Genetic Structure and Expression of the Surface Glycoprotein GP82, the Main Adhesin of Trypanosoma cruzi Metacyclic Trypomastigotes

T. cruzi improves the likelihood of invading or adapting to the host through its capacity to present a large repertoire of surface molecules. the metacyclic stage-specific surface glycoprotein GP82 has been implicated in host cell invasion. GP82 is encoded by multiple genes from the trans-sialidase superfamily. GP82 shows a modular organization, with some variation of N-terminal region flanking a conserved central core where the binding sites to the mammalian cell and gastric mucin are located. the function of GP82 as adhesin in host cell invasion process could expose the protein to an intense conservative and selective pressure. GP82 is a GPI-anchored surface protein, synthesized as a 70 kDa precursor devoid of N-linked sugars. GPI-minus variants accumulate in the ER indicating that GPI anchor acts as a forward transport signal for progressing along the secretory pathway as suggested for T. cruzi mucins. It has been demonstrated that the expression of GP82 is constitutive and may be regulated at post-transcriptional level, for instance, at translational level and/or mRNA stabilization. GP82 mRNAs are mobilized to polysomes and consequently translated, but only in metacyclic trypomastigotes. Analysis of transgenic parasites indicates that the mechanism regulating GP82 expression involves multiple elements in the 3'UTR.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

Validation of Interaction Between the GlyT2 and the NSF

The Glycine Transporter 2 (GlyT2) is a presynaptic membrane transporter in charge of re-uptake of glycine from the synaptic cleft back into the presynaptic neuron. GlyT2 is present only in vertebrate neurons and possesses a 200 amino- acid long N-terminal tail of unknown function. We hypothesize that such domain serve as a docking site for a network of presynaptic proteins, such as NSF that plays a pivotal role during vesicle transport. Therefore, our goal in this project is to analyze protein-protein interaction between GlyT2 and NSF. We have used co-immunoprecipitation experiments from rat brainstem with GlyT2 and NSF antibodies as bait, and heterologous expression of both genes in porcine aortha epithelial (PAE) cells. Preliminary results showed that both antibodies are capable of precipitating the corresponding protein from rat brain; at the same time, blotting of a GlyT2 immunoprecipitate with NSF antibodies readily detected a protein at the predicted molecular weight for NSF (about 110 kDa for GlyT2, and 82kDa for NSF). These results suggest that NSF is a potential GlyT2 interacting protein. To study this interaction in vitro, we tagged NSF gene with the green fluorescent protein at the N-terminus and the final construct was expressed in PA cells. We immunoprecipitated GFP-NSF from transfected cells with NSF or GFP antibodies and after separation in SDS-PAGE and western blot analysis with NSF antibodies, we identified an reactive band of ~110 kDa corresponding to the fusion protein. Future experiments to analyze the interaction will include the additional expression of GlyT2 and studies of localization and interaction by fluorescence microscopy

The regulation of the Glycine Transporter 1 by phosphorylation

The plasma membrane glycine transporters 1 (GlyT1) is the carrier molecule responsible for the fast removal of the inhibitory neurotransmitter glycine from the synaptic cleft back into the presynaptic terminals of glycinergic neurons by an electrogenic, Na+-Cl- transport couple mechanism maintained by the Na+,K+-ATPase. GlyTs belong to SLC6 family that includes other neurotransmitter transporters, such as the dopamine, norepinephrine, serotonin, and GABA transporters. In order to determine the role of PKC-dependent phosphorylation on GlyT1, we have analyzed the effect of PKC activation by phorbol ester (PMA) on the activity, post-translational modifications and the levels of cell surface transporter in mutants where several replacements of Ser/Thr to Ala were made. We found that, similar to other transporters like DAT, NET and SERT, activation of protein kinase C by phorbol ester led to increased phosphorylation and ubiquitination of GlyT1. These modifications were accompanied by a reduction in transport capacity and enhanced transporter endocytosis. Interestingly, replacement of serine/threonine residues to alanine at both tails abolished PKC-dependent phosphorylation but dramatically enhanced ubiquitination and degradation. These results suggest that transporter modifications represent a mechanism of GlyT1 regulation. This work was supported by a grant from NIMH 5SC1MH 086070-04 to MM, AA is a recipient from the bridges program, grant NIH 5R25GM049011-1

Remote Sensing via ℓ 1-Minimization

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis

Analysis and chromosomal mapping of Leishmania ( Leishmania) amazonensis amastigote expressed sequence tags

The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania ( Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis

Partial protective responses induced by a recombinant cysteine proteinase from Leishmania (Leishmania) amazonensis in a murine model of cutaneous leishmaniasis

A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. in Western blots of L (L) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8(+) lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4(+) lymphocytes was observed in these animals. the cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L (L) amazonensis compared to those exhibited by control mice. (C) 2009 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Div Parasitol, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilUniv Fed Piaui, Picos, Piaui, BrazilUniv Bandeirante São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Div Parasitol, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc