Characterization of Entamoeba histolytica α-actinin2

Abstract

We have cloned and characterized a second alpha-actinin isoform in Entamoeba histolytica. This protein, alpha-actinin2, has a N-terminal actin-binding domain, a C-terminal calcium-binding domain and an intervening rod domain containing two spectrin repeats. The protein binds and cross-links actin filaments in a calcium-dependent manner. Therefore alpha-actinin2 is a genuine alpha-actinin except for the shorter rod domain compared to the rod domain of isoforms of higher organisms. A alpha-actinin-like protein has previous been implicated in the adherence to the host cell and infection. It is therefore possible that alpha-actinin2 is involved in mechanism of infection, and in particular in reorganisation of the parasite's cytoskeleton that follows on adherence. E. histolytica alpha-actinin2 represents one of the first members of the spectrin superfamily where well defined spectrin repeats are found. The isolation and characterization of this second alpha-actinin isoform is valuable not only into the study of E. histolytica infection mechanisms, but also for understanding the evolution processes of the spectrin superfamily.

The domain structure of Entamoeba α-actinin2

“…When passed through a Sephacryl S400 HR column, full-length Entamoeba α-actinin2 eluted very close to a globular reference protein of 440 kDa, with a Stokes radius of 6.1 nm. Considering the shorter rod domain [28], this value compares favourably with the previously determined radii of chicken skeletal muscle and sea urchin α-actinins (7.7 nm) [39] and that of Acanthamoeba (7.4 nm) [40], which all have a longer rod domain composed of four spectrin repeats. The expressed peptides, with the exception of the ABD, eluted much earlier from the gel filtration column than expected for globular molecules of similar size (Fig.…”

“…At all the tested ratios of ABD-ROD to actin, bundles were observed, but the higher the ratio, the denser the bundles. These very dense bundles appeared very similar to those observed with full length α-actinin2 [28]. When actin filaments were incubated with ABD, no bundles could be seen.…”

“…This group of proteins is characterized by the presence of a highly conserved N-terminal actin-binding domain, a central rod domain and a calcium-binding C-terminus [25][26][27]. Previously, we identified, cloned and characterized two Entamoeba α-actinins (63 kDa and 70 kDa) [28,29]. These two isoforms display an unusual feature in their domain architecture: the rod domain is shorter and contains only one or two spectrin repeats, not four repeats as in all previous characterized α-actinins.…”

α-Actinin structure and regulation

“…The number of the repeats has changed during evolution and the rod appears to be the least conserved region of the a-actinin: all known vertebrate a-actinins contain four SR, while protozoan Entamoeba histolytica, the fungus Schizosaccharomyces pombe and the parasite Trichomonas vaginalis have one [25,26], two [27,28] or five [29] such repeats, respectively. The phylogenetic analysis shows that an a-actinin like precursor has given rise to the members of spectrin superfamily (A) Representation of the cytoskeleton in focal contacts showing a-actinin (red) linking actin (blue) to membrane-associated structures, like vinculin (dark green), talin (light green), integrin (brown) and tensin (purple).…”

“…The actin-binding property of the non-muscle aactinins is reduced or even abolished at calcium concentration of > 10 -7 M and pH > 7 [4, 107 -109]; at lower concentrations the viscosity of F-actin is significantly increased by a-actinin non-muscle isoforms [3, 16]. Recent biochemical and genetic studies on ancestral a-actinins from E. histolytica have shown that the calcium interferes with the ability of binding to actin filaments [26,27] and corroborated the thesis that the calcium binding and calcium non-binding forms evolved at the point of the vertebrate-invertebrate divergence during evolution [25,110]. Nevertheless, the atomic detail on the mechanism of calcium and a-actinin interaction is still unknown.…”