Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis , a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis

Background Clonorchis sinensis , the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase ( Cs PK), a crucial glycolytic enzyme, was characterized in this research. Results Differences were observed in the sequences and spatial structures of Cs PK and PKs from humans, rats, mice and rabbits. Cs PK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of Cs PK was 53.1 kDa. Recombinant Cs PK (r Cs PK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of Cs PK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. Cs PK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with r Cs PK. Also, rat anti-r Cs PK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. Conclusions The sequences and spatial structures, molecular mass, and expression profile of Cs PK have been characterized. r Cs PK was indicated to be a homopentamer. Rat anti-r Cs PK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. Cs PK is worthy of further study as a promising target for drug and vaccine development.