Cystatins from the Human Liver Fluke : Molecular Characterization and Functional Analysis.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, . Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of . Six cDNA coding sequences of cystatins, Cys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, Cys1 and Cys2 are classified as type 1 cystatins, while Cys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins Cys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, Cys1, 3 and 4 were selected for inhibition analysis and immune reactivity with -infected hamster sera. Cys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that cystatins are not major antigenic components in the ES product of this parasite and that other effects of cystatins should be investigated.