Secondary structure of a calcium binding protein (CaBP) from Entamoeba histolytica.

A calcium binding protein from Entamoeba histolytica, (EhCaBP, M(r) approximately 15 kDa) is the causative agent for amoebiosis and has a very low sequence homology (approximately 30%) with other known CaBPs. Almost complete sequence specific resonance assignments for (1)H, (13)C and (15)N spins in EhCaBP were obtained using double and triple resonance NMR experiments. Qualitative interpretation of the nuclear Overhauser enhancements, chemical shift indices and of hydrogen exchange rates threw valuable light upon the secondary structure of this protein. CaBP is found to have two globular domains each of which consists of two pairs of helix-loop-helix motifs. Though this protein has a very small sequence homology with calmodulins, the topological arrangement of the alpha-helices and beta-strands in EhCaBP resemble them.     

Ehhmbp45 is a novel hemoglobin-binding protein identified in Entamoeba histolytica

Hemoglobin-binding proteins are necessary for pathogens to obtain iron from Hb. Entamoeba histolytica can grow using Hb as source of iron, but the underlying mechanism has not previously been established. In this work, we identified a 45 kDa Hb-binding protein of E. histolytica, which we named Ehhmbp45. In silico analysis showed that Ehhmbp45 contains the conserved domains needed for Hb-binding, while overlay assays demonstrated that Ehhmbp45 is able to bind Hb. In addition, we found that Ehhmbp45 mRNA levels were up-regulated under iron starvation conditions and were subsequently restored to basal levels when Hb was added to the cell cultures. These findings provide the first insights on the role of Ehhmbp45 in iron acquisition from Hb.     

The Calmodulin-like Calcium Binding Protein EhCaBP3 of Entamoeba histolytica Regulates Phagocytosis and Is Involved in Actin Dynamics

Phagocytosis is required for proliferation and pathogenesis of Entamoeba histolytica and erythrophagocytosis is considered to be a marker of invasive amoebiasis. Ca²⁺ has been found to play a central role in the process of phagocytosis. However, the molecular mechanisms and the signalling mediated by Ca²⁺ still remain largely unknown. Here we show that Calmodulin-like calcium binding protein EhCaBP3 of E. histolytica is directly involved in disease pathomechanism by its capacity to participate in cytoskeleton dynamics and scission machinery during erythrophagocytosis. Using imaging techniques EhCaBP3 was found in phagocytic cups and newly formed phagosomes along with actin and myosin IB. In vitro studies confirmed that EhCaBP3 directly binds actin, and affected both its polymerization and bundling activity. Moreover, it also binds myosin 1B in the presence of Ca²⁺. In cells where EhCaBP3 expression was down regulated by antisense RNA, the level of RBC uptake was reduced, myosin IB was found to be absent at the site of pseudopod cup closure and the time taken for phagocytosis increased, suggesting that EhCaBP3 along with myosin 1B mediate the closure of phagocytic cups. Experiments with EhCaBP3 mutant defective in Ca²⁺ -binding showed that Ca²⁺ binding is required for phagosome formation. Liposome binding assay revealed that EhCaBP3 recruitment and enrichment to membrane is independent of any cellular protein as it binds directly to phosphatidylserine. Taken together, our results suggest a novel pathway mediating phagocytosis in E. histolytica, and an unusual mechanism of modulation of cytoskeleton dynamics by two calcium binding proteins, EhCaBP1 and EhCaBP3 with mostly non-overlapping functions.     

Entamoeba histolytica: transferrin binding proteins

Entamoeba histolytica trophozoites depend on iron for their growth; thus, they must use some host iron-containing molecules to fulfill this requirement. In this work we report that amoebas are able to utilize human holo-Tf as iron source and to recognize it through transferrin binding proteins. By use of an anti-human transferrin antiserum in an immunoblotting assay, two main polypeptides with apparent molecular masses of 70 and 140 kDa were found in total extract of trophozoites cultured in vitro. However, when a monoclonal anti-human transferrin receptor antibody was used, only one band with molecular mass of 140 kDa was observed. Both the human transferrin and the monoclonal antibody recognized a protein on the amoebic surface, demonstrated by confocal microscopy. Furthermore, the complex transferrin-transferrin binding protein was internalized by an endocytic process and probably dissociated inside the cell. This mechanism could be one manner in which E. histolytica acquires iron from the human host transferrin.     

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 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.     

Diversity of phosphoinositide binding proteins in Entamoeba histolytica

Phosphatidylinositol phosphates (PIPs, phosphoinositides) are localized to the membranes of all cellular compartments, and play pivotal roles in multiple cellular events. To fulfill their functions, PIPs that are located to specific organelles or membrane domains bind to and recruit various proteins in spatiotemporal specific manner via protein domains that selectively bind to either a single or an array of PIPs. In Entamoeba histolytica, the human intestinal protozoan parasite, PIPs and PIP-binding proteins have been shown to be involved in their virulence-associated mechanisms such as cell motility, vesicular traffic, trogo- and phagocytosis. In silico search of the domains and the signatures implicated in PIP binding in the E. histolytica proteome allows identification of dozens of potential PIP-binding proteins. However, such analysis is often misleading unless the protein domain used as query is cautiously selected and the binding specificity of the proteins are experimentally validated. This is because all the domains initially presumed to bind PIPs in other systems are not always capable of PIP binding, but rather involved in other biological roles. In this review, we carried out in silico survey of proteins which have PIP-binding domains in the E. histolytica genome by utilizing only validated PIP-binding domains that had been experimentally proven to be faithful PIP-binding bioprobes. Our survey has identified that FYVE (Fab1, YOTB1, Vac1, EEA1) and PH (pleckstrin homology) domain containing proteins are the most expanded families in E. histolytica. A few FYVE domain-containing proteins (EhFP4 and 10) and phox homology (PX) domain containing proteins (EhSNX1 and 2) were previously studied in depth in E. histolytica. Furthermore, most of the identified PH domain-containing proteins are annotated as protein kinases and possess protein kinase domains. Overall, PIP-binding domain-containing proteins that can be identified by in silico survey of the genome using the domains from well characterized bioprobes are limited in E. histolytica. However, their domain architectures are often unique, suggesting unique evolution of PIP-binding domain-containing proteins in this organism.     

Growth stimulation by serum in Entamoeba histolytica is associated with protein tyrosine dephosphorylation

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2-60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.     

Apolipoprotein B is a calcium binding protein

Human hepatocarcinoma Hep G2 cells were grown in culture medium containing [45Ca2+]. The secreted lipoproteins of d less than 1.063 g/ml and d 1.063-1.21 g/ml were isolated from the culture media and analyzed by 3.3% and 7% SDS-polyacrylamide gel electrophoresis. Radioactivity profiles of [45Ca] from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated [45Ca2+] and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein.     

Entamoeba histolytica: biochemical characterization of a protein disulfide isomerase

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy.     

Two isotypes of phosphatidylinositol 3‐phosphate‐binding sorting nexins play distinct roles in trogocytosis in Entamoeba histolytica

Phosphatidylinositol phosphates (PIPs) function as important second messengers in many cellular events. In the human intestinal protist Entamoeba histolytica, where phagocytosis/trogocytosis plays an indispensable role in proliferation and pathophysiology during infection, various PIPs are involved in multiple steps of phago/trogocytosis. PI3‐phosphate (PI3P) plays a pivotal role in the biogenesis of phagosome/trogosomes via recruitment of PI3P effectors. Because no known PI3P downstream effectors are conserved in E. histolytica, we exploited a unique method to identify the proteins PI3P dependently recruited to phagosomes. We rationalised that overexpression of PI3P‐binding GFP‐HrsFYVE competes for PI3P on phagosomal membranes and results in dissociation of PI3P effectors from phagosomes. EhVps26 and EhVps35, but not sorting nexins (SNXs), of the retromer complex were detected from phagosomes only without GFP‐HrsFYVE overexpression. Two potential SNXs, EhSNX1 and EhSNX2, identified in the genome, possess only phox homology domain and specifically bound to PI3P, but retromer components, EhVps26 and EhVps35, did not bind to PI3P. Live and immunofluorescence imaging showed that EhSNX1 was recruited to the trogocytic cup and tunnel‐like structures, and subsequently, EhSNX2 was recruited to trogosomes. Furthermore, EhSNX1, but not EhSNX2, specifically bound to Arp2/3 and EhVps26, which were localised to the tunnel‐like structures and the trogosomes, respectively. EhSNX2 gene silencing increased trogocytosis, suggesting that EhSNX2 plays an inhibitory role in trogocytosis.     

Crystal structure of calcium binding protein-1 from Entamoeba histolytica: a novel arrangement of EF hand motifs

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Several EF-hand containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though these proteins have very high sequence similarity, they bind to different target proteins in a Ca2+ dependent manner, leading to different functional pathways (Yadava et al., Mol Biochem Parasito 1997;84:69-82; Chakrabarty et al., J Biol Chem 2004;279:12898-12908) The crystal structure of the Entamoeba histolytica calcium binding protein-1 (EhCaBP1) has been determined at 2.4 A resolution. The crystals were grown using MPD as precipitant and they belong to P6(3) space group with unit cell parameters of a = 95.25 A, b = 95.25 A, c = 64.99 A. Only two out of the four expected EF hand motifs could be modeled into the electron density map and the final model refined to R factor of 25.6% and Free_R of 28%. Unlike CaM, the first two EF hand motifs in EhCaBP1 are connected by a long helix and form a dumbbell shaped structure. Owing to domain swapping oligomerization three EhCaBP1 molecules interact in a head to tail manner to form a triangular trimer. This arrangement allows the EF-hand motif of one molecule to interact with that of an adjacent molecule to form a two EF-hand domain similar to that seen in the N-terminal domain of the NMR structure of CaBP1, calmodulin and troponin C. The oligomeric state of EhCaBP1 results in reduced flexibility between domains and may be responsible for the more limited set of targets recognized by EhCaBP1.     

Analysis of the protein kinome of Entamoeba histolytica

Protein kinases play important roles in almost all major signaling and regulatory pathways of eukaryotic organisms. Members in the family of protein kinases make up a substantial fraction of eukaryotic proteome. Analysis of the protein kinase repertoire (kinome) would help in the better understanding of the regulatory processes. In this article, we report the identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica. Using a combination of various sensitive sequence search methods and manual analysis, we have identified a set of 307 protein kinases in E. histolytica genome. We have classified these protein kinases into different subfamilies originally defined by Hanks and Hunter and studied these kinases further in the context of noncatalytic domains that are tethered to catalytic kinase domain. Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organization and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like typical calcium/calmodulin dependent kinases as they lack noncatalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of kinases in E. histolytica. Interestingly, a PKA/PKG-like subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclin-dependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual features recognized in our analysis include the absence of MEK as a part of the Mitogen Activated Kinase signaling pathway and identification of transmembrane region containing Src kinase-like kinases. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are Cysteine-rich and to domains known to be involved in protein-protein interactions. Our kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual kinases.     

Structural characterization of the apo form of a calcium binding protein from Entamoeba histolytica by hydrogen exchange and its folding to the holo state

One of the calcium binding proteins from Entamoeba histolytica (EhCaBP) is a 134 amino acid residue long (M(r) approximately 14.9 kDa) double domain EF-hand protein containing four Ca(2+) binding sites. CD and NMR studies reveal that the Ca(2+)-free form (apo-EhCaBP) exists in a partially collapsed form compared to the Ca(2+)-bound (holo) form, which has an ordered structure (PDB ID ). Deuterium exchange studies on the partially structured apo-EhCaBP reveal that the C-terminal domain is better structured than the N-terminal domain. The protein can be reversibly folded and unfolded upon addition of Ca(2+) and EGTA, respectively. Titration shows a slow initial folding of the apo form with increasing Ca(2+) concentration, followed by a highly cooperative folding to its final state at a certain threshold of Ca(2+). Ca(2+) and the EGTA titration taken together show that site II in the N-terminal domain has the highest affinity for Ca(2+) contrary to earlier studies. Further, this study has thrown light on the relative Ca(2+) binding affinity and specificity of each site in the intact protein. A structural model for the partially collapsed form of apo-EhCaBP and its equilibrium folding to its completely folded holo state has been suggested. Large conformational changes seen in transforming from the apo to holo form of EhCaBP suggest that this protein should be functioning as a sensor protein and might have a significant role in host-parasite recognition.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.     

The actin cytoskeleton orchestra in Entamoeba histolytica

Years of evolution have kept actin conserved throughout various clades of life. It is an essential protein starring in many cellular processes. In a primitive eukaryote named Entamoeba histolytica, actin directs the process of phagocytosis. A finely tuned coordination between various actin-binding proteins (ABPs) choreographs this process and forms one of the virulence factors for this protist pathogen. The ever-expanding world of ABPs always has space to accommodate new and varied types of proteins to the earlier existing repertoire. In this article, we report the identification of 390 ABPs from Entamoeba histolytica. These proteins are part of diverse families that have been known to regulate actin dynamics. Most of the proteins are primarily uncharacterized in this organism; however, this study aims to annotate the ABPs based on their domain arrangements. A unique characteristic about some of the ABPs found is the combination of domains present in them unlike any other reported till date. Calponin domain-containing proteins formed the largest group among all types with 38 proteins, followed by 29 proteins with the infamous BAR domain in them, and 23 proteins belonging to actin-related proteins. The other protein families had a lesser number of members. Presence of exclusive domain arrangements in these proteins could guide us to yet unknown actin regulatory mechanisms prevalent in nature. This article is the first step to unraveling them.     

EhMAPK, the mitogen-activated protein kinase from Entamoeba histolytica is associated with cell survival

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H(2)O(2) treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H(2)O(2) treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H(2)O(2) treatment reduced parasite viability significantly but heat shock and 0.5 mM H(2)O(2) treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified.     

Entamoeba histolytica Encodes a Highly Divergent β-Tubulin

ABSTRACT. The microtubules of the amitochondrial parasite Entamoeba histolytica are atypical in certain respects. Consistent with this, we report that E. histolytica encodes the most divergent β -tubulin identified to date, with only 54% to 58% identity to β -tubulins from various species. A similarly divergent β -tubulin is encoded by the related Entamoeba invadens ; single gene copies appear to be present in both organisms. The Entamoeba sequences were compared with a database of 101 β -tubulins, including the highly divergent sequence from another amitochondrial protozoan, Trichomonas vaginalis. A total of 81 residues were universally conserved, and 76 residues varied only once. Correlations with previous studies indicate that microtubule function is altered when most, but not all, conserved residues are mutated.