Because of the important contributions of parasites with respect to human morbidity and mortality, and because proteases are implicated in normal and pathological processes, parasite proteases are key biomolecules. Proteases of helminth parasites are essential to the maintenance of parasitism, and adversely affect hosts in a number of ways. In particular, proteases are employed by parasitic worms to degrade the extracellular dermal matrix during skin penetration, to digest host tissue components and hemoglobin, and to inhibit of host protective immune responses through cleavage of immunoglobulin and complement subunits. Therefore, proteases may be suitable targets for immuno- or chemotherapy. The aims of this study were to isolate and characterize protease-encoding genes from parasitic helminths. Novel cysteine and aspartic protease genes were identified and characterized from a panel of helminth species, including Strongyloides stercoralis, S. ratti, Ancylostoma caninum, Schistosoma japonicum, S. mansoni and Caenorhabditis elegans. The identification of these genes and their products has provided clues to their biological form and function. To isolate novel protease genes from parasitic nematodes by polymerase chain reaction (PCR) -based techniques, a pair of consensus oligonucleotide primers were employed which were designed to anneal to the active-site cysteine- and asparagine­ coding regions of cysteine proteases. The primers were biased toward the nucleotide and codon usages of cysteine protease genes of nematodes, rather than eukaryotic organisms in general. In particular, these primers, ncpC and ncpN, were based on consensus nucleotide sequences encoding the active site Cys and Asn residues and flanking residues, from Haemonchus contortus, Ostertagia ostertagi, and C. elegans. By using the nucleic acid sequences of these nematode genes only, it was possible to reduce the degeneracy of the primers, as well as increase their specificity in comparison to primers pioneered by previous workers. Using ncpC and ncpN, PCR products that represented fragments of cysteine protease genes were amplified and cloned from the rodent parasite, Strongyloides ratti, the human pathogen, S. stercoralis, the canine hookworm Ancylostoma caninum, and C. e/egans, and their nucleotide and deduced amino acid sequences were determined. To characterize genes encoding cysteine protease(s) secreted by A. caninum, an adult hookworm cDNA library was constructed and screened with the cloned hookworm cysteine protease gene fragment cDNAs encoding two proteases were obtained from the library and sequenced. The first gene, AcCP-1, encoded a cathepsin B-like zymogen cysteine protease of 343 amino acids, predicted to be processed in vivo into a mature cysteine protease of 255 amino acids. Closest nucleotide identities were to Haemonchus contortus cysteine protease (61%) and human cathepsin B (60%). The second gene, AcCP-2, encoded a mature cysteine protease of 254 aa, that showed 86% identity to AcCP-1, and 58% and 47% identity to bovine cathepsin B and human cathepsin B, respectively. These are the first reported protease genes from hookworms. AcCP-1 and AcCP-2 were expressed in Escherichia coli using the pQE expression vector, and the recombinant proteases were subsequently purified by nickel-chelate affinity chromatography. Monospecific antibodies were raised in rabbits to the recombinant antigens, and immunohistolocalization studies were performed on paraffin­ embedded, formalin-fixed worm sections to localize the site of expression of AcCP-1. The anti-protease antiserum hybridized to the esophageal, excretory and amphidial glands, all of which open into the buccal region of the hookworm, suggesting that AcCP- 1 is a secreted enzyme. Recombinant Ac\CP-1 was not recognized on western blots by sera from A. caninum-infected people, but the antiserum recognized a ~40 kDa band in hookworm excretory/secretory products. Proteases encoded by AcCP-1 and AcCP-2 may be responsible for the cysteine protease activity that has been shown previously to be secreted by adult A. caninum. This suggestion was supported by preliminary three dimensional homology modelling of AcCP-1, which indicated that AcCP-1 showed distinct substrate preferences to mammalian cathepsin B. Homology PCR, employing ncpC and ncpN, was undertaken with cDNA from the trematode, Schistosoma mansoni, but no cysteine protease genes were isolated. Subsequently, using the nematcxie-specific, consensus primers in tandem with a generic primer designed to hybridize to the asparagine active site of cysteine proteases, gene fragments were amplified from adult S. mansoni cDNA by PCR, and cloned. One of these fragments showed marked identity to Sm31, the cathepsin B cysteine protease of adult S. mansoni, whereas another differed from Sm31 and was employed as a probe to isolate two cDNAs from an adult S. mansoni gene library. Together, these cDNAs encoded a novel preprocathepsin L of 319 amino acids; this zymogen is predicted to be processed in vivo into a mature, active cathepsin L protease of 215 amino acids. Closest homologies to the schistosome cathepsin L (SmCLl) were cathepsins L from rat, mouse, and chicken (46-47% identity). Since aspartic proteases are considered the most closely conserved group of proteases, and because they appear to be ubiquitous in eukaryotes, an aspartic protease consensus primer, napD, was designed on the conserved first active site coding region of 16 eukaryotic aspartic proteases. Primer napD, in tandem with a lambda phage cloning vector-specific primer (M13-20), was used in homology PCR experiments designed to locate aspartic protease genes in the nematode, A. caninum, and the trematode, S. japonicum. An aspartic protease gene fragment was amplified from the hookworm cDNA library by homology PCR. The gene fragment was cloned, sequenced, and found to encode a zymogen of 422 amino acids. A dendrogram showing the functional relationships of the zymogens to subclasses of other aspartic proteases revealed that the A. caninum zymogen showed most identity (~50%) to the lysosomal aspartic protease of the mosquito, Aedes aegypti, and to vertebrate cathepsin Ds. Furthermore, it showed partial identity with cyprosin, an aspartic protease from the flower of the cardoon, Cynara cardunculus. The hookworm enzyme, Acasp, showed a ~30 amino acid residue carboxy1 tenninal extension, similar to that seen in the cysteine proteases, cruzipain and congopain, from trypanosomes. Acasp has one potential Asn-glycosylation site, located in the mature enzyme sequence. This asparagine residue in Acasp was in an analogous position to that of human cathepsin E. A pilot, 3D homology model of Acasp was constructed based upon the crystal co-ordinates of human cathepsin D, but was found to differ markedly from human cathepsin D within loop 2 and at the P2 binding pocket. Additionally, the β -hairpin (loop 3) region was absent, being substituted by a pair of cysteine residues which possibly form an extra disulphide connection. In like fashion, homology PCR using the primer napD amplified gene fragments encoding an aspartic protease from S. japonicum cDNA. One of the fragments was used to probe an adult S. japonicum cDNA library. A cDNA was isolated that, when sequenced, was found to encode a zymogen of 380 amino acid residues. Identity comparisons showed that the enzyme (Sjpasp) was most closely related to mammalian cathepsin Ds. The deduced amino acid sequence of Sjpasp showed four potential Asn­glycosylation sites, two of which were in identical positions to the two glycosylation sites of human kidney lysosomal cathepsin D. Further, all four disulfide bonds found in mammalian cathepsin D sequences were present in Sjpasp, although the β-hairpin (loop 3) which is cleaved during maturation of vertebrate cathepsin Ds to yield light and heavy chain subunits was absent from Sjpasp. While most residues involved in substrate specificity and catalysis of aspartic proteases were preserved in Sjpasp, several residues in these regions exhibit changes which may result in a novel substrate specificity. In summary, homology PCR strategies were of pivotal value in the isolation of novel protease gene fragments from nematodes, and from schistosomes. These genes included AcCP-1 and AcCP-2, which encode cathepsin B-like proteases from adult hookworms, Acasp, which encodes a cathepsin D-like protease also from adult A. caninum, Sjpasp and SmCLl which encode cathepsins D and L from schistosomes, and novel cysteine protease encoding-genes from Strongyloides stercoralis and S. ratti and from C.elegans. Future research can now proceed to investigate the importance of enzymes encoded by these genes, both in the maintenance of parasitism in these helminths potential targets for anti-parasitic therapies.