On the three-finger protein domain fold and CD59-like proteins in Schistosoma mansoni

Background: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. Methodology/Principal Findings: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. Conclusions: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Grant Number:04/12872-3)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institute of Health, National Institute of Allergy and Infectious Diseases (NIH-NIAID), Grant AI-095893NIH-NIAID Grant AI-056273FAPESP 00/11624-

Key Topics in Molecular Docking for Drug Design

Molecular docking has been widely employed as a fast and inexpensive technique in the past decades, both in academic and industrial settings. Although this discipline has now had enough time to consolidate, many aspects remain challenging and there is still not a straightforward and accurate route to readily pinpoint true ligands among a set of molecules, nor to identify with precision the correct ligand conformation within the binding pocket of a given target molecule. Nevertheless, new approaches continue to be developed and the volume of published works grows at a rapid pace. In this review, we present an overview of the method and attempt to summarise recent developments regarding four main aspects of molecular docking approaches: (i) the available benchmarking sets, highlighting their advantages and caveats, (ii) the advances in consensus methods, (iii) recent algorithms and applications using fragment-based approaches, and (iv) the use of machine learning algorithms in molecular docking. These recent developments incrementally contribute to an increase in accuracy and are expected, given time, and together with advances in computing power and hardware capability, to eventually accomplish the full potential of this area

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

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

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

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