排序方式: 共有44条查询结果,搜索用时 0 毫秒
1.
Sialidases are hydrolytic enzymes present from virus to highereukaryotes, catalyzing the removal of sialic acid from glycoconjugates.Some protozoa Trypanosomatidae secrete high levels of sialidaseinto the medium. We have now purified the secreted sialidasefrom Trypanosoma rangeli Its N-terminal sequence reveals 100%identity with the corresponding region of the trans-sialidasefrom T.cruzi Trans-sialidase, although homologous to viral andbacterial sialidases, displays a novel sialyltransferase activityand is involved in host cell invasion. Several homologous trans-sialidase-likegenes were cloned from genomic DNA of T.rangeli, and groupedin three subfamilies. Active siali-dase-encoding genes werefound in one of them. The re-combinant sialidase shows similarproperties to those of the native enzyme, including undetectabletrans-sialidase activity. Nevertheless, it has an overall identityof 68.9% with the catalytic domain of T.cruzi trans-sialidase,increasing to 86.7% admitting conservative substitutions. Onlythree other eukaryotic sialidases have been previously cloned,none of them showing significant homology to trans-sialidase.The isolation of a highly similar sialidase is relevant to furtheridentify the molecular determinants allowing trans-sialidaseactivity. As a first approach, chimeric constructs between sialidaseand trans-sialidase were generated, one of them rendering asialidase with three times lower Km than the natural enzyme. eukaryotic sialidase gene family glycosidase parasite sialic acid 相似文献
2.
3.
Andrés B. Lantos Giannina Carlevaro Beatriz Araoz Pablo Ruiz Diaz María de los Milagros Camara Carlos A. Buscaglia Mariano Bossi Hai Yu Xi Chen Carolyn R. Bertozzi Juan Mucci Oscar Campetella 《PLoS pathogens》2016,12(4)
Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form. 相似文献
4.
Mucci J Mocetti E Leguizamón MS Campetella O 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(8):4545-4550
Sialylation is emerging as an important issue in developing thymocytes and is considered among the most significant cell surface modifications, although its physiologic relevance is far from being completely understood. It is regulated by the concerted expression of sialyl transferases along thymocyte development. After in vivo administration of trans-sialidase, a virulence factor from the American trypanosomatid Trypanosoma cruzi that directly transfers the sialyl residue among macromolecules, we found that the alteration of the sialylation pattern induces thymocyte apoptosis inside the "nurse cell complex." This suggests a glycosylation survey in the development of the T cell compartment. In this study, we report that this thymocyte apoptosis mechanism requires the presence of androgens. No increment in apoptosis was recorded after trans-sialidase administration in females or in antiandrogen-treated, gonadectomized, or androgen receptor mutant male mice. The androgen receptor presence was required only in the thymic epithelial cells as determined by bone marrow chimeric mouse approaches. The presence of the CD43 surface mucin, a molecule with a still undefined function in thymocytes, was another absolute requirement. The trans-sialidase-induced apoptosis proceeds through the TNF-alpha receptor 1 deathly signaling leading to the activation of the caspase 3. Accordingly, the production of the cytokine was increased in thymocytes. The ability of males to delete thymocytes altered in their sialylation pattern reveals a sexual dimorphism in the glycosylation survey during the development of the T cell compartment that might be related to the known differences in the immune response among sexes. 相似文献
5.
Tribulatti MV Figini MG Carabelli J Cattaneo V Campetella O 《Journal of immunology (Baltimore, Md. : 1950)》2012,188(7):2991-2999
Galectins, a family of mammalian lectins, have emerged as key regulators of the immune response. We previously demonstrated that galectin (Gal)-8, from the tandem-repeat subgroup, exerts two well-defined effects on mouse naive peripheral CD4 T cells: Ag-specific costimulation and Ag-independent proliferation. These stimulatory signals on naive T cells have not been described for any other Gal. Therefore, we investigated whether Gal-1 and Gal-3, two prominent members of the Gal family, share the stimulatory effects exerted by Gal-8 on naive T cells. We found that Gal-1 costimulated Ag-specific T cell responses similarly to Gal-8, as evaluated in the DO11.10 TCR(OVA)-transgenic mouse model, by acting simultaneously on APCs and target CD4 T cells. In contrast, Gal-3 failed to costimulate Ag-specific T cell responses; moreover, it antagonized both Gal-1 and Gal-8 signals. We observed that both Gal-1 and Gal-3 were unable to induce Ag-independent proliferation; however, when two Gal-1 molecules were covalently fused, the resulting chimeric protein efficiently promoted proliferation. This finding indicates that Gal-1 might eventually induce proliferation and, moreover, stresses the requirement of a tandem-repeat structure. Remarkably, a single dose of recombinant Gal-1 or Gal-8 administered together with a suboptimal Ag dose to DO11.10 mice strengthened weak responses in vivo. Taken together, these findings argue for the participation of Gals in the initiation of the immune response and allow the postulation of these lectins as enhancers of borderline Ag responses, thus representing potential adjuvants for vaccine formulations. 相似文献
6.
Santiago J. Carmona Paula A. Sartor María S. Leguizamón Oscar E. Campetella Fernán Agüero 《PloS one》2012,7(12)
The availability of complete pathogen genomes has renewed interest in the development of diagnostics for infectious diseases. Synthetic peptide microarrays provide a rapid, high-throughput platform for immunological testing of potential B-cell epitopes. However, their current capacity prevent the experimental screening of complete “peptidomes”. Therefore, computational approaches for prediction and/or prioritization of diagnostically relevant peptides are required. In this work we describe a computational method to assess a defined set of molecular properties for each potential diagnostic target in a reference genome. Properties such as sub-cellular localization or expression level were evaluated for the whole protein. At a higher resolution (short peptides), we assessed a set of local properties, such as repetitive motifs, disorder (structured vs natively unstructured regions), trans-membrane spans, genetic polymorphisms (conserved vs. divergent regions), predicted B-cell epitopes, and sequence similarity against human proteins and other potential cross-reacting species (e.g. other pathogens endemic in overlapping geographical locations). A scoring function based on these different features was developed, and used to rank all peptides from a large eukaryotic pathogen proteome. We applied this method to the identification of candidate diagnostic peptides in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We measured the performance of the method by analyzing the enrichment of validated antigens in the high-scoring top of the ranking. Based on this measure, our integrative method outperformed alternative prioritizations based on individual properties (such as B-cell epitope predictors alone). Using this method we ranked 10 million 12-mer overlapping peptides derived from the complete T. cruzi proteome. Experimental screening of 190 high-scoring peptides allowed the identification of 37 novel epitopes with diagnostic potential, while none of the low scoring peptides showed significant reactivity. Many of the metrics employed are dependent on standard bioinformatic tools and data, so the method can be easily extended to other pathogen genomes. 相似文献
7.
8.
trans-sialidase is a unique sialidase in that, instead of hydrolizing sialic acid, it preferentially transfers the monosaccharide to a terminal beta-galactose in glycoproteins and glycolipids. This enzyme, originally identified in Trypanosoma cruzi, belongs to a large family of proteins. Some members of the family lack the enzymatic activity. No function has been yet assigned to them. In this work, the gene copy number and the possible function of inactive members of the trans -sialidase family was studied. It is shown that genes encoding inactive members are not a few, but rather, are present in the same copy number (60-80 per haploid genome) as those encoding active trans -sialidases. Recombinant inactive proteins were purified and assayed for sialic acid and galactose binding activity in agglutination tests. The enzymatically inactive trans -sialidases were found to agglutinate de-sialylated erythrocytes but not untreated red blood cells. Assays made with mouse and rabbit red blood cells suggest that inactive trans -sialidases bind to beta, rather than alpha, terminal galactoses, the same specificity required by active trans -sialidases. A recombinant molecule that was made enzymatically inactive through a mutation in a single amino acid also retained the galactose binding activity. The binding was competed by lactose and was dependent on conservation of the protein native conformation. Therefore, at least some molecules in the trans -sialidase family that have lost their enzymatic function still retain their Gal-binding properties and might have a function as lectins in the parasite-host interaction. 相似文献
9.
Santiago J. Carmona Morten Nielsen Claus Schafer-Nielsen Juan Mucci Jaime Altcheh Virginia Balouz Valeria Tekiel Alberto C. Frasch Oscar Campetella Carlos A. Buscaglia Fernán Agüero 《Molecular & cellular proteomics : MCP》2015,14(7):1871-1884
Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.Detailed knowledge of antigens and epitopes recognized in the context of naturally acquired human infections has important implications for our understanding of immune system responses against pathogens, and of the immunopathogenesis of infectious diseases. This knowledge is also important for practical clinical applications such as the development of improved vaccines, intervention strategies, and diagnostics.In the last decades, significant progress has been made in the discovery of antigens and epitopes thanks to a number of methodologies such as cDNA expression libraries (1), combinatorial peptide libraries (2), and peptide and protein microarrays (3, 4). However, current knowledge of the B-cell antigens and the epitope repertoire recognized by the immune system in human infections is still scarce. Indeed, the Immune Epitope Database (5) currently contains an average of only 10 antigens with mapped B-cell epitopes recognized from naturally acquired human infections for bacterial or eukaryotic pathogens. The reasons for this are many, but can be largely attributed to different limitations in the mentioned screening technologies. Heterologous expression of cDNA libraries has been used to guide antigen discovery, but mapping of epitopes most often lags behind as it is a much more costly exercise. Similarly, combinatorial peptide libraries greatly facilitate the identification of peptides that are specifically recognized by antibodies, but these peptides have sequences that can greatly differ from those of the native epitopes (they are mimotopes), thus making it difficult to identify the original antigens. As a result, we currently have only limited detailed information on the fine specificities of the antibody response against complex pathogens.The number of tools for studying immune responses has recently expanded with the inclusion of peptide and protein microarrays, which have been used to identify pathogen-specific antigens and linear epitopes (6–13). Although whole-protein arrays can successfully identify antigens recognized by antibodies, they present the typical difficulties associated with the production of recombinant proteins in heterologous or in vitro systems, do not provide information on the nature and precise location of the epitope(s) in a protein, and are more likely to suffer from nonspecific antibody binding because of the exposure of a large number of potentially antigenic regions. In contrast, peptide arrays can provide exquisite detail of epitope localization, but until now had other limitations mostly associated with their reduced capacity, preventing the complete scanning of large numbers of candidate proteins.Recent advances in computerized photolithography and photochemistry have led to the development of a novel high-density peptide microarray technology, where individual peptides can be synthesized in situ on a glass slide at high densities (14–17). This technology makes the production of high-density peptide arrays highly cost effective compared with previous approaches, while allowing the interrogation of complex immune responses with unprecedented throughput and mapping precision. Previous applications of this technology were limited to the fine mapping of epitopes in single proteins, using monoclonal antibodies, or using immunized animal sera as the source of polyclonal antibodies (16–18).Using these high-density peptide arrays, we here describe the first large-scale study of fine antibody specificities associated with Chagas Disease, which is an exemplar of a chronic human infectious disease. Chagas Disease, caused by the protozoan Trypanosoma cruzi, is an endemic disease of the Americas, affecting ∼8 million people (19). The parasite invades and replicates within host cells, and briefly enters the bloodstream to reach other target tissues. Initially, the disease goes through an acute stage, characterized by patent parasitaemia and the appearance of antibodies against acute-phase antigens, such as SAPA (20), followed by a delayed specific humoral response. In general, the parasite-specific immune response mounted during T. cruzi infections is insufficient to completely eradicate the pathogen, leading to chronic infection (19). In this chronic stage circulating parasites are difficult to detect, even by extremely sensitive methods such as PCR. Therefore, detection of antibodies against whole-parasite extracts or defined antigens (21, 22) remains the standard for diagnosis of Chagas Disease.In this work, we screened high-density microarray slides containing peptides derived from T. cruzi proteins with mixtures of immunoglobulins purified directly from blood samples of Chagas Disease patients. This led to the identification of novel antigens and the simultaneous mapping of their linear B-cell epitopes, thus demonstrating the capacity and performance of this platform for studying antibody specificities associated with human infectious diseases. 相似文献
10.
The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood. 相似文献