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

Abstract

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, Opisthorchis viverrini. 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 Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1–6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3–6 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 OvCys1, 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, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 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 Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated.

Molecular and biochemical characterizations of a Fasciola gigantica retinoid X receptor-α isoform A (FgRXRα-A)

“…All collected fractions were run on 12.5% SDS-PAGE and stained with Coomassie brilliant blue G-250 for evaluation. At the same time, the recombinant thioredoxin (rTrx) was produced from BL21(DE3) E. coil containing pET32a(+) and purified under native conditions as previously described 17 , 35 , 36 . The purified r Fg RXRα-A + Trx and rTrx were transferred to a micro-dialysis bag (Sigma-Aldrich, St. Louis, MO, USA) for dialysis against PBS, pH 7.4.…”