[Show abstract][Hide abstract] ABSTRACT: Giardia lamblia is a multiflagellated protozoan that inhabits the small intestine of vertebrates, causing giardiasis. To colonize the small intestine, the trophozoites form of the parasite remains attached to intestinal epithelial cells by means of cytoskeletal elements that form a structure known as the ventral disc. Previous studies have shown that the ventral disc is made of tubulin and giardins.
To obtain further information on the composition of the ventral disc, we developed a new protocol and evaluated the purity of the isolation by transmission electron microscopy. Using 1D- and 2D-PAGE and mass spectrometry, we identified proteins with functions associated with the disc. In addition to finding tubulin and giardin, proteins known to be associated with the ventral disc, we also identified proteins annotated in the Giardia genome, but whose function was previously unknown.
The isolation of the ventral disc shown in this work, compared to previously published protocols, proved to be more efficient. Proteomic analysis showed the presence of several proteins whose further characterization may help in the elucidation of the mechanisms involved in the attachment of the protozoan to epithelial cells.
[Show abstract][Hide abstract] ABSTRACT: A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies.
Journal of Proteomics 02/2009; 72(2):241-55. · 4.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains alpha and beta. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family.