RESUMEN
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.
Asunto(s)
Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Proteínas del Helminto/metabolismo , Proteómica , Animales , Electroforesis en Gel de Poliacrilamida , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Estadios del Ciclo de VidaRESUMEN
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.
Asunto(s)
Exosomas/fisiología , Proteínas/química , Semen/fisiología , Espermatozoides/fisiología , Porcinos/fisiología , Reacción Acrosómica , Animales , Electroforesis en Gel de Poliacrilamida , Exosomas/metabolismo , Metabolismo de los Lípidos , Masculino , Semen/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
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.
Asunto(s)
Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica , Giardia lamblia/genética , Giardiasis/parasitología , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Membrana Celular , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/inmunología , Femenino , Giardia lamblia/clasificación , Giardia lamblia/crecimiento & desarrollo , Giardia lamblia/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Alineación de Secuencia , Trofozoítos/química , Trofozoítos/crecimiento & desarrollo , Trofozoítos/metabolismoRESUMEN
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.
Asunto(s)
Enfermedades de los Bovinos/inmunología , Equinococosis/veterinaria , Echinococcus granulosus/inmunología , Infertilidad/veterinaria , Animales , Bovinos , Enfermedades de los Bovinos/metabolismo , Enfermedades de los Bovinos/patología , Equinococosis/inmunología , Equinococosis/metabolismo , Echinococcus granulosus/metabolismo , Células HeLa , Humanos , Inmunidad Humoral , Infertilidad/inmunología , Microscopía FluorescenteRESUMEN
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.
Asunto(s)
Glicoproteínas/química , Linfocitos/metabolismo , Neuraminidasa/química , Proteínas Protozoarias/química , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/patogenicidad , Factores de Virulencia/química , Animales , Glicoproteínas/metabolismo , Glicosilación , Interacciones Huésped-Parásitos , Humanos , Células Jurkat , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Neuraminidasa/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Factores de Virulencia/metabolismoRESUMEN
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.
Asunto(s)
Células Epiteliales/metabolismo , Trompas Uterinas/metabolismo , Proteínas de Choque Térmico/metabolismo , Interacciones Espermatozoide-Óvulo , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Adulto , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Femenino , Humanos , Inmunohistoquímica , Masculino , Ciclo Menstrual , Persona de Mediana Edad , Unión Proteica , Proteínas Recombinantes/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
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.
Asunto(s)
Proliferación Celular , Galectinas/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/metabolismo , Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Galectinas/genética , Galectinas/farmacología , Humanos , Inflamación/inmunología , Inflamación/fisiopatología , Células Jurkat , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Equinococosis , Echinococcus granulosus/fisiología , Fertilidad/fisiología , Proteínas del Helminto/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Reparación del ADN , Echinococcus granulosus/anatomía & histología , Proteínas del Helminto/genética , Humanos , Peróxido de Hidrógeno/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxidantes/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Filogenia , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Fabaceae , Semillas/enzimología , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación , Animales , Células Cultivadas , Eritrocitos/efectos de los fármacos , Calor , Concentración de Iones de Hidrógeno , Peso Molecular , Conejos , Espectrometría de Fluorescencia , Inhibidores de Tripsina/farmacologíaRESUMEN
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.
Asunto(s)
Conotoxinas/toxicidad , Receptores Nicotínicos/fisiología , Marcadores de Afinidad , Animales , Sitios de Unión , Ligandos , Mapeo Peptídico , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Receptores Colinérgicos , Receptores Nicotínicos/química , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/aislamiento & purificación , TorpedoRESUMEN
In 2001, a University of California, Davis-University of the Republic, Montevideo, partnership created a Fogarty ITREOH program to exploit the potential of ELISA to provide a low-cost environmental analysis attractive to economically distressed countries of temperate South America. This paper describes the development and validation of an ELISA method for the determination of Cyanobacteria microcystin toxins in algal blooms, which release hepatotoxic metabolites that can reach toxic levels in rivers, lakes, or coastal estuaries used for recreation or water supplies. The assay made possible the first systematic monitoring of water from the Rio de la Plata at Montevideo over two summers. The project has been integrated into a bi-national effort to monitor the Rio de la Plata.
Asunto(s)
Recreación , Abastecimiento de Agua/normas , Toxinas Bacterianas/análisis , Cianobacterias/química , Ensayo de Inmunoadsorción Enzimática , Microbiología del AguaAsunto(s)
Histonas/fisiología , Proteínas Protozoarias/fisiología , Toxoplasma/química , Secuencia de Aminoácidos , Animales , Ciclo Celular , Reparación del ADN , ADN Protozoario/genética , Expresión Génica , Histonas/genética , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Alineación de Secuencia , Toxoplasma/citología , Toxoplasma/genética , Toxoplasma/crecimiento & desarrolloRESUMEN
Protoscolices of the parasitic helminth Echinococcus granulosus contain two malate dehydrogenases (EC 1.1.1.37), one cytosolic and one mitochondrial. The latter has been separated from the other isoform and purified to protein homogeneity. Sequencing of tryptic peptides by Edman degradation allowed the design of oligonucleotide primers for PCR, leading to the cloning and sequencing of a full length cDNA. The encoding gene is present as a single copy per haploid genome and codes for a protein with high sequence identity (56-58%) with the similar enzymes from mammals, Caenorhabditis elegans and yeast. Active recombinant mitochondrial malate dehydrogenase was expressed in Escherichia coli, as protein fusions with glutathione S-transferase or a poly-His tail. The purified recombinant enzymes had a kinetic behaviour similar to that of the native enzyme, being inhibited by excess of the substrate oxaloacetate and unaffected by excess L-malate. The results indicate that E. granulosus contains two typical eukaryotic malate dehydrogenases, with relative levels quite different from those present in mammalian tissues like heart, in good agreement with the predominantly fermentative metabolism of the protoscolices.
Asunto(s)
Echinococcus granulosus/enzimología , Echinococcus granulosus/genética , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/genética , Expresión Génica , Genes de Helminto , Cinética , Malato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.
Asunto(s)
Cromatina/química , Echinococcus/química , Fasciola hepatica/química , Histonas/química , Histonas/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Aminoácidos/química , Aminoácidos/metabolismo , Animales , Bovinos , Cromatina/metabolismo , Cromatografía Líquida de Alta Presión , Echinococcus/metabolismo , Electroforesis en Gel de Poliacrilamida , Fasciola hepatica/metabolismo , Histonas/genética , Histonas/metabolismo , Erizos de Mar/química , Erizos de Mar/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Timo/química , Timo/metabolismo , Trypanosoma cruzi/química , Trypanosoma cruzi/metabolismoRESUMEN
MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and proline-directed kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas/química , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Animales , Anticuerpos Monoclonales , Sitios de Unión , Encéfalo/citología , Encéfalo/embriología , Diferenciación Celular , Embrión de Pollo , Lipoproteínas/metabolismo , Lipoproteínas/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neuronas/química , Neuronas/citología , Fosfoproteínas , Fosforilación , Retina/química , Retina/citología , Retina/embriología , Serina/inmunología , Serina/metabolismoRESUMEN
Trypanosoma cruzi, the flagellate protozoan which is the causative agent of the American trypanosomiasis, Chagas disease has carboxypeptidase activity. The enzyme has been purified to protein homogeneity, and shown to be a lysosomal monomeric glycoprotein with a molecular mass of about 54kDa. The enzyme has an optimum acidic pH (4.5 with furyl acryloyl-Phe-Phe as substrate), is highly specific for hydrophobic C-terminal amino acid residues, and is strongly inhibited by 3,4-dichloroisocoumarin (IC(50) value 0.3 microM). The enzyme is encoded by a number of genes arrayed in head-to-tail tandems; one of these genes has been cloned and sequenced. Sequence comparisons indicate that the enzyme belongs to the C group of serine carboxypeptidases, within the S10 serine peptidase family, and shows the higher similarity to plant and yeast enzymes. The residues involved in catalysis and most of those involved in substrate binding are conserved in the T. cruzi enzyme as well as 8 out of 10 Cys residues known to be involved in disulfide bridges in the yeast enzyme. This is the first report of an S10 family enzyme in trypanosomatids. The presence of serine carboxypeptidases is not restricted to T. cruzi, being possibly a general character of trypanosomatids.
Asunto(s)
Carboxipeptidasas , Lisosomas/enzimología , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Carboxipeptidasas/química , Carboxipeptidasas/genética , Carboxipeptidasas/aislamiento & purificación , Carboxipeptidasas/metabolismo , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.
Asunto(s)
Apoptosis/efectos de los fármacos , Fabaceae , Linfoma/patología , Inhibidores de Tripsina/farmacología , Secuencia de Aminoácidos , Animales , Supervivencia Celular/efectos de los fármacos , Cromatografía de Afinidad , Lectinas , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Fitoterapia , Ratas , Semillas , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38 kDa subunits. Whereas the 22 kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38 kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the O gamma atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.
Asunto(s)
Antígenos Helmínticos/metabolismo , Echinococcus/inmunología , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/química , Secuencia de Bases , Catálisis , Adhesión Celular , Cartilla de ADN , ADN Complementario , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Tripsina/químicaRESUMEN
Vespid venoms contain Antigen 5, an important allergen whose primary structure and immunological behavior have been extensively studied from venoms of vespids of the Northern Hemisphere. We report herein structural and immunological aspects of Antigen 5 from Polybia scutellaris subspecies rioplatensis (vulgar name: camoati) found in South America. Mast cell degranulation, histamine release, and IgE induction experiments performed in mice allow us to suggest that P. scutellaris Antigen 5 is a variant with reduced IgE response and anaphylactic activity. Sequence data indicate that the protein has a 72.5-90.3% similarity to that of members of the vespid Antigen 5 family with an already known primary structure. Moreover, results suggest that the protein-a new member of an extracellular protein superfamily-could be a good candidate for immunotherapy related to vespid allergy.