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Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein.

ABSTRACT: A novel 22.8 kDa of Opisthorchis viverrini (Ov) calcium-binding EF-hand protein (Ov CaBP) was identified and isolated from an immunoscreening of the adult stage Ov cDNA library by using a human cholangiocarcinoma (CCA) serum. This protein was related to other calcium-binding proteins and conserved among the trematodes. Ov CaBP shared 98% amino acid identity to 22.8 kDa of Clonorchis sinensis CaBP and both were classified as a new group of CaBP EF-hand protein by multiple sequence alignment and phylogenetic tree analysis. The open reading frame of Ov CaBP was 585 bp which encoded for 194 amino acids. The N-terminal part is composed of two calcium-binding EF-hand motifs whereas the C-terminal part contains a dynein light chain motif (DLC). In addition, transcription analysis by RT-PCR revealed that it was constitutively transcribed in all stages, including metacercariae, juvenile, and adult. Furthermore, recombinant Ov CaBP protein (rOv CaBP) was expressed as a soluble protein and antibody generated against this rOv CaBP protein was capable of detecting Ov CaBP in the Ov somatic extracts but not in Ov ES products. This anti-rOv CaBP serum was also used to localize Ov CaBP in Ov infected hamster's liver sections which the distribution of Ov CaBP was located in gut epithelium, miracidia in eggs and slightly in parenchyma. Moreover, rOv CaBP protein showed a calcium-binding property in non-denaturing gel mobility shift assay.

SUBMITTER: Senawong G

PROVIDER: S-EPMC3728565 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein.

Senawong Gulsiri G Laha Thewarach T Loukas Alex A Brindley Paul J PJ Sripa Banchob B

Parasitology international 20110719 1

A novel 22.8 kDa of Opisthorchis viverrini (Ov) calcium-binding EF-hand protein (Ov CaBP) was identified and isolated from an immunoscreening of the adult stage Ov cDNA library by using a human cholangiocarcinoma (CCA) serum. This protein was related to other calcium-binding proteins and conserved among the trematodes. Ov CaBP shared 98% amino acid identity to 22.8 kDa of Clonorchis sinensis CaBP and both were classified as a new group of CaBP EF-hand protein by multiple sequence alignment and p ...[more]

PMID: 21782972

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Project description:The EF hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca(2+) and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca(2+) regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-hand domain serve as coupled Ca(2+) sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to define their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.

Dataset Information

Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein.

Publications

Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein.

Similar Datasets

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