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

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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+- dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca2+ 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.