Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica

• Published in: The Journal of biological chemistry 2004-03, Vol.279 (13), p.12898-12908
• Main Authors: Chakrabarty, Paramita, Sethi, Dhruv K, Padhan, Narendra, Kaur, Kanwal J, Salunke, Dinakar M, Bhattacharya, Sudha, Bhattacharya, Alok
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - metabolism; Cell Division; Circular Dichroism; Entamoeba histolytica; Entamoeba histolytica - metabolism; Gene Library; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Peptides - chemistry; Phylogeny; Polymerase Chain Reaction; Protein Binding; Protein Conformation; Protein Isoforms; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Sequence Homology, Amino Acid; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfur-Sulfur Bond Isomerases - chemistry; Sulfur-Sulfur Bond Isomerases - metabolism; Surface Plasmon Resonance
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M304716200

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.