Evaluation of the ability of SmCD59.1 and SmCD59.2 to modulate complement activity.

<p>Hemolytic assays were performed after incubating normal human serum (NHS) with different amounts of SmCD59.1 (produced in <i>P. pastoris</i>), SmCD59.2 (produced in <i>P. pastoris</i> or <i>E. coli</i>), or BSA (negative control). (A) Treated NHS was then incubated with rabbit erythrocytes (Alternative Pathway) or (B) antibody-sensitized sheep erythrocytes (Classical Pathway). The percentage of hemolysis was calculated in comparison with erythrocytes suspensions completely lysed with water (100% lysis). The volume of NHS used in these assays corresponds to the amount that promotes 50% lysis of erythrocytes. Each column represents the mean of three independent experiments ± SD.</p

Fluorescence confocal microscopy images showing immunolocalization of SmCD59.1 and SmCD59.2 in whole mount and in transverse sections of <i>S. mansoni</i> adult worms.

<p>(A and B) Confocal projections of SmCD59.1 and SmCD59.2 protein in the tegument of whole mount adult male worms. (D and E) Fluorescence detection of SmCD59.1 and SmCD59.2 in transverse sections of <i>S. mansoni</i> adult worms. (C and F) Negative control, serum from naïve rat. Secondary antibody coupled to Alexa 488 (green) was used for SmCD59 localization. DAPI (blue) was used for nucleus localization (E and F) and Rhodamine Phalloidin (red) was used for actin localization (A, B and C). Arrows – tegument tubercules; M – male; F – female; p – parenchyma; mc – muscle cells.</p

ClustalX multiple sequence alignment of the mature protein sequence (excluding the signal peptide) of TFPDs from platyhelminthes, CD59 and Ly6.

<p>The regions with high identity and similarity between sequences are shown as black and gray columns, according to the ClustalX algorithm. Arrows indicate highly conserved Cysteines and Asparagines with a C and an N, respectively. Dashed lines represent pairs of cysteine residues forming disulfide bonds determined from Hs-CD59 (red) and predicted for SmCD59.2 (black). Only for SmCD59 sequences, potential sites for N-glycosylation are shown in blue with asparagine (N) in red, and potential sites for GPI anchor are shown in yellow. Human CD59 active sites are shaded in red. The sequences abbreviation are: <i>Schistosoma mansoni</i> (SmCD59.1-7), <i>Schistosoma japonicum</i> (Sj1, Sj2.3, Sj3, Sj4.1, Sj6), <i>Schistosoma hematobium</i> (Sh1-3 and Sh5-7), <i>Clonorchis sinensis</i> (Cs-757, Cs-8328, Cs-8627), <i>Opisthorchis viverrini</i> (Ov-8524, Ov-3995 and Ov-6738), <i>Fasciola hepatica</i> (Fh-6273), <i>Fasciola gigantica</i> (Fg-25430 and Fg-15245), <i>Schmidtea mediterranea</i> (Smed), <i>Equus caballus</i> (Ec-Ly6), <i>Pongo abelii</i> (Pa-Ly6 and Pa-CD59), <i>Macaca mulatta</i> (Mam-Ly6), <i>Mus musculus</i> (Mm-Ly6 and Mm-CD59), <i>Monodelphis domestica</i> (Md-Ly6), <i>Ornithorhynchus anatinus</i> (Oa-Ly6), <i>Homo sapiens</i> (Hs-Ly6 and Hs-CD59), <i>Saimiriine herpesvirus</i> (Sah-CD59), <i>Rattus norvegicus</i> (Rn-CD59) (the accession numbers are listed in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002482#pntd.0002482.s006" target="_blank">Table S2</a>).</p

Complement resistance of CHO cells expressing SmCD59.1, SmCD59.2 and hCD59 (positive control).

<p>Viable cells were quantified by flow cytometry following exposure to anti-CHO antibodies and normal human serum (NHS) or heat-inactivated serum (iNHS). Black histograms are CHO cells transfected with hCD59 (A), SmCD59.1 (B) or SmCD59.2 (C), respectively. Red histograms are control cells transfected with empty pcDNA vector (A, B and C). The first peak in both black and red histograms represents viable cells and the second peak represents dead cells stained by propidium iodide (PI). (D) Cell viability of CHO cells transfected with hCD59 or pcDNA treated with anti-CHO antibodies and NHS or iNHS in three independent experiments (mean ± SD). P values are indicated.</p

Homology modeling of SmCD59.2.

<p>Ribbon display, side view (A) and top view (B). The models were generated with Modeller 5.1 using 3-D structure of human CD59 as a template (2UWR). The quality of the model was assessed with Procheck. Disulfide bridges are visualized as magenta sticks, and the three finger-shaped backbone is visualized as projections emerging from the disulfide bridges.</p

Immunoblotting of protein extracts from <i>S. mansoni</i> stages using anti-rSmCD59.1 or anti-rSmCD59.2 polyclonal antibodies.

<p>Protein extracts (20 µg) from different <i>S. mansoni</i> stages: EGG (eggs), MIR (miracidia), CER (cercariae), SCH (<i>in vitro</i> 7-day-old schistosomula), ♂ (male adult worm), ♀ (female adult worm) and P (positive control, 100 ng of rSmCD59.1) were analyzed using (A) anti-rSmCD59.1 antiserum; or (B) anti-rSmCD59.2 antiserum, P (positive control, 100 ng of rSmCD59.2 expressed in <i>E. coli</i>). Extracts of stripped worms (Strip) and Tegument (Teg) of adult worms were probed with (C) anti-rSmCD59.1 or (D) anti-rSmCD59.2 antisera. Insoluble (Ins) and soluble (Sol) protein extracts of stripped worms; (Tsm) enriched tegument surface membranes fraction; (Twm) tegument extract without surface membranes. Positions of molecular mass standard are indicated.</p