RESUMO
BACKGROUND: Expression of the neuronal membrane glycoprotein M6a (GPM6A), the proteolipid protein (PLP/DM20) family member, is downregulated in the hippocampus of chronically stressed animals. Its neuroplastic function involves a role in neurite formation, filopodium outgrowth and synaptogenesis through an unknown mechanism. Disruptions in neuroplasticity mechanisms have been shown to play a significant part in the etiology of depression. Thus, the current investigation examined whether GPM6A expression is also altered in human depressed brain. METHODS: Expression levels and coexpression patterns of GPM6A, GPM6B, and PLP1 (two other members of PLP/DM20 family) as well as of the neuroplasticity-related genes identified to associate with GPM6A were determined using quantitative polymerase chain reaction (qPCR) in postmortem samples from the hippocampus (n = 18) and the prefrontal cortex (PFC) (n = 25) of depressed suicide victims and compared with control subjects (hippocampus n = 18; PFC n = 25). Neuroplasticity-related proteins that form complexes with GPM6A were identified by coimmunoprecipitation technique followed by mass spectrometry. RESULTS: Results indicated transcriptional downregulation of GPM6A and GPM6B in the hippocampus of depressed suicides. The expression level of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) and coronin1A (CORO1A) was also significantly decreased. Subsequent analysis of coexpression patterns demonstrated coordinated gene expression in the hippocampus and in the PFC indicating that the function of these genes might be coregulated in the human brain. However, in the brain of depressed suicides this coordinated response was disrupted. CONCLUSIONS: Disruption of coordinated gene expression as well as abnormalities in GPM6A and GPM6B expression and expression of the components of GPM6A complexes were detected in the brain of depressed suicides.
Assuntos
Transtorno Depressivo Maior/metabolismo , Expressão Gênica , Hipocampo/metabolismo , Plasticidade Neuronal/genética , Córtex Pré-Frontal/metabolismo , Suicídio , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transtorno Depressivo Maior/genética , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Adulto JovemRESUMO
Developmental stages of the trypanosome life cycle differ in their morphology, biology, and biochemical properties. Consequently, several proteins have to be tightly regulated in their expression to allow trypanosomes to adapt rapidly to sudden environmental changes, a process that might be of central importance for parasite survival. However, in contrast to higher eukaryotic cells, trypanosomes do not seem to regulate gene expression through regulation of transcription initiation. These parasites make use of post-transcriptional regulatory mechanisms and modification of mRNA half-life is a relevant one. Trans-acting factors binding to cis-elements that affect mRNA stability of mature transcripts have not been identified in these cells. In this work, a novel U-rich RNA-binding protein (TcUBP-1) from Trypanosoma cruzi, the agent of Chagas disease, was identified. Its structure includes an RNA recognition motif, a nuclear export signal, and auxiliary domains with glycine- and glutamine-rich regions. TcUBP-1 recognizes the 44-nucleotide AU-rich RNA instability element located in the 3'-untranslated region of mucin SMUG mRNAs (Di Noia, J. M., D'Orso, I., Sanchez, D. O., and Frasch, A. C. (2000) J. Biol. Chem. 275, 10218-10227) as well as GU-rich sequences. Over-expression of TcUBP-1 in trypanosomes decreases the half-life of SMUG mucin mRNAs in vivo but does not affect the stability of other parasite mRNAs. Because TcUBP-1 is developmentally regulated, it might have a relevant role in regulating protein expression during trypanosome differentiation, allowing a correct expression pattern of U-rich-containing mRNAs.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Protozoários/fisiologia , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/fisiologia , Trypanosoma cruzi/química , Regiões 3' não Traduzidas , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Mucinas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Trypanosoma cruzi/genéticaRESUMO
Sialidases are present on the surface of several trypanosomatid protozoan parasites. They are highly specific for sialic acid linked in alpha-(2,3) to a terminal beta-galactose and include the strictly hydrolytic enzymes and trans-sialidases (sialyl-transferases). Based on the structural comparison of the sialidase from Trypanosoma rangeli and the trans-sialidase from T. cruzi (the agent of Chagas' disease in humans), we have explored the role of specific amino acid residues sought to be important for substrate specificity. The substitution of a conserved tryptophanyl residue in the two enzymes, Trp312/313-Ala, changed substrate specificity, rendering the point mutants capable to hydrolyze both alpha-(2,3)- and alpha-(2,6)-linked sialoconjugates. The same mutation abolished sialyl-transferase activity, indicating that transfer (but not hydrolysis) requires a precise orientation of the bound substrate. The exchange substitution of another residue that modulates oligosaccharide binding, Gln284-Pro, was found to significantly increase the hydrolytic activity of sialidase, and residue Tyr119 was confirmed to be part of a second binding site for the acceptor substrate in trans-sialidase. Together with the structural information, these results provide a consistent framework to account for the unique enzymatic properties of trypanosome trans-sialidases.
Assuntos
Neuraminidase/química , Neuraminidase/metabolismo , Mutação Puntual/genética , Trypanosoma/enzimologia , Substituição de Aminoácidos/genética , Aminoácidos/análise , Animais , Sítios de Ligação , Ligação Competitiva , Hidrólise , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neuraminidase/genética , Conformação Proteica , Especificidade por Substrato , Trypanosoma/genéticaRESUMO
Post-transcriptional regulatory mechanisms have been suggested to be the main point of control of gene expression in kinetoplastid parasites. We have previously shown that Trypanosoma cruzi SMUG mucin mRNA steady-state level is developmentally regulated by post-transcriptional mechanisms, being stable in the epimastigote insect vector stage, but unstable in the trypomastigote infective stage of the parasite. Its turnover is controlled by an AU-rich element (ARE) localized in the 3'-untranslated region, since a reporter gene lacking this sequence was stable in the trypomastigote stage (Di Noia, J. M., D'Orso, I., Sanchez, D. O., and Frasch, A. C. (2000) J. Biol. Chem. 275, 10218-10227). Here, we show by gel mobility shift assay that the 44-nt ARE sequence interacts with a set of stage-specific AU-rich element RNA-binding proteins (ARE-BPs). The epimastigote stage AU-rich element RNA-binding protein, named E-ARE-BP, and the trypomastigote stage ARE-BPs, named T-ARE-BPs, are efficiently competed by poly(U). UV cross-linking analysis showed that E-ARE-BP has an apparent molecular mass of 100 kDa and is different from the 45-50-kDa ARE-BPs present in other stages of the parasite. Transfection experiments allowed the identification of a novel cis-element that might be responsible for a positive effect on mRNA stability. It is a G-rich element, named GRE, composed by two contiguous CGGGG pentamers. The factors that recognize GRE were different from the ones that bind to ARE, in both molecular masses and subcellular localization. Thus, ARE and GRE are functionally different cis-elements, which might regulate mucin expression throughout the parasite life cycle.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , RNA de Protozoário/genética , Proteínas de Ligação a RNA/metabolismo , Trypanosoma cruzi/genética , Adenina , Animais , Composição de Bases , Sequência de Bases , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Guanina , Dados de Sequência Molecular , Transfecção , Trypanosoma cruzi/crescimento & desenvolvimento , UracilaRESUMO
Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.
Assuntos
Brucella abortus/genética , DNA Bacteriano/química , Genoma BacterianoRESUMO
A random sequence survey of the genome of Trypanosoma cruzi, the agent of Chagas disease, was performed and 11,459 genomic sequences were obtained, resulting in approximately 4.3 Mb of readable sequences or approximately 10% of the parasite haploid genome. The estimated total GC content was 50.9%, with a high representation of A and T di- and trinucleotide repeats. Out of the estimated 5000 parasite genes, 947 putative new genes were identified. Another 1723 sequences corresponded to genes detected previously in T. cruzi through expression sequence tag analysis. 7735 sequences had no matches in the database, but the presence of open reading frames that passed Fickett's test suggests that some might contain coding DNA. The survey was highly redundant, with approximately 35% of the sequences included in a few large sequence families. Some of them code for protein families present in dozens of copies, including proteins essential for parasite survival and retrotransposons. Other sequence families include repetitive DNA present in thousands of copies per haploid genome. Some families in the latter group are new, parasite-specific, repetitive DNAs. These results suggest that T. cruzi could constitute an interesting model to analyze gene and genome evolution due to its plasticity in terms of sequence amplification and divergence. Additional information can be found at http://www.iib.unsam.edu.ar/tcruzi.gss. html.
Assuntos
DNA de Protozoário/análise , Genes de Protozoários/genética , Genoma de Protozoário , Família Multigênica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA/métodos , Trypanosoma cruzi/genética , Animais , Composição de Bases , Sequência de Bases , Sequências Repetitivas Dispersas , Dados de Sequência Molecular , Alinhamento de Sequência , SoftwareRESUMO
Trypanosomes are unable to synthesize the monosaccharide sialic acid, but some African trypanosomes and the American Trypanosoma cruzi can incorporate sialic acid derived from the host. To do so, T. cruzi expresses a trans-sialidase, an enzyme that catalyzes the transfer of sialic acid from host glycoconjugates to mucin-like molecules located on the parasite surface membrane. The importance of the process is indicated by the fact that T. cruzi has hundreds of genes encoding trans-sialidase, trans-sialidase-like proteins and mucin core proteins. Sequence divergence of members of these families has resulted in some molecules having functions unrelated to the acquisition of sialic acid. In this article, Alberto Frasch reviews the structure and possible function of the proteins making up these families.
Assuntos
Mucinas/metabolismo , Neuraminidase/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Animais , Doença de Chagas/parasitologia , Humanos , Mucinas/química , Mucinas/genética , Neuraminidase/química , Neuraminidase/genéticaRESUMO
The protozoan parasite Trypanosoma cruzi, the agent of Chagas disease, has a large number of mucin molecules on its surface, whose expression is regulated during the life cycle. These mucins are the main acceptors of sialic acid, a monosaccharide that is required by the parasite to infect and survive in the mammalian host. A large mucin-like gene family named TcMUC containing about 500 members has been identified previously in T. cruzi. TcMUC can be divided into two subfamilies according to the presence or absence of tandem repeats in the central region of the genes. In this work, T. cruzi parasites were transfected with one tagged member of each subfamily. Only the product from the gene with repeats was highly O-glycosylated in vivo. The O-linked oligosaccharides consisted mainly of beta-d-Galp(1-->4)GlcNAc and beta-d-Galp(1-->4)[beta-d-Galp(1-->6)]-d-GlcNAc. The same glycosyl moieties were found in endogenous mucins. The mature product was anchored by glycosylphosphatidylinositol to the plasma membrane and exposed to the medium. Sera from infected mice recognized the recombinant product of one repeats-containing gene thus showing that they are expressed during the infection. TcMUC genes encode a hypervariable region at the N terminus. We now show that the hypervariable region is indeed present in the exposed mature N termini of the mucins because sera from infected hosts recognized peptides having sequences from this region. The results are discussed in comparison with the mucins from the insect stages of the parasite (Di Noia, J. M., D'Orso, I., Sánchez, D. O., and Frasch, A. C. C. (2000) J. Biol. Chem. 275, 10218-10227) which do not have variable regions.
Assuntos
Mucinas/química , Mucinas/genética , Trypanosoma cruzi/fisiologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/imunologia , Sequência de Bases , Sequência de Carboidratos , Doença de Chagas/imunologia , Epitopos/química , Variação Genética , Glicosilação , Glicosilfosfatidilinositóis/metabolismo , Camundongos , Dados de Sequência Molecular , Mucinas/imunologia , Oligossacarídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Transfecção , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma cruzi has a complex mucin gene family of 500 members with hypervariable regions expressed preferentially in vertebrate associated stages of the parasite. In this work, a novel mucin-type gene family is reported, composed of two groups of genes organized in independent tandems and having very short open reading frames. The structures of deduced proteins share the N and C termini but differ in central regions. One group has repeats with the consensus Lys-Asn-Thr(7)-Ser-Thr(3)-Ser(Ser/Lys)-Ala-Pro and the other a Thr-rich sequence of the type Asp-Gln-Thr(17-20)-Asn-Ala-Pro-Ala-Lys-Asp-Thr(5-7)-Asn-Ala-Pro-Ala-L ys. In both cases, expected mature core proteins are around 7 kDa. Both groups, named L and S, respectively, differ in the structure of genomic loci and mRNA, with differential blocks in the 3'-untranslated region. The highest mRNA level for S and L groups are in the epimastigote stage but they show distinct developmentally regulated patterns. Transcripts are short lived and their steady-state abundance is regulated post-transcriptionally with increased mRNA stability in insect stage epimastigote. AU-rich sequences, similar to ARE motives known to cause mRNA instability in higher eukaryotes, are present in the 3'-untranslated region of the transcripts. In transfection experiments this sequence is shown to be functional for the L group destabilizing its mRNA in a stage-specific manner. Furthermore, an effect of this AU-rich region on translation efficiency is shown. To our knowledge, this is the first time that a functional ARE sequence-dependent post-transcriptional regulation mechanism is reported in a lower eukaryote.
Assuntos
Regiões 3' não Traduzidas/química , Regiões 3' não Traduzidas/genética , Genes de Protozoários , Mucinas/genética , Família Multigênica , Biossíntese de Proteínas , RNA Mensageiro/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Composição de Bases , Pareamento de Bases , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Glicosilação , Dados de Sequência Molecular , Mucinas/química , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Processamento Pós-Transcricional do RNA , RNA de Protozoário/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase, which is involved in key aspects of parasite-host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic beta-propeller fold tightly associated with a lectin-like domain. Comparison with the modeled structure of T.cruzi trans-sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure-based drug design.
Assuntos
Neuraminidase/metabolismo , Sialiltransferases/metabolismo , Trypanosoma/enzimologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neuraminidase/genética , Conformação Proteica , Sialiltransferases/genética , Trypanosoma/genéticaRESUMO
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.
Assuntos
Galactose/metabolismo , Neuraminidase/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Sondas de DNA , Testes de Hemaglutinação , Camundongos , Dados de Sequência Molecular , Neuraminidase/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por SubstratoRESUMO
Proteins containing amino acid repeats are widespread among protozoan parasites. It has been suggested that these repetitive structures act as immunomodulators, but other functional aspects may be of primary importance. We have recently suggested that tandem repeats present in Trypanosoma cruzi trans-sialidase stabilize the catalytic activity in blood. Because the parasite releases trans-sialidase, this delayed clearance of the enzyme might have implications in vivo. In the present work, the ability of repetitive units from different T. cruzi molecules in stabilizing trans-sialidase activity in blood was evaluated. It is shown that repeats present on T. cruzi shed proteins (antigens 13 and Shed-Acute-Phase-Antigen [SAPA]) increase trans-sialidase half-life in blood from 7 to almost 35 hours. Conversely, those repeats present in intracellular T. cruzi proteins only increase the enzyme half-life in blood up to 15 hours. Despite these results, comparative analysis of structural and catalytic properties of both groups of chimeric enzymes show no substantial differences. Interestingly, antigens 13 and SAPA also increase the persistence in blood of chimeric glutathione S-transferases, thus suggesting that this effect is inherent to these repeats and independent of the carrier protein. Although the molecular basis of this phenomenon is still uncertain, its biotechnological potential can be envisaged.
Assuntos
Glicoproteínas/química , Glicoproteínas/farmacocinética , Neuraminidase/química , Neuraminidase/farmacocinética , Sequências de Repetição em Tandem , Trypanosoma cruzi/enzimologia , Animais , Estabilidade Enzimática , Glutationa Transferase/genética , Glicoproteínas/genética , Meia-Vida , Cinética , Camundongos , Camundongos Endogâmicos C3H , Neuraminidase/genética , Proteínas Recombinantes , Relação Estrutura-AtividadeRESUMO
Five years ago the Special Programme for Research and Training in Tropical Diseases (TDR) from the World Health Organization (WHO) launched the Parasite Genome Project. The aims were to obtain information on genome organization and gene discovery in five parasites, namely, Schistosoma, Filaria, Leishmania and Trypanosomas brucei and cruzi. Organization of research networks for each parasite under study, promotion of international collaboration and training of researchers in developing countries, were also main objectives of the programme. After five years, a large amount of information has been obtained, which is now available to researchers in the field.
Assuntos
Redes de Comunicação de Computadores , Bases de Dados como Assunto/organização & administração , Genoma de Protozoário , Trypanosoma cruzi/genética , Animais , DNA de Protozoário/análise , Biblioteca Genômica , Desenvolvimento de ProgramasRESUMO
Analysis of expressed sequence tags (ESTs) constitutes a useful approach for gene identification that, in the case of human pathogens, might result in the identification of new targets for chemotherapy and vaccine development. As part of the Trypanosoma cruzi genome project, we have partially sequenced the 5' ends of 1, 949 clones to generate ESTs. The clones were randomly selected from a normalized CL Brener epimastigote cDNA library. A total of 14.6% of the clones were homologous to previously identified T. cruzi genes, while 18.4% had significant matches to genes from other organisms in the database. A total of 67% of the ESTs had no matches in the database, and thus, some of them might be T. cruzi-specific genes. Functional groups of those sequences with matches in the database were constructed according to their putative biological functions. The two largest categories were protein synthesis (23.3%) and cell surface molecules (10.8%). The information reported in this paper should be useful for researchers in the field to analyze genes and proteins of their own interest.
Assuntos
Mapeamento Cromossômico/métodos , Etiquetas de Sequências Expressas , Genes de Protozoários , Análise de Sequência de DNA/métodos , Trypanosoma cruzi/genética , Animais , DNA Complementar/genética , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência do Ácido NucleicoRESUMO
A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.
Assuntos
Antígenos de Bactérias/genética , Vacinas Bacterianas/genética , Brucella abortus/genética , Vetores Genéticos , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Vacinas Bacterianas/imunologia , Sequência de Bases , Brucella abortus/imunologia , Brucella abortus/fisiologia , Brucelose/microbiologia , Clonagem Molecular , DNA Bacteriano , Feminino , Expressão Gênica , Genes Reporter , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Trypanosoma cruzi/genéticaRESUMO
In previous works we have identified genes in the protozoan parasite Trypanosoma cruzi whose structure resemble those of mammalian mucin genes. Indirect evidence suggested that these genes might encode the core protein of parasite mucins, glycoproteins that were proposed to be involved in the interaction with, and invasion of, mammalian host cells. We now show that the mucin gene family from T. cruzi is much larger and diverse than expected. A minimal number of 484 mucin genes per haploid genome is calculated for a parasite clone. Most, if not all, genes are transcribed, as deduced from cDNA analysis. Comparison of the cDNA sequences showed evidences of a high mutation rate in localized regions of the genes. Sequence conservation among members of the family is much higher in the untranslated (UTR) regions than in the sequences encoding the mature mucin core protein. Transcription units can be classified into two main subfamilies according to the sequence homologies in the 5'-UTR, whereas the 3'-UTR is highly conserved in all clones analyzed. The common origin of members of this gene family as well as their relationships can be defined by sequence comparison of different domains in the transcription units. The regions encoding the N and C termini, supposed to correspond to the leader peptide and membrane-anchoring signal, respectively, (Di Noia, J. M., Sánchez, D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146-24149) are highly conserved. Conversely, the central regions are highly variable. These regions encode the target sites for O-glycosylation and are made of a variable number of repetitive units rich in Thr and Pro residues or are nonrepetitive but still rich in Thr/Ser and Pro residues. The region putatively coding for the N-terminal domain of the mature core protein is hypervariable, being different in most of the transcripts sequenced. Nonrepetitive central domains are unique to each gene. Gene-specific probes show that the relative abundance of different mRNAs varies greatly within the same parasite clone.
Assuntos
Mucinas/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Genes de Protozoários , Dados de Sequência Molecular , Mutação , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
trans-Sialidase inhibition assay (TIA) was employed in a population at high risk of Trypanosoma cruzi infection. From 20 serum samples that were negative by conventional serologic and parasitologic assays, 18 (90%) were reactive in TIA, providing further evidence of the higher sensitivity of TIA and suggesting that the actual prevalence of T. cruzi infection might be underestimated.
Assuntos
Doença de Chagas/sangue , Testes Sorológicos/métodos , Trypanosoma cruzi/isolamento & purificação , Animais , Humanos , Neuraminidase , Sensibilidade e EspecificidadeRESUMO
NADP-linked glutamate dehydrogenase (NADP+-GluDH, EC 1.4.1.4) has been purified to homogeneity from epimastigotes of Trypanosoma cruzi by an improved procedure, and the amino acid sequences of 11 internal peptides obtained by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg-C or CNBr have been obtained. Using oligonucleotide primers synthesized according to the amino acid sequence of the N-terminus of the mature enzyme and to the nucleotide sequence of a clone corresponding to the C-terminus, obtained by immunological screening of an expression library, two complete open reading frames (TcGluDH1 and TcGluDH2) were isolated and sequenced. The sequences obtained are most similar to that of the NADP+-GluDH of Escherichia coli (70-72% identity), and less similar (50-56%) to those of lower eukaryotes. Using TcGluDH1 as a probe, evidence for the presence of several genes and developmental regulation of the expression of NADP+-GluDH in different parasite stages was obtained. TcGluDH1 encodes an enzymically active protein, since its expression in E. coli resulted in the production of a GluDH activity with kinetic parameters similar to those of the natural enzyme.
Assuntos
DNA de Protozoário/química , Desidrogenase de Glutamato (NADP+)/genética , Desidrogenase de Glutamato (NADP+)/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Citosol/enzimologia , Escherichia coli/enzimologia , Evolução Molecular , Desidrogenase de Glutamato (NADP+)/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Trypanosoma cruzi trans-sialidase consists of a C-terminal domain composed essentially of immunodominant amino acid repeat units (SAPA-repeats) and an amino region responsible for the enzymatic activity (catalytic domain). To investigate the possible function(s) of SAPA-repeats, recombinant trans-sialidases either containing or lacking the C-terminal region were tested in mice. The presence of SAPA-repeats in the intravenously injected protein has two consequences. First, they enhance the persistence of the trans-sialidase activity in blood. Second, SAPA-repeats promoted the production of antibodies directed to the catalytic domain that inhibit trans-sialidase activity. These results suggest that SAPA-repeats modulate the trans-sialidase activity in blood.
Assuntos
Doença de Chagas/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Neuraminidase/genética , Neuraminidase/imunologia , Trypanosoma cruzi/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/sangue , Clonagem Molecular , Feminino , Epitopos Imunodominantes/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologia , Sequências Repetitivas de Ácido Nucleico/fisiologiaRESUMO
Both, culture-derived and metacyclic trypomastigotes of Trypanosoma cruzi shed a glycoprotein, the shed acute phase antigen, that is responsible for the trans-sialidase activity. In the present work the structure of the glycosylphosphatidylinositol membrane anchor of the trans-sialidase isolated from metacyclic forms was determined. Parasites were metabolically labelled with [9, 10(n)3H]-palmitic acid and the glycoprotein was purified by immunoprecipitation with a monoclonal antibody directed against the repetitive aminoacid sequence. Treatment of the glycoprotein with phosphatidylinositol phospholipase C (PI-PLC) from Bacillus thuringiensis rendered a lipid that comigrated in TLC with a standard of ceramide. No alkylglycerol was detected in contrast with the results previously obtained for the trans-sialidase isolated from culture-derived trypomastigotes where both lipids were found. Chemical and chromatographic analysis showed that the lipid moiety is palmitoyldihydrosphingosine with a minor amount of stearoyldihydrosphingosine. The glycan constituent of the glycosylphosphatidylinositol-anchor was analysed by nitrous acid deamination of the aqueous phase of the PI-PLC treatment, followed by reduction with NaBH4 and hydrolysis of the phosphodiester with aqueous hydrofluoric acid. A major oligosaccharide was obtained and enzymatic treatment with exoglycosidases and further chromatography in a high pH anion exchange system showed that the trimannosyl core backbone is substituted by an alpha-galactose. A comparison between the lipid constituent of the glycosylphosphatidylinositol anchor of several proteins and their spontaneous shedding by the action of an endogenous PI-PLC was made.