ABSTRACT
Toxoplasma gondii belongs to the Apicomplexa phylum that caused a widespread zoonotic infection in wide range of intermediate hosts. Over one-third of the world's population are latently infected with T. gondii and carry it. The complex life cycle of T. gondii indicates the presence of a plurality of antigenic epitopes. During the recent years, continuous efforts of scientists have made precious advances to elucidate the different aspects of the cell and molecular biology of T. gondii. Despite of great progresses, the development of vaccine candidates for preventing of T. gondii infection in men and animals is still remains a challenge. The calcium-dependent protein kinases (CDPKs) belongs to the superfamily of kinases, which restricted to the apicomplexans, ciliates, and plants. It has been documented that they contribute several functions in the life cycle of T. gondii such as gliding motility, cell invasion, and egress as well as some other critical developmental processes. In current paper, we reviewed the recent progress concerning the development of CDPK-based vaccines against acute and chronic T. gondii.
Subject(s)
Animals , Humans , Male , Apicomplexa , Cell Movement , Epitopes , Immunization , Life Cycle Stages , Molecular Biology , Phosphotransferases , Protein Kinases , Toxoplasma , Vaccines , ZoonosesABSTRACT
LeCPK2 (GenBank GQ205414), a versatile calcium-dependent protein kinase (CDPK or CPK) gene was isolated from tomato in our previous study. In this study, the biochemical properties of LeCPK2 were further investigated. To examine the role of the C-terminal calmodulin-like domain (CLD) of LeCPK2 with respect to Ca2+ activation, the kinase activities of recombinant full-length and truncated LeCPK2 were measured by Kinase-Glo® Luminescent kinase assay (Promega). The results showed that LeCPK2 activity was Ca2+-dependent and the C-terminal CLD of 161 residues was essential for the activation of LeCPK2. The activity of LeCPK2 was sharply stimulated by Ca2+ with K0.5 (concentration of Ca2+ for half-maximal activity) of 48.8 and 45.5 nM with substrate histone IIIs and syntide 2, respectively. The optimal concentration of Mg2+ for LeCPK2 activity was 20 and 10 mM for substrate histone IIIs and syntide 2, respectively. The Km value of LeCPK2 towards histone IIIs and syntide 2 was 44.9 μg/ml and 89.52 μM, respectively. The determination of biochemical properties of LeCPK2 would provide some clues on how its activity was regulated in vivo.
Subject(s)
Amino Acid Sequence , Calcium Chloride/chemistry , Solanum lycopersicum/enzymology , Solanum lycopersicum/genetics , Magnesium Chloride/chemistry , Molecular Sequence Data , Protein Kinases/analysis , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Kinases/metabolism , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
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.