RESUMO
Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense glycocalix mainly composed by glycoproteins called mucins which are also the acceptors of sialic acid in a reaction catalyzed by a trans-sialidase (TcTS). Sialylation of trypomastigote mucins protects the parasite from lysis by the anti α-Galp antibodies from serum. The TcTS is essential for the infection process since T. cruzi is unable to biosynthesize sialic acid. The enzyme specifically transfers it from a terminal ß-d-Galp unit in the host glycoconjugate to terminal ß-d-Galp units in the parasite mucins to construct the d-NeuNAc(α2â3)ß-d-Galp motif. On the other hand, although galactose is the most abundant sugar in mucins of both, the infective trypomastigotes and the insect stage epimastigotes, α-d-Galp is only present in the infective stage whereas ß-d-Galf is characteristic of the epimastigote stage of the less virulent strains. Neither α-d-Galp nor d-Galf is acceptor of sialic acid. In the mucins, some of the oligosaccharides are branched with terminal ß-d-Galp units to be able to accept sialic acid in the TcTS reaction. Based on previous reports showing that anti α-Galp antibodies only partially colocalize with sialic acid, we have undertaken the synthesis of the trisaccharide α-d-Galp(1â3)-[ß-d-Galp(1â6)]-d-Galp, the smallest structure containing both, the antigenic d-Galp(α1â3)-d-Galp unit and the sialic acid-acceptor ß-d-Galp unit. The trisaccharide was obtained as the 6-aminohexyl glycoside to facilitate further conjugation for biochemical studies. The synthetic approach involved the α-galactosylation at O-4 of a suitable precursor of the reducing end, followed by ß-galactosylation at O-6 of the same precursor and introduction of the 6-aminohexyl aglycone. The fully deprotected trisaccharide was successfully sialylated by TcTS using either 3'-sialyllactose or fetuin as donors. The product, 6-aminohexyl α-d-NeuNAc(2â3)-ß-d-Galp(1â6)-[α-d-Galp(1â3)]-ß-d-Galp, was purified and characterized.
Assuntos
Anticorpos/química , Glicoproteínas/metabolismo , Neuraminidase/metabolismo , Trissacarídeos/síntese química , Trypanosoma cruzi/metabolismo , Anticorpos/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Sequência de Carboidratos , Técnicas de Química Sintética , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas Periplásmicas de Ligação/imunologia , Trissacarídeos/metabolismoRESUMO
The CLEC16A gene has an important role in the immune activation and regulation inflammatory. This gene encodes to C-type lectin domain that is involved in the recognition of DAMPS. The aim of this study was assess the CLEC16A gene polymorphisms in the risk of developing ACS in a group of patients. Four rs12708716, rs12917716, rs6498142 and rs9925481 (positions 146529 A>G, 155804 G>C, 47905 C>G and 64135 C>T, respectively) single nucleotide polymorphisms of CLEC16A gene were analyzed by TaqMan assays in a group of 452 patients with ACS and 456 healthy controls. The analysis was performed on the total group of individuals and then in groups of men and women separately. Under co-dominant model adjusted by cardiovascular risk factors the rs12708716 (146529 A>G) and rs12917716 (155804 G>C) polymorphisms were significantly associated with decrease risk of ACS in men (OR=0.16, PCo-dom=0.027 and OR=0.37, PCo-dom=0.016, respectively). In summary, our data suggests that two polymorphisms of the CLEC16A gene play an important role in the developing of ACS in men.
Assuntos
Síndrome Coronariana Aguda/genética , Lectinas Tipo C/genética , Modelos Genéticos , Proteínas de Transporte de Monossacarídeos/genética , Polimorfismo Genético , Caracteres Sexuais , Síndrome Coronariana Aguda/etnologia , Síndrome Coronariana Aguda/imunologia , Idoso , Feminino , Humanos , Lectinas Tipo C/imunologia , Masculino , México , Pessoa de Meia-Idade , Proteínas de Transporte de Monossacarídeos/imunologia , Fatores de RiscoRESUMO
Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.
Assuntos
Proteínas de Ligação ao Cálcio , Membrana Eritrocítica/metabolismo , Proteínas de Helminto/isolamento & purificação , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Proteínas Periplásmicas de Ligação , Trypanosoma cruzi/fisiologia , Animais , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/metabolismo , Endotélio Vascular/parasitologia , Membrana Eritrocítica/parasitologia , Imunofluorescência , Galactose/metabolismo , Coração/parasitologia , Proteínas de Helminto/imunologia , Proteínas de Helminto/fisiologia , Humanos , Soros Imunes/imunologia , Imuno-Histoquímica , Lectinas , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas de Transporte de Monossacarídeos/fisiologia , Coelhos , Trypanosoma cruzi/químicaRESUMO
An immunogenic nontoxic protein (TsNTxP) was purified from the venom of the scorpion Tityus serrulatus (Ts). This peptide is composed of 63 amino acid residues with a high degree of structural homology with the toxins isolated from Ts. The nucleotide sequence of the gene that encodes TsNTxP was obtained and also showed a high degree of similarity with genes encoding Tityus toxins [Guatimosim, S.C.F., Prado, V.F., Diniz, C.R., Chávez-Olórtegui, C.. Kalapothakis, E., 1999. Molecular cloning and genomic analysis of TsNTxP; an immunogenic protein from Tityus serrulatus scorpion venom. Toxicon 37, 507-517]. In the present study the TsNTxP gene was expressed in E. coli BL21DE3 cells as a fusion protein with maltose-binding protein. The recombinant protein (TsNTxPrec) was purified by affinity chromatography and used as an immunogen in rabbits. The antigenic specificity of anti-TsNTxPrec antibodies was compared by an enzyme-linked immunosorbent assay using TsNTxP, TstFG50 (the fraction of Ts venom that represents most of the toxicity of the crude venom) and the crude venom, to coat microtitration plates. Anti-TsNTxPrec antibodies had a comparable high cross-reactivity for all antigens tested. Concentrations of Ts venom equivalent to 20 LD50 were effectively neutralized by 1 ml of the anti-TsNTxPrec serum. This result provides basic data for the use of such recombinant scorpion protein as an immunogen in the development of antivenoms for clinical use.
Assuntos
Antivenenos/biossíntese , Imunização , Proteínas de Transporte de Monossacarídeos/imunologia , Neurotoxinas/imunologia , Venenos de Escorpião/imunologia , Vacinas Sintéticas/imunologia , Animais , Antivenenos/isolamento & purificação , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Feminino , Vetores Genéticos , Camundongos , Neurotoxinas/toxicidade , Testes de Neutralização , Coelhos/imunologia , Proteínas Recombinantes/imunologia , Venenos de Escorpião/toxicidade , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
OBJECTIVE: To investigate the effect of weight loss on GLUT 4 content of insulin sensitive tissues of obese mice. SUBJECTS: Mice were made obese by neonatal treatment with monosodium glutamate (MSG). In addition, one group of obese animals was submitted to a caloric restriction to promote 20% weight loss (MSG-L). Both groups were compared to age-matched control mice. MEASUREMENTS: Anthropometric data, glycaemia and insulinaemia were measured. The GLUT 4 protein was assessed by Western blotting analysis in white (WAT) and brown (BAT) adipose tissue, and skeletal (SM) and cardiac (CM) muscles. RESULTS: The MSG mice were very obese according to their morphometric analysis, showing moderate hyperglycaemia with severe hyperinsulinaemia, and reduced (P < 0.001) glucose/insulin (G/I) ratio. The procedure for weight loss promoted a significant (P < 0.001) reduction of both glycaemic and insulinaemic levels, and an increase in G/I ratio. Compared to control animals, the GLUT 4 content in obese MSG mice, was decreased by 30% (P < 0.05) in SM and CM, by 80% (P < 0.001) in BAT and in different subcellular membrane fractions of WAT. On the other hand, transporter protein content was restored to normal levels in MSG-L animals. CONCLUSION: The reduced GLUT 4 content of insulin sensitive tissues from MSG-treated obese mice is recovered by a 20% loss in weight. This mechanism can be involved in the observed increase of insulin sensitivity.