EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane inEntamoeba histolytica

Abstract

The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.Many cellular functions are dependent on specific proteinprotein interactions. These protein-protein interactions are governed by a variety of protein binding domains one of which is the LIM domain. Since first described nearly 2 decades ago (21, 28, 57), LIM domain-containing proteins have been identified in a wide range of eukaryotes, including protozoa (16,30,45), plants (5,20,40), and yeast (Saccharomyces cerevisiae) (39), stressing the evolutionary importance of these proteins. In the parasitic protozoan Entamoeba histolytica, a few studies have revealed the presence of such proteins (17, 36), although to date, no LIM domain-containing proteins have been characterized in this organism. The name LIM is derived from the three developmentally regulated transcription factors, LIN-11 of Caenorhabditis elegans (21), Isl1 of rat (28), and MEC-3 of C. elegans (57) in which the LIM domain was initially identified. The LIM domain is a cysteine-rich motif consisting of two zinc finger-like modules and displaying the consensus sequence CX 2 CX 16-23 HX 2 CX 2 CX 2 CX 16-21 CX 2 (C/H/D/) (31). This domain is found in a variety of different proteins with diverse functions, including transcription factors and cytoskeleton-associated proteins, and may be found in association with other functional domains, such as homeodomains, protein kinase domains, or other protein binding domains (...

New insights into Entamoeba histolytica pathogenesis

“…LIM domains are protein–protein interaction domains. E. histolytica LIM protein A (EhLIMA), the only LIM-domain protein in E. histolytica , is in both lipid raft and cytoskeletal fractions, suggesting that it may play a role in connecting the two, which may be important in regulating raft formation and movement [30]. …”

Entamoeba histolytica Encodes Unique Formins, a Subset of Which Regulates DNA Content and Cell Division

“…The E. histolytica genome encodes several cytoskeleton-associated proteins and signaling molecules that could regulate actin reorganization (35; TIGR Entamoeba histolytica Genome Project [http://www.tigr.org/tdb/e2k1/eha1/]; Pathema Bioinformatics Resource Center, Entamoeba [http: //pathema.jcvi.org/cgi-bin/Entamoeba/PathemaHomePage.cgi]). Some of them have been shown to participate in motility through pseudopod and uroid formation, capping, phagocytosis, interaction with extracellular matrix proteins, and signal transduction mechanisms (3,5,15,20,22,30,47,53,56,58,63,65). However, unlike those of many metazoan systems, the cytoskeleton components involved in cell division in E. histolytica are not fully understood.…”

Pathogenic Entamoeba histolytica, but not Entamoeba dispar, induce neutrophil extracellular trap (NET) formation

“…Another protein more highly expressed in E. histolytica than in E. dispar is EhLimA . Its function is not clear, but it is known to be expressed on the plasma membrane and to connect with the actin cytoskeleton . This molecule is an interesting candidate for the possible virulence factor responsible for triggering NETosis, because a functional actin cytoskeleton in E. histolytica was found to be relevant for this response (Fig.…”