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The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a con-

A cDNA encoding the complete precursor of a Fasciola heputicu cathepsin L protease was isolated and sequenced. Functionally active enzyme was expressed and secreted by Saccharoinyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature-sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, which lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express funtionally active enzyme. The yeast-expressed enzyme exhibited physicochemical properties in common with the native enzyme including, pH optimum for activity, stability at 37°C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P, position. This is the first report of the functional expression of a cathepsin L proteinase in S. cerevisiue that did not require the use of yeast secretory signal sequences.Keywords: Fasciola hepatica ; trematode ; parasite ; cathepsin ; protease.Cathepsin L proteases of parasitic helminths were first characterised in our laboratory when we showed that Fasciola heputicu, the causative agent of human and animal liver fluke disease, actively secreted these into culture medium [l, 21. Moreover, we showed that cathepsin L could cleave IgG specifically in the hinge region and thereby separate the Fab and the Fc regions [3]. In addition, since the cathepsin L could prevent the antibody-mediated attachment of immune effector cells to the parasite, we proposed that secretion of this enzyme in vivo by the parasites protects them from host immune responses [4].More recent studies have shown that cathepsin L proteases are secreted by other helminths, such as the human pathogens Schistosonzu mansoni [5, 61 and Aizcylostoma caninum (hookworm) [7]. In these parasitic infections cathepsin L species have been implicated in roles such as hemoglobin digestion and tissue degradation [S]. Furthermore, in the case of hookworm infections, cathepsin L may induce allergenic-like reactions [7]. Because of their involvement in crucial biological functions, cathepsin L proteases are considered important candidates to which novel anti-parasitic chemotherapeutic or immunoprophylactic reagents could be directed [S]. In most cases the paucity of available parasitic material precludes the isolation of these proteases in quantities necessary for detailed biochemical and physico-chemical analyses. However, this problem can be overcome by expressing these proteases using recombinant DNA systems that produce functional enzyme. In the present study we have isolated a cDNA clone by Sciences, Dublin City University, Dublin 9, Republic of Ireland screening an F: hepatica cDNA library with anti-(cathepsin L1) serum. Nucleotide sequence analysis revealed that the clone encoded a complete cathepsin L protease precursor (preprocathepsin L). Functional cathepsin L proteas...

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To investigate the developmentally programmed telomere addition that accompanies chromosome fragmentation during macronuclear differentiation in Tetrahymena thermophila, five representative telomeric regions from the macronucleus were cloned and characterized in detail. The sequences adjacent to the telomeric (C4A2:T2G4) repeats on these five macronuclear ends had no significant sequence homology or shared secondary structure. Two developmentally independent examples of one macronuclear telomere had a 5 base pair difference in the position of the junction between the telomeric repeats and the adjacent sequences. A telomere-adjacent sequence, in the form of a synthetic oligonucleotide, was unable to prime the addition of telomeric repeats in vitro. The implications of these results for the mechanisms underlying developmentally programmed chromosome fragmentation and telomere addition in Tetrahymena are discussed.