Characterization of Entamoeba histolytica α-actinin

Abstract

We have cloned, expressed and characterized a α-actinin-like protein of Entamoeba histolytica. Analysis of the primary structure reveals that the essential domains of the α-actinin protein family are conserved: an N-terminus actin-binding domain, a C-terminus calcium-binding domain and a central helical rod domain. However, the rod domain of this Entamoeba protein is considerably shorter than the rod domain in α-actinins of higher organisms.

The cloned Entamoeba 63 kDa protein is recognized by conventional α-actinin antibodies as well as binds and cross-links filamentous actin and calcium ions in the same manner as α-actinins. Despite the shorter rod domain this protein has conserved the most important functions of α-actinins. Therefore, it is suggested that this 63 kDa protein is an atypical and ancestral α-actinin.

Introduction

Entamoeba histolytica infection is estimated to cause between 50,000 and 100,000 deaths every year primarily in certain tropical and subtropical areas. Thereby is E. histolytica induced amoebiasis the third most common parasitic disease in humans after malaria and schistosomiasis [1], [2], [3]. Only one of the Entamoeba species humans can host is known to cause disease. The clinical manifestation of parasite infection is amoebic colitis as well as amoebic liver abscess and other extraintestinal lesions due to the spread of the parasite via the blood to other organs [4], [5], [6].

Due to the high incident of infection the study of the E. histolytica life cycle as well as the strategies and regulation of the invasion is of a great importance. The sequencing of the entire E. histolytica genome has been one of the most important approaches to understand and characterize the mechanism and proteins involved in infection [7].

The attachment and penetration of the parasite into the host cells requires not only a Gal/GalNAc lectin [8] but also reorganisation of cytoskeletal and actin-binding proteins [9], [10], [11] as well as extracellular cysteine proteases [12]. Some of these E. histolytica proteins have been characterized, such as the calmodulin-like protein [13], myosin [14], [15], [16] and actin-binding proteins EhABPH and ABP-120 [17], [18]. Although these proteins share some of the structural and functional properties with isoforms of higher eukaryotes they also have some atypical features. Indicating that some of its proteins may have remained unique or invariable during evolution.

Previously we have found a possible actin-binding protein in the genome of E. histolytica, with high similarity to α-actinins. Phylogenetic analysis placed this α-actinin-like protein at the bottom of the evolution and identifying it as a possible ancestor to modern α-actinins [19]. α-Actinins is a ubiquitous actin-binding protein, present in most eukaryotic cells with the exception of plants and baker's yeast (Saccharomyces cervisiae) [19]. The hallmark of α-actinin is the presence of three distinct structural domains: an N-terminus actin-binding domain composed of two calponin homology domains, a central rod domain with four spectrin repeats and a C-terminus calcium-binding domain with EF-hand motifs [20], [21]. The actin-binding domain, and to a slightly lesser extent, the calcium-binding domain have been conserved during evolution. The rod domain is the most variable part; the amino acid sequence varies not only among the different isoforms but also among the different spectrin repeats. In some primitive organisms like protozoa and yeast (Schizosaccharomyces pombe) the rod domain contains only one or two spectrin repeats instead of the usual four repeats [19].

Due to the ability to form antiparallel dimers, α-actinin is able to cross-link actin filaments in a wide number of structures such as Z-disc, adhesion plaques, stress fibers and at the leading edge in motile cells [21]. In protozoan such as Acantamoeba and Trichomonas vaginalis, α-actinin is located mainly in the cortical areas mediating morphological changes during the infection [22], [23] whereas in yeast (S. pombe), α-actinin appears to play an important role in the formation of the medial ring in cytokinesis [24]. Recently it has been shown that an Entamoeba α-actinin is attached to the Gal/GalNAc lectin, which is known to be important for the pathogenesis of the parasite [8].

Structure analysis of the E. histolytica α-actinin-like protein have predicted a protein with a conserved actin-binding domain with two calponin homology domains and a calcium-binding domain with three putative EF-hands. The predicted rod domain connecting these two domains is considerably shorter (ca. 145 residues) than in typical α-actinins.

The phylogeny placed this α-actinin-like protein as one of the most ancestral α-actinins and suggested that the four spectrin repeats present in modern α-actinins arose after two consecutive intragenic duplications from a single repeat α-actinin. In addition, the phylogenetic analysis suggested that the rod domain is most similar to the first spectrin repeat of chicken α-actinin [19].

Here we report the cloning, expression and characterization of this α-actinin-like protein of E. histolytica. Biochemical analysis showed that the cloned 63 kDa protein is recognized by conventional α-actinin antibodies as well as binds filamentous actin and calcium ions in the same manner as α-actinins. Thus, despite the shorter rod domain this protein has conserved the most important functions of α-actinins.