Molecular and biochemical analysis of 8 kDa calcium-binding protein in Clonorchis sinensis
간흡충 8 kDa 칼슘 결합 단백질의 분자·생화학적 분석
Dept. of Medical Science/석사
Clonorchis sinensis is a parasitic helminth distributed through East Asia including Korea, Japan, China, Taiwan, and Vietnam. The infection with the flukes induces biliary cirrhosis followed by fibrosis of surrounding tissues, and even occasionally cholangiocarcinoma. Calcium-binding protein is common, and pivotal protein which is implicated in the muscle contraction and signal transduction in most of organisms. However, calcium-binding protein of C. sinensis has not reported thus far. In this study, a cDNA clone encoding a calcium-binding protein was isolated from C. sinensis and entitled CsCaBP. Deduced amino acid sequence of CsCaBP showed around 50% of sequence homologies with calcium-binding proteins of the other trematodes. The Pfam database search showed that the CsCaBP sequence has two canonical EF hands commonly seen in calmodulin. To understand the structural basis of CsCaBP, homology modeling was performed by Geno3D and SWISS-MODEL. Both of calcium-binding site I and II were located on the surface of CsCaBP. Estimated structure of calcium-loaded state was different from that of apo-state. The recombinant CsCaBP (rCsCaBP) was produced in Escherichia coli and purified by Ni-NTA affinity chromatography to homogeneity. Molecular mass of rCsCaBP confirmed by SDS-PAGE was 12 kDa as predicted by sequence analysis. Native gel mobility shift assay performed with rCsCaBP incubated with metal ions showed calcium-specific binding affinity of rCsCaBP. The rCsCaBP-immunized mouse sera reacted with rCsCaBP and native CsCaBP in adult C. sinensis worm extract. Distribution of native CsCaBP in adult C. sinensis worm was visualized by immunostaining. The most of CsCaBP was found in oral and ventral suckers, vitelline follicles and subtegumental regions.Collectively, the CsCaBP might be concerned with parasite’s movement and muscle contraction of suckers for the attachment to its habitat in the host. The present study provides structural and biological basis for the development of new anthelmintic molecules.