title: Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica.
abstract: Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.
authors.affiliation: School of Environmental Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi-110067, India.
authors.forename: Paramita
authors.initials: P
authors.lastname: Chakrabarty
authors.forename: Dhruv K
authors.initials: DK
authors.lastname: Sethi
authors.forename: Narendra
authors.initials: N
authors.lastname: Padhan
authors.forename: Kanwal J
authors.initials: KJ
authors.lastname: Kaur
authors.forename: Dinakar M
authors.initials: DM
authors.lastname: Salunke
authors.forename: Sudha
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Alok
authors.initials: A
authors.lastname: Bhattacharya
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002455:Cell Division; D002942:Circular Dichroism; D004748:Entamoeba histolytica / Q000378:metabolism*; D015723:Gene Library; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019749:Sulfur-Sulfur Bond Isomerases / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D002135:Calcium-Binding Proteins; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; C085578:Ca-binding protein 2; D019749:Sulfur-Sulfur Bond Isomerases