RESUMO
Echinococcus granulosus protoscolex proteins were separated using two-dimensional electrophoresis and then identified using mass spectrometry; we identified 61 proteins, 28 which are newly described of which 4 could be involved in hydatid cyst fertility molecular mechanisms.
Assuntos
Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Proteínas de Helminto/metabolismo , Proteômica , Animais , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Estágios do Ciclo de VidaRESUMO
Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a direct interaction between these vesicles and sperm, whereas inhibition of some capacitation-dependent features suggests a stabilizing function for exosomes in boar semen.
Assuntos
Exossomos/fisiologia , Proteínas/química , Sêmen/fisiologia , Espermatozoides/fisiologia , Suínos/fisiologia , Reação Acrossômica , Animais , Eletroforese em Gel de Poliacrilamida , Exossomos/metabolismo , Metabolismo dos Lipídeos , Masculino , Sêmen/metabolismo , Análise de Sequência de ProteínaRESUMO
BACKGROUND: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B). RESULTS: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites. CONCLUSIONS: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.
Assuntos
Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Giardia lamblia/genética , Giardíase/parasitologia , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Membrana Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Feminino , Giardia lamblia/classificação , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Alinhamento de Sequência , Trofozoítos/química , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/metabolismoRESUMO
Echinococcus granulosus, the agent of hydatid disease, presents an indirect life cycle, with canines (mainly dogs) as definitive hosts, and herbivores and human as intermediary ones. In intermediary hosts fertile and infertile cysts develop, but only the first ones develop protoscoleces, the parasite form infective to definitive hosts. We report the presence of bovine IgGs in the germinal layer from infertile cysts (GLIC), in an order of magnitude greater than in the germinal layer from fertile cysts (GLFC). When extracted with salt solutions, bovine IgGs from GLIC are associated with low or with high affinity (most likely corresponding to non specific and antigen specific antibodies, respectively). Specific IgGs penetrate both the cells of the germinal layer and HeLa cultured cells and recognize parasitic proteins. These results, taken together with previous ones from our laboratory, showing induction of apoptosis in the germinal layer of infertile hydatid cysts, provide the first coherent explanation of the infertility process. They also offer the possibility of identifying the parasite antigens recognized, as possible targets for immune modulation.
Assuntos
Doenças dos Bovinos/imunologia , Equinococose/veterinária , Echinococcus granulosus/imunologia , Infertilidade/veterinária , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Equinococose/imunologia , Equinococose/metabolismo , Echinococcus granulosus/metabolismo , Células HeLa , Humanos , Imunidade Humoral , Infertilidade/imunologia , Microscopia de FluorescênciaRESUMO
Trypanosoma cruzi, the agent of the American trypanosomiasis or Chagas disease, bypasses its lack of de novo synthesis of sialic acids by expressing a surface-anchored trans-sialidase. This enzyme transfers sialic acid residues from the host's sialylglycoconjugates to the parasite's galactosylglycoconjugates. In addition to carrying out a pivotal role in parasite persistence/replication within the infected mammal, the trans-sialidase is shed into the bloodstream and induces alterations in the host immune system by modifying the sialylation of the immune cells. A major obstacle to understand these events is the difficulty to identify the transferred sialic acid among all those naturally occurring on the cell surface. Here, we report the use of azido-modified unnatural sialic acid to identify those molecules that act as cell surface acceptors of the sialyl residue in the trans-sialidase-catalyzed reaction, which might then be involved in the immune alterations induced. In living parasites, we readily observed the transfer of azido-sialic acid to surface mucins. When evaluating mouse thymocytes and splenocytes as acceptors of the azido-sugar, a complex pattern of efficiently tagged glycoproteins was revealed. In both leukocyte populations, the main proteins labeled were identified as different CD45 isoforms. Disruption of the cell architecture increased the number and the molecular weight distribution of azido-sialic acid tagged proteins. Nevertheless, CD45 remained to be the main acceptor. Mass spectrometry assays allowed us to identify other acceptors, mainly integrins. The findings reported here provide a molecular basis to understand the abnormalities induced in the immune system by the trans-sialidase during T. cruzi infection.
Assuntos
Glicoproteínas/química , Linfócitos/metabolismo , Neuraminidase/química , Proteínas de Protozoários/química , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/química , Animais , Glicoproteínas/metabolismo , Glicosilação , Interações Hospedeiro-Parasita , Humanos , Células Jurkat , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Fatores de Virulência/metabolismoRESUMO
OBJECTIVE: To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction. DESIGN: Prospective study. SETTING: Basic research laboratory. SUBJECT(S): Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET. INTERVENTION(S): Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78). MAIN OUTCOME MEASURE(S): Assessment of protein expression and secretion by immunohistochemistry and Western immunoblotting, respectively. Evaluation of rec-Grp78 binding to human spermatozoa by immunocytochemistry, and analysis of its effect upon gamete interaction using the hemizona assay. RESULT(S): Grp78 was found in the surface of oviduct epithelial cells. Soluble Grp78 was detected in oviductal fluids from women in the periovulatory period and in oviductal tissue conditioned medium. Rec-Grp78 was able to bind to the sperm acrosomal cap, and its presence during gamete interaction led to a decrease in the number of spermatozoa bound to the zona pellucida (ZP). When calcium ions from the incubation medium were replaced by strontium, rec-Grp78 enhanced sperm-ZP interaction. CONCLUSION(S): Grp78 is expressed and secreted by oviduct epithelial cells. The protein would bind to the gametes and may modulate their interaction in a calcium-dependent manner.
Assuntos
Células Epiteliais/metabolismo , Tubas Uterinas/metabolismo , Proteínas de Choque Térmico/metabolismo , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Adulto , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Imuno-Histoquímica , Masculino , Ciclo Menstrual , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes/metabolismo , Técnicas de Cultura de TecidosRESUMO
Galectin (Gal) constitute a family of carbohydrate-recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal-8 splice variants described previously in the thymus. Gal-8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4(+) T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCR(OVA). The antigen-independent proliferation induced by Gal-8 displayed increased expression of pro- and anti-inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen-specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal-8 counter-receptors. Gal-8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen-dependent and -independent T cell-proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal-8 as an enhancer of otherwise borderline immune responses and also suggest that Gal-8 might fuel the reactivity at inflammatory foci.
Assuntos
Proliferação de Células , Galectinas/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Galectinas/genética , Galectinas/farmacologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Células Jurkat , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Equinococose , Echinococcus granulosus/fisiologia , Fertilidade/fisiologia , Proteínas de Helminto/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Reparo do DNA , Echinococcus granulosus/anatomia & histologia , Proteínas de Helminto/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxidantes/metabolismo , Oxirredução , Estresse Oxidativo , Filogenia , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de SequênciaRESUMO
A trypsin inhibitor was purified from Calliandra selloi Macbride seeds (CSTI). SDS-PAGE under non-reducing conditions showed a single band of approximately 20,000 Da, while under reducing conditions two bands of 16,000 and 6000 Da were observed, indicating that CSTI consists of two polypeptide chains. Molecular masses of 20,078 and 20,279 were obtained by mass spectrometry, although only one pI of 4.0 was observed and one peak was obtained by reversed phase chromatography. Amino-terminal sequence analysis showed homology to Kunitz-type inhibitors. CSTI was able to inhibit trypsin (Ki 2.21 x 10(-7)M), alpha-chymotrypsin (Ki 4.95 x 10(-7)M) and kallikrein (Ki 4.20 x 10(-7)M) but had no effect on elastase. Trypsin inhibitory activity was stable over a wide range of pH and temperature. CSTI was particularly susceptible to DTT treatment, followed by addition of iodoacetamide. Far-UV circular dichroism measurements revealed that CSTI is a beta-II protein. Thermal unfolding showed a two-state transition with a midpoint at 68 degrees C. Far-UV CD spectra of CSTI at pH extremes showed little changes, while more pronounced differences in near-UV CD spectra were detected. Remarkably, treatment with 1mM DTT caused very slight changes in the far-UV CD spectrum, and only after carbamidomethylation was there was a marked loss observed in secondary structure.
Assuntos
Fabaceae , Sementes/enzimologia , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificação , Animais , Células Cultivadas , Eritrócitos/efeitos dos fármacos , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Coelhos , Espectrometria de Fluorescência , Inibidores da Tripsina/farmacologiaRESUMO
The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.