Identification and functional characterization of CsStefin-1, a cysteine protease inhibitor of Clonorchis sinensis

Cathepsin Fs of Clonorchis sinensis (CsCFs) are major secreted proteins that are expressed in the intestine of the parasite and play pivotal roles in parasite nutrition and host-parasite interactions. However, strict regulation of their activities is also essential to minimize inadequate superfluous damage to the parasite and host. In this study, we identified and characterized a novel cysteine protease inhibitor of C. sinensis, CsStefin-1, as a modulator of CsCFs. CsStefin-1 was shown to be a typical cysteine protease inhibitor of family 1 cystatins that lacks the N-terminal signal peptide and C-terminal cysteine residues required for disulfide bond formation. Phylogenetic and structural analyses also showed that CsStefin-1 is a family 1 intracellular cystatin. Bacterially expressed CsStefin-1 effectively inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, papain, and CsCFs. CsStefin-1 was active over a wide pH range and was highly stable under physiological conditions. CsStefin-1 also inhibited the processing of CsCFs. CsStefin-1 was expressed throughout various developmental stages of the parasite from metacercaria to adult worm and the protein was detected in worm extract, but not in the excretory and secretory products of adult worm. Immunolocalization analysis showed that CsStefin-1 was mainly localized to the intestinal epithelium, where CsCFs are actively synthesized. Our results collectively suggest the regulatory functions of CsStefin-1, modulation of CsCFs activity and processing, to protect the parasite from superfluous damage by the endogenous cysteine proteases.

199Highlights► CsStefin-1, a cysteine protease inhibitor of Clonorchis sinensis, was shown to be a typical cysteine protease inhibitor of family 1 cystatins. ► Bacterially expressed CsStefin-1 effectively inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, papain, and CsCFs. ► CsStefin-1 was active over a wide pH range and was highly stable under physiological conditions and inhibited the processing of CsCFs. ► CsStefin-1 was expressed throughout various developmental stages of the parasite from metacercaria to adult worm and the protein was detected in worm extract, but not in the excretory and secretory products of adult worm. ► Immunolocalization analysis showed that CsStefin-1 was mainly localized to the intestinal epithelium.