenetic tree of the relatedness of the S. stercoralis enzyme to other aspartic proteases reported from other organisms. For this unrooted tree, we chose aspartic proteases from H. contortus (Z72490) and C. elegans (U34889) because these sequences gave the best matches to the SsPep using the BLAST algorithm. In like fashion, we included plasmepsin II from the malarial parasite P. falciparum (P46925), oryzasin from rice, and pepsinogens C from human and monkey (P20142, P03955) because they gave very high database matches. In addition, we included an aspartic protease from another parasitic nematode, A. caninum (U34888), cathepsin D- like enzymes from human blood flukes S. mansoni (U60995) and S. japonicum (L41346), cathepsin D from chicken (Q05744), a pepsin-like protease from the

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coccidian parasite Eimeria acer6ulina (Z24676), and a lysosomal, cathepsin D-like enzyme from the mosquito Aedes aegypti (M95187). The phylogenetic analysis supported the classification of SsPep as a pepsinogen-like enzyme (Fig. 2). Ss-pep appeared to be closely related to the nematode pepsinogen A-like proteases of H. contortus and C. elegans, confirming the qualitative analysis of Glx/Asx and Lue/Ile ratios described above.

3.5. Concluding remarks The role of the novel aspartic protease remains to be determined. Given that it appears to be similar to mammalian pepsinogens, and to have substituted Lys-203 residue of many lysosomal-associated aspartic proteases, it may be expected to be secreted rather than retained in lysosomes or other cellular compartments. The related HcPep-1 from H. contortus appears to be secreted into the gut of the adult stage of this parasite where it may play a role in hemoglobin proteolysis of ingested host blood. The cathepsin D-like proteases of schistosomes also play central roles in hemoglobin proteolysis (Brindley et al., 1997). On a practical level, whatever the physiological role of SsPep turns out to be, it may be of value in diagnosis given that at least one other protease from S. stercoralis shows potential for serodiagnosis of human strongyloidiasis (Brindley et al., 1995). Finally, we suggest that this novel pepsinogen-like aspartic protease be named strongyloidespepsin, abbreviated as SsPep, based on the convention used to name related enzymes such as rhizopuspepsin (EC 3.4.23.6) (Chen et al., 1991).

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