A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6.5 and 7.5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52-76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.

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A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.Paragonimus westermani is a trematode parasite that causes chronic inflammatory lung disease as well as systemic infection including cerebral invasion in humans and carnivorous mammals. Human infection occurs by ingesting undercooked freshwater crayfish or crabs containing metacercaria or eating raw boar meat. The metacercariae excyst in the duodenum, penetrate the peritoneal cavity, and finally, migrate to the lung, in which they become adults and are surrounded by a thick granulomatous wall (1, 18). The adult worms can survive for approximately 5 years.Parasite cysteine proteases are known to play critical roles in parasitic infections. They participate in a broad range of biological processes including egg hatching and subsequent stage transitions, invasion and migration through host tissues, and immune modulation and nutrient uptake (4,25,28,29,39,43). Recent studies have shown that these enzymes might be good targets for the development of vaccines (20, 33) and antiparasitic drugs (7, 9).In P. westermani, at least six different species of cysteine protease with molecular sizes of 53, 34, 28, 27, 17, and 15 kDa, respectively, have been characterized in eggs, metacercariae, juveniles, and adults (4,5,14,22,45). The 28-and 27-kDa proteases released by the metacercariae have been shown to share biochemical features with cathepsin L of F. hepatica (8) or with cathepsin S of sparganum (25) and are believed to play important roles in metacercarial excystment, tissue migration, and immune evasion (4, 6, 14, 45).It is of particular interest that P. westermani possesses multiple cysteine proteases of similar sizes ranging from 29 to 27 kDa (4...