Abstract
Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.
Highlights
Vector, aflagellated amastigote forms primarily exist within the phagolysosomal complex of the mammalian macrophages [2]
It has recently been shown that depletion of the cytoskeleton protein, actin, in the bloodstream form of Trypanosoma brucei inhibited endocytosis that subsequently resulted in cell death, suggesting that actin is indispensable during this process [6]
This protein is well characterized in a number of organisms including protozoans, like Entamoeba [8], Tetrahymena [9, 10], Paramecium [11], Dictyostelium [12], Plasmodium [13, 14], and Toxoplasma [15], little is known about the biochemical properties and cellular functions of actin present in trypanosomatid parasites, such as Leishmania and Trypanosoma [6, 16]
Summary
Sf9 insect cells (Invitrogen) were maintained at 27 °C as monolayer cultures in TNM-FH medium (Invitrogen). Transfected Sf9 cells were assayed 96 h postinfection for the expression of LdACT-His (rLdACT) by 10% SDSPAGE and Western blot analysis using mouse monoclonal antibodies against hexahistidine (Amersham Biosciences) as well as rabbit monospecific polyclonal antibodies against LdACT, followed by HRP-conjugated goat anti-mouse IgG/anti-rabbit IgG. Transfected cells were further selected in the growth medium containing tunicamycin up to 20 g/ml and maintained in the same conditions Lysates of both p6.5-LdACT-and p6.5-transfected promastigotes prepared by lysing equal number of cells were separated by 10% SDS-PAGE, transblotted onto polyvinylidene fluoride membrane, washed, and blocked, as described previously [16, 23], and probed with rabbit antiLdACT antibodies as well as mice anti-GRP78 antibodies (protein loading control) followed by HRP-conjugated goat antimouse IgG and anti-rabbit IgG. Cells were treated with rabbit anti-LdACT antibodies, followed by fluorescein isothiocyanate-conjugated antirabbit IgG, and analyzed by using a FACS Calibur (BD Biosciences) flow cytometer using Cell Quest support software for acquisition and analysis
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