Molecular identification, immunolocalization, and characterization of Clonorchis sinensis calmodulin.

One cDNA clone (Cs18h09) encoding Clonorchis sinensis calmodulin (CsCaM) was isolated from our adult cDNA plasmid library. The open reading frame of CsCaM contains 450 bp which encodes 149 amino acids. CsCaM protein comprises four calcium-binding EF-hand motifs. The amino acid sequence of CsCaM shares very high homology with other species. Quantitative RT-polymerase chain reaction (PCR) revealed that CsCaM mRNA was constitutively transcribed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, recombinant CsCaM (rCsCaM) was expressed as a soluble protein and anti-rCsCaM rat serum could detect CsCaM in the C. sinensis somatic extracts but not in the C. sinensis excretory-secretory products (ESPs). Moreover, immunolocalization assay showed that CsCaM was located in tegument, intestine, pharynx, and eggs. Furthermore, rCsCaM was found to bind calcium ion (Ca2+) and magnesium (Mg2+) in electrophoretic mobility shift assay. Ca2+ binding increased the ability of rCsCaM to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, causing a blue shift in the fluorescence emission from 540 to 515 nm with an excitation wavelength of 380 nm and substantial increase in fluorescence intensity but not Mg2+. Collectively, here we showed the basic characterization of CsCaM and inferred that CsCaM could be a Ca2+ sensor protein, and CsCaM may possibly participate in growth and development of adult worm and egg of C. sinensis through binding Ca2+.