Navegando por Autor "Curwen, Rachel S."
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Item Abundance of tegument surface proteins in the human blood fluke Schistosoma mansoni determined by QconCAT proteomics.(2011) Borges, William de Castro; Simpson, Deborah M.; Dowle, Adam A.; Curwen, Rachel S.; Oates, Jane Thomas; Beynon, Robert J.; Wilson, R. AlanThe schistosome tegument provides a major interface with the host blood stream in which it resides. Our recent proteomic studies have identified a range of proteins present in the complex tegument structure, and two models of protective immunity have implicated surface proteins as mediating antigens. We have used the QconCAT technique to evaluate the relative and absolute amounts of tegument proteins identified previously. A concatamer comprising R- or K-terminated peptides was generated with [13C6] lysine/arginine amino acids. Two tegument surface preparations were each spiked with the purified SmQconCAT as a standard, trypsin digested, and subjected to MALDI ToF-MS. The absolute amounts of protein in the biological samples were determined by comparing the areas under the pairs of peaks, separated by 6 m/z units, representing the light and heavy peptides derived from the biological sample and SmQconCAT, respectively. We report that aquaporin is the most abundant transmembrane protein, followed by two phosphohydrolases. Tetraspanin Tsp-2 and Annexin-2 are also abundant but transporters are scarce. Sm200 surface protein comprised the bulk of the GPI-anchored fraction and likely resides in the secreted membranocalyx. Two host IgGs were identified but in amounts much lower than their targets. The findings are interpreted in relation to the models of protective immunity.Item Enzymatic shaving of the tegument surface of live schistosomes for proteomic analysis : a rational approach to select vaccine candidates.(2011) Borges, William de Castro; Dowle, Adam A.; Curwen, Rachel S.; Oates, Jane Thomas; Wilson, R. AlanBackground: The membrane-associated and membrane-spanning constituents of the Schistosoma mansoni tegument surface, the parasite’s principal interface with the host bloodstream, have recently been characterized using proteomic techniques. Biotinylation of live worms using membrane-impermeant probes revealed that only a small subset of the proteins was accessible to the reagents. Their position within the multilayered architecture of the surface has not been ascertained. Methodology/Principal Findings: An enzymatic shaving approach on live worms has now been used to release the most accessible components, for analysis by MS/MS. Treatment with trypsin, or phosphatidylinositol-specific phospholipase C (PiPLC), only minimally impaired membrane integrity. PiPLC-enriched proteins were distinguished from those released in parasite vomitus or by handling damage, using isobaric tagging. Trypsin released five membrane proteins, Sm200, Sm25 and three annexins, plus host CD44 and the complement factors C3 and C4. Nutrient transporters and ion channels were absent from the trypsin fraction, suggesting a deeper location in the surface complex; surprisingly, two BAR-domain containing proteins were released. Seven parasite and two host proteins were enriched by PiPLC treatment, the vaccine candidate Sm29 being the most prominent along with two orthologues of human CD59, potentially inhibitors ofc omplement fixation. The enzymes carbonic anhydrase and APD-ribosyl cyclase were also enriched, plus Sm200 and alkaline phosphatase. Host GPI-anchored proteins CD48 and CD90, suggest ‘surface painting’ during worm peregrination in the portal system. Conclusions/Significance: Our findings suggest that the membranocalyx secreted over the tegument surface is not the inert barrier previously proposed, some tegument proteins being externally accessible to enzymes and thus potentially located within it. Furthermore, the detection of C3 and C4 indicates that the complement cascade is initiated, while two CD59 orthologues suggest a potential mechanism for its inhibition. The detection of several host proteins is a testimonial to the acquisitive properties of the tegument surface. The exposed parasite proteins could represent novel vaccine candidates for combating this neglected disease.Item The 20S proteasome of Schistosoma mansoni : a proteomic analysis.(2007) Borges, William de Castro; Cartwright, Jared; Ashton, Peter D.; Braschi, Simon; Cota, Renata Guerra de Sá; Rodrigues, Vanderlei; Wilson, R. Alan; Curwen, Rachel S.Proteasomes are molecular machines found in virtually all cells that provide one of the mechanisms for protein turnover. We have analysed the 20S proteasome of Schistosoma mansoni, the first multimeric complex isolated from this helminth parasite. Three chromatographic steps were employed to yield a highly homogeneous preparation. 2-DE of the purified complex revealed 58 spots, of which 46 could be assigned either an a or a b proteasome signature by MS. Most of the 14 transcripts (7a and 7b) encoded by the parasite genome were represented by multiple spots and we suggest that this diversity is due to PTMs of subunits. For most of the isoforms, variations in pI predominated although alterations in mass were also observed. 2-DE separations of extracts from infective cercariae and blood-dwelling adult worms probed by Western blotting, using a human anti-a subunit antibody, revealed different patterns of reactivity, most probably in a3 and a6 subunits, on the basis of sequence conservation. This difference was rapidly lost following transformation of the cercaria to the skin schistosomulum stage, suggesting that changes in the proteasome structure, likely caused by the introduction of a new set of PTMs, precede remodelling of the parasite body prior to intravascular migration.