Crystal and solution structures of fragments of the human leucocyte common antigen-related protein. the supplemental material. ABSTRACT Lymphatic filariasis is usually a debilitating ML327 disease that afflicts over 70 million people worldwide. It is caused by the parasitic nematodes worms as you possibly can therapeutic targets. Tfpi Using short interfering RNA (siRNA), we successfully targeted four candidate gene transcripts: Bma-Serpin, Bma-ShTK, Bma-Reprolysin, and Bma-LAD-2. Of those, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was decided to be essential for adult worm survival. We observed a 70.42% knockdown in Bma-LAD-2 transcript levels 1?day post-siRNA incubation and an 87.02% reduction in protein expression 2?days post-siRNA incubation. This inhibition of Bma-LAD-2 expression resulted in an 80% decrease in worm motility over 6?days, a 93.43% reduction in microfilaria release (Mf) by day 6 post-siRNA incubation, and a dramatic decrease in (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Transmission electron microscopy revealed the loss of microvilli and unraveling of mitochondrial cristae in the intestinal epithelium of Bma-LAD-2 siRNA-treated worms. Strikingly, Bma-LAD-2 siRNA-treated worms exhibited an almost complete loss of pseudocoelomic fluid. A luciferase immunoprecipitation system assay did not detect anti-Bma-LAD-2 IgE in the serum of 30 LF patients, indicating that LF exposure does not result in IgE sensitization to this antigen. These results indicate that Bma-LAD-2 is an essential protein for adult and may be an effective therapeutic target. or because the drugs can precipitate severe side effects by rapid killing of Mf (11,C14). To avoid side effects from killing of microfilariae (Mf) in coendemic populations and to potentially enable a single treatment remedy of filarial infections, our group has focused on identifying drug and/or vaccine targets specific to adult filarial worms. Because adult worms contain a complete intestinal tract, whereas microfilariae do not, our group evaluated the intestinal tract of adult filarial worms as a possible source of therapeutic targets. Already, this strategy appears to be promising against other helminths. Numerous studies have demonstrated protection against hookworm and barber pole worm contamination using nematode intestinal antigens as vaccine candidates (15,C20). Furthermore, there seems to be little specific IgE against intestinal antigens in the sera of infected animal models as well as in previously exposed individuals (21, 22), suggesting that intestinal antigens are safe to administer as vaccines in areas where the disease is usually endemic. Our lab previously performed a proteomic analysis of the body wall, gut, and reproductive tract of adult worms (23). We identified 396 proteins specific for the intestine and then selected 9 for evaluation as potential drug and therapeutic targets. The selection criteria were having (i) high homology with orthologs in other filarial species and low homology to humans, (ii) a large extracellular domain potentially accessible to drugs and antibody, and (iii) a predicted function likely essential for adult filarial survival. Previous work we have conducted found that a filarial intestinal antigen, Bm-UGT (UDP-glucuronosyl transferase), was essential for adult survival and could be targeted with probenecid to achieve death of adult worms (24). Using short interfering RNA (siRNA) inhibition, we successfully knocked down 4 target proteins. Of these, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was found to be essential for adult worm survival. Suppression of Bma-LAD-2 expression resulted in decreased worm motility, metabolism, and Mf release. Electron microscopy revealed that inhibition of Bma-LAD-2 resulted in almost complete loss of pseudocoelomic fluid, suggesting that disrupting the tight junctions between filarial intestinal cells and causing subsequent disruption of the worms hydrostatic skeleton is usually a ML327 novel mechanism to kill filarial parasites. RESULTS Structural analysis of Bma-LAD-2. The Bma-LAD-2 protein is usually 1,171 amino acids (aa) in length (molecular mass of 133,310.4?Da), with a signal peptide, aa 1 to 18, a large extracellular segment at position 19 to 1120, a transmembrane portion at aa 1121 to 1143, and a small cytoplasmic domain name at position 1143 ML327 to 1171 (see Fig.?S1 in the supplemental material). The putative domain name organization and model of the structure of the extracellular domain name (residues 18 to 1120) is usually shown in Fig.?1 for both the Bma-LAD-2 monomer and dimer. The Bma-LAD-2 monomer is usually predicted to fold into 6 immunoglobulin domains (Ig1-Ig6) followed by 5 fibronectin-type domains (FN1 to -5) (Fig.?1A). The outermost N-terminal Ig domains are predicted to homodimerize to ML327 form tight junctions. The Bma-LAD-2 dimer model, based on dimerization mode of the homologous protein neurofascin (25), is usually stabilized by contacts between the ML327 domains of Ig1 and Ig2 paired in an orthogonal side-to-side stacking mode (Fig.?1B). Open in a separate windows FIG?1 Molecular business of Bma-LAD-2 extracellular domain. (A) Bma-LAD-2 monomer. Schematic domain name organization (top) and model of monomer structure (bottom) assembled based on sequence similarity and available crystal structures of homologous proteins as described in Materials and Methods. (B) Putative structure of Bma-LAD-2 dimer. Expanded view shows the dimer interface.
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