calcium-binding protein 1 of entamoeba histolytica transiently associates with phagocytic cups in a calcium-independent manner

EhCaBP1, a calcium-binding protein of the parasite Entamoeba histolytica, is known to participate in cellular processes involving actin filaments. This may be due to its direct interaction with actin. In order to understand the kinetics of EhCaBP1 in such processes, its movement was studied in living cells expressing GFP-EhCaBP1. The results showed that EhCaBP1 accumulated at phagocytic cups and pseudopods transiently. The time taken for appearance and disappearance of EhCaBP1 was found to be around 12 s. Site-directed mutagenesis was used to generate an EhCaBP1 mutant with reduced Ca2+- and G-actin binding ability without any defect in its ability to bind F-actin. The overexpression of this mutant EhCaBP1 in the E. histolytica trophozoites resulted in the impairment of erythrophagocytosis, uptake of bacterial cells, killing of target cells but not fluid-phase pinocytosis. However, the mutant protein was still found to transiently localize with f-actin at the phagocytic cups and pseudopods. The mutant protein displayed reduced ability to activate endogenous kinase(s) suggesting that phagosome formation may require Ca2+-EhCaBP1 transducing downstream signalling but initiation of phagocytosis may be independent of its intrinsic ability to bind Ca2+. The results suggest a dynamic association of EhCaBP1 with F-actin-mediated processes

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

Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V

Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported

Prediction and Analysis of Canonical EF Hand Loop and Qualitative Estimation of Ca²⁺ Binding Affinity

<div><p>The diversity of functions carried out by EF hand-containing calcium-binding proteins is due to various interactions made by these proteins as well as the range of affinity levels for Ca²⁺ displayed by them. However, accurate methods are not available for prediction of binding affinities. Here, amino acid patterns of canonical EF hand sequences obtained from available crystal structures were used to develop a classifier that distinguishes Ca²⁺-binding loops and non Ca²⁺-binding regions with 100% accuracy. To investigate further, we performed a proteome-wide prediction for <i>E. histolytica</i>, and classified known EF-hand proteins. We compared our results with published methods on the E. <i>histolytica</i> proteome scan, and demonstrated our method to be more specific and accurate for predicting potential canonical Ca²⁺-binding loops. Furthermore, we annotated canonical EF-hand motifs and classified them based on their Ca²⁺-binding affinities using support vector machines. Using a novel method generated from position-specific scoring metrics and then tested against three different experimentally derived EF-hand-motif datasets, predictions of Ca²⁺-binding affinities were between 87 and 90% accurate. Our results show that the tool described here is capable of predicting Ca²⁺-binding affinity constants of EF-hand proteins. The web server is freely available at <a href="http://202.41.10.46/calb/index.html" target="_blank">http://202.41.10.46/calb/index.html</a>.</p></div

The calcium binding protein EhCaBP6 is a microtubular-end binding protein in Entamoeba histolytica

The genome of Entamoeba histolytica encodes several calcium binding proteins and those characterized thus far have been shown to participate predominantly in phagocytosis and endocytosis. Our study showed that EhCaBP6 has two EF-hand domains EFI and EFIII; it can bind Ca Ca 2+ in vitro and undergoes conformational transition on binding Ca 2+ suggesting that it can serve as a calcium signal sensor. EhCaBP6 is localized in the nucleus, cytoplasm and plasma membrane and is sensitive to heat stress. Unlike other Ca 2+ binding proteins that have been studied in E. histolytica, EhCaBP6 is found at microtubule ends and at the intercellular bridge with the microtubules during cytokinesis. Furthermore, increased expression of EhCaBP6 was correlated with a significant increase in the number of microtubular structures suggesting that this protein may regulate chromosome segregation and cytokinesis in E. histolytica

The Performance of SVM Models with different learning parameters on D1 and D2 dataset.

<p>Using binary patterns and AA (amino acid) composition [γ <b>(g)</b> (in RBF kernel), c: parameter for trade-off between training error & margin] where SN–sensitivity, SP–specificity, ACC-accuracy, MCC–Matthews Correlation Coefficient.</p

Amino acid composition of the 12-mer long Ca²⁺-binding region (“Interacting”) and the non-binding region (“Non-Interacting”) of EF-hand proteins.

<p>Amino acid composition of the 12-mer long Ca²⁺-binding region (“Interacting”) and the non-binding region (“Non-Interacting”) of EF-hand proteins.</p

The Performance of SVM Models on validation dataset with experimentally derived binding affinity from EhCaBPs (D7).

<p>The Performance of SVM Models on validation dataset with experimentally derived binding affinity from EhCaBPs (D7)with different learning parameters on various hybrid models [γ (g) (in RBF kernel), c: parameter for trade-off between training error & margin] where SN–sensitivity, SP–specificity, ACC-accuracy, MCC–Matthews Correlation Coefficient, AUC/ROC-Area under curve/ Receiver Operating Curve.</p

The Performance of SVM Models on PSSM based training dataset D3 & D4.

<p>The Performance of SVM Models on PSSM based training dataset D3 & D4 with different learning parameters on various hybrid models [γ (g) (in RBF kernel), c: parameter for trade-off between training error & margin] where SN–sensitivity, SP–specificity, ACC-accuracy, MCC–Matthews Correlation Coefficient, AUC/ROC-Area under curve/ Receiver Operating Curve.</p

Summary of macroscopic binding constants and thermodynamic parameters obtained from the ITC studies of Ca²⁺-binding isotherm of EhCaBPs at 25°C.

<p>Summary of macroscopic binding constants and thermodynamic parameters obtained from the ITC studies of Ca²⁺-binding isotherm of EhCaBPs at 25°C.</p