Molecular and biochemical analysis of novel serine protease inhibitors from the human liver fluke Opisthorchis viverrini

Serine protease inhibitors or serpins are a broadly distributed superfamily of protease inhibitors. Serpins play key roles in a variety of physiological and cellular functions and are, for example, associated with the blood coagulation cascade, complement activation, inflammation and immune response in humans. In helminthiases, parasite serpins might be utilized to alter or disrupt host immune functions in order to survive. In this study two serpin genes were identified in the genome of the human liver fluke Opisthorchis viverrini and their coding sequences were molecular cloned by reverse transcription PCR. The deduced amino acid sequences of Ov-SLS (Serpin Late Stage) and Ov-SIS (Serpin Infectious Stage) contain 405 and 403 amino acid residues, respectively. A multiple sequence alignment with serpins from other parasite species confirmed conservation of residues essential for function and folding, e.g., reactive loop center (RCL). Ov-SLS RNA was detected in 4-week-old juveniles and in mature parasites while Ov-SIS was found in newly excysted juvenile worms. Recombinant Ov-SLS and Ov-SIS were successfully expressed in Escherichia coli, purified by affinity chromatography, and used to generate polyclonal antibodies in mice. In immunoblot analysis, Ov-SLS was found in soluble crude worm extract of mature parasites and Ov-SIS in insoluble extract of 2-week-old juveniles by dot blot analysis. Functional analysis of recombinant Ov-SLS and Ov-SIS with human serine proteases found low binding of Ov-SLS to kallikrein and moderate binding of Ov-SIS to thrombin as well as low binding with kallikrein and chymotrypsin. Additionally, human opisthorchiasis sera showed significant reactivity to recombinant Ov-SIS, whereas they were not reactive to recombinant Ov-SLS. Moreover, both recombinant serpins interfered slightly the intrinsic coagulation pathway. These results suggest that Ov-SIS is important in the early stage of infection and may serve as a diagnostic marker for opisthorchiasis. Both serpins may play a minor role in interfering with the host’s blood coagulation, possibly aiding parasite survival.