Comparative Characterization of Four Calcium-Binding EF Hand Proteins from Opisthorchis viverrini

Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2–42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21–23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca²⁺, Mg²⁺, Zn²⁺, and Cu²+. All OvCaBPs showed mobility shifts with Ca²⁺ and Zn²⁺. OvCaBP1 showed also positive results with Mg²⁺ and Cu²⁺. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.

Molecular Cloning of Plasmodium vivax Calcium-Dependent Protein Kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.