Immunolocalization of Falstatin

<div><p>(A) Immunofluorescence microscopy. Erythrocytes infected with synchronized 3D7 or W2 parasites were collected every 8 h, stained with DAPI and anti-falstatin antibodies and FITC-second antibody, and then evaluated by immunofluorescence microscopy.</p><p>(B) Immunoelectron microscopy. Late-schizont stage parasites were incubated with anti-falstatin antibodies and gold-conjugated second antibody and then evaluated by electron microscopy. Labels show individual merozoites (M) and erythrocyte cytosol (EC).</p></div

Inhibitor Competition

<p>The indicated amounts of falstatin and anti-falstatin antibody were incubated with lysates from asynchronous parasite cultures before addition of [¹²⁵I] DCG04, electrophoresis, and analysis by autoradiography. Results with increasing concentrations of falstatin (A), increasing concentrations of antibody (B), and increasing falstatin in the presence of antibody (C) are shown. Labels above the gels represent concentrations of falstatin and antibody (μg/ml). Proteins are labeled based on known migration patterns that were previously confirmed by mass spectrometry. FP, falcipain; DPAP1, dipeptidyl aminopeptidase1[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020117#ppat-0020117-b028" target="_blank">28</a>].</p

Stage-Specific Expression of Falstatin

<p>Extracts from highly synchronized parasites were collected every 8 h, separated by SDS-PAGE, and evaluated by immunoblotting with anti-falstatin antibodies. Each sample of early-ring, late-ring, early-trophozoite, late-trophozite, early-schizont, or late-schizont extracts corresponded to 1.3 × 10⁷ parasitized cells. The positions of molecular weight markers (kDa) are indicated. ER, early-ring; LR, late-ring; ET, early-trophozite; LT, late-trophozite; ES, early-schizont; LS, late-schizont.</p

Expression and Purification of Falstatin

<p>Falstatin was expressed in E. coli and purified by Ni-NTA affinity chromatography and ion exchange (IEX) chromatography. Protein was resolved by SDS-PAGE and stained with Coomassie blue. The positions of molecular weight markers (kDa) are indicated.</p

Activity of Falstatin against Different Classes of Proteases

<p>Equal amounts (4 μg) of proteases (FP2, falcipain-2; FP3, falcipain-3; trypsin; α-chymo, α-chymotrypsin; pepsin; renin; collagenase; MM-2, matrix-metalloprotease-2) were mixed with 350 μl of appropriate buffers containing falstatin (1.5 μg) for 15 min, FITC-casein (20 μg) was added, and hydrolysis of the substrate with and without falstatin was compared for each protease. Error bars represent the standard deviations of results from two different assays, each performed in duplicate.</p

Release of Falstatin with Schizont Lysis

<p>Synchronized late-schizont-infected erythrocytes were cultured in Albumax-free medium. At the indicated time points culture media were collected and concentrated, falstatin was immunoprecipitated with anti-falstatin antiserum and anti-rat IgG beads, the beads were washed, bound proteins were solubilized in sample buffer, and falstatin was resolved by SDS-PAGE and identified by immunoblotting. Controls with pre-immune serum did not immunoprecipitate any detectable proteins. The positions of molecular weight markers are indicated (kDa). Schizont and ring parasitemias at the indicated time points are shown below the gel.</p

Inhibition of Falstatin Function by Anti-Falstatin Antibodies

<p>Hydrolysis of the peptide substrate Z-Leu-Arg-AMC by falcipain-2 (FP2; 19.8 nM), falcipain-3 (FP3; 27.1 nM), or trophozoite extract (TE; corresponding to 5.5 × 10⁶ parasites per reaction) was evaluated in the absence or presence of falstatin (31 nM) and the indicated quantities of anti-falstatin antibodies in 350 μl of 100 mM sodium acetate, 8 mM DTT (pH 6.0). Reaction components were incubated for 15 min before addition of substrate, and activity was measured as arbitrary fluorescence units over time (FU/min). Error bars represent the standard deviations of results from two different assays, each performed in duplicate.</p

Mechanism of Interaction of Falstatin with Falcipain-2

<p>Activity (arbitrary fluorescent units (FU) per minute) against Z-Leu-Arg-AMC is shown for indicated mixtures of falcipain-2 (FP2) and inactivated falcipain-2 (FP2^E-64) with falstatin. Reaction components were incubated for 15 min before the addition of substrate and measurement of fluorescence over time. Error bars represent the standard deviations of results from two different assays, each performed in duplicate.</p

Effect of Anti-Falstatin Antibodies on Cultured Parasites

<p>Schizont-infected erythrocytes were combined with fresh erythrocytes in culture medium with 0.5% DMSO or PBS, pre-immune serum, or the indicated concentration of E-64d (in DMSO) or antibody (in PBS). Smears were then made and stained with Giemsa, and percentages of schizonts after 12 h (A), rings after 20 h (B), and total parasites (C) were counted. In a separate experiment (D), purified schizonts were incubated for 20 h with PBS, control pre-immune serum (50 μg/ml), rat antiserum against P. falciparum farnesyl pyrophosphate synthetase (control Ab; 50 μg/ml), or the indicated concentrations of antibodies (Ab; μg/ml) in PBS with or without preincubation for 10 min with 2 μg falstatin (F). Errors bars indicate standard deviations from means of two different assays, each done in triplicate. PI, pre-immune serum.</p

Alignment of Falstatin with Chagasin and Putative Cysteine Protease Inhibitors of Other Plasmodial Species

<p>Sequences were aligned using the CLUSTALW program. Amino acid identities with falstatin are in blue, and amino acids of falstatin are numbered. The predicted N-terminal signal sequence is underlined.</p