RESUMEN
BACKGROUND: Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) are parasite features that have been suggested to influence the acquisition of protective immunity against malaria. This study sought to assess the relationship between MOI and parasite density (PD) in malaria patients living in the Central Region of Ghana and to determine whether naturally occurring antibody levels against P. falciparum GLURP (PF3D7_1035300) and MSP3 (PF3D7_1035400) antigens are associated with decreased parasite load. METHODS: Dried filter paper blood blots were obtained from children and adults diagnosed with uncomplicated P. falciparum malaria. Microscopy was used to estimate P. falciparum parasite density and polymerase chain reaction (PCR) amplification of the polymorphic regions of msp1 (PF3D7_0930300) and msp2 (PF3D7_0206800) was used for parasite genotyping and MOI determination. ELISA was used to measure the serum IgG concentration of R0 fragment of GLURP (GLURP(R0)) and MSP3 antibodies. RESULTS: All 115 samples were positive for P. falciparum by PCR using either the msp1 or msp2 genotyping primer sets. The most prevalent msp1 and msp2 alleles were KI and 3D7, respectively. The geometric mean (GM) for MOI determined by both msp1 and msp2 genotyping was 1.3 for the entire population and was generally higher in children than in adults. Seropositivity was estimated at 67 and 63% for GLURP(R0) and MSP3 antibodies, respectively, and antibody titers were negatively correlated with parasite density. CONCLUSIONS: The negative correlation between naturally occurring GLURP(R0) and MSP3 antibody levels and parasite density observed in this study suggest that augmenting the antibody response with the GMZ2 vaccine could enhance protection in the Central Region of Ghana.
Asunto(s)
Antígenos de Protozoos/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Carga de Parásitos , Plasmodium falciparum/inmunología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/inmunología , Adolescente , Adulto , Animales , Antígenos de Protozoos/genética , Niño , Preescolar , Pruebas con Sangre Seca , Ensayo de Inmunoadsorción Enzimática , Femenino , Variación Genética , Genotipo , Ghana/epidemiología , Humanos , Inmunoglobulina G/sangre , Lactante , Vacunas contra la Malaria/inmunología , Malaria Falciparum/epidemiología , Masculino , Proteína 1 de Superficie de Merozoito/genética , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Adulto JovenRESUMEN
During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.
Asunto(s)
Antimaláricos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Leucina/análogos & derivados , Oocistos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Animales , Culicidae , Cisteína Endopeptidasas/biosíntesis , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente Indirecta , Gametogénesis/efectos de los fármacos , Humanos , Immunoblotting , Técnicas In Vitro , Leucina/farmacología , Plasmodium falciparum/metabolismoRESUMEN
Antibodies raised against an Escherichia coli-produced recombinant protein encoding a 76-kDa section (region C) of malaria transmission-blocking vaccine candidate, Pfs230, have previously been shown to significantly reduce the ability of Plasmodium falciparum parasites to infect mosquitoes (71.2-89.8%). To further define the region of the Pfs230 required for transmission-blocking activity, four recombinant proteins each encoding a section of region C (Pfs230 amino acids 443-1132) were produced using the same E. coli expression system and tested for immunogenicity in mice: (i) r230/MBP.C5' encodes the first half of region C (amino acids 443-791, six cysteines); (ii) r230/MBP.CM1 encodes only cysteine motif (CM) 1 (amino acids 583-913, eight cysteines); (iii) r230/MBP.C1.6 (amino acids 453-913, eight cysteines) also includes all of CM1; and (iv) r230/MBP.C2 encodes only CM2 (amino acids 914-1268, 11 cysteines). All the recombinant proteins induced antibodies that recognized parasite-produced Pfs230, but the titre of the Pfs230 specific-antibodies generated varied, C = C1.6 = C5' > CM1 > CM2. Two recombinants, r230/MBP.C5' and r230/MBP.C1.6, induced antibody titres that were equivalent to or greater than the titre generated by r230/MBP.C. However, in contrast to r230/MBP.C, none of the recombinants induced antibodies that effectively blocked parasite infectivity to mosquitoes. This suggests that the inclusion of amino acids 914-1132 is important for the production of the transmission-blocking epitope present in region C.
Asunto(s)
Anopheles/parasitología , Anticuerpos Antiprotozoarios/biosíntesis , Vacunas contra la Malaria/inmunología , Malaria Falciparum/transmisión , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Malaria Falciparum/inmunología , Ratones , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunación , Vacunas Sintéticas/inmunologíaRESUMEN
Soluble epoxide hydrolase (sEH) is suggested to alter the mode of action and increase the toxic potency of fatty acid epoxides. To characterize the structural features necessary for sEH-dependent epoxy fatty acid toxicity, 75 aliphatic compounds were assayed for cytotoxicity in the presence and absence of sEH. Three groups of aliphatic epoxide-diol pairs were described by their observed differential toxicity. Group I compounds were typified by terminal epoxides whose toxicity was reduced in the presence of sEH. Group II compounds were toxic in either their epoxide or diol form, but toxicity was unaffected by sEH. Group III compounds exhibited sEH-dependent toxicity and were therefore used to investigate the structural elements required for cytotoxicity in this study. The optimal structure for group III compounds appeared to be a fatty acid 18-20 atoms long (e.g., a carbon backbone plus a terminal heteroatom) with an epoxide positioned between C-7 and C-12. In the absence of sEH, replacement of epoxides with a vicinal diol was required for toxicity. While diol stereochemistry was unimportant, vicinal diol-induced toxicity exhibited fewer positional constraints to toxicity than sEH-dependent epoxide toxicity. Tested fatty acids and esters with neither an epoxide nor a vicinal diol were not toxic. These data support the hypothesis that long-chain epoxy fatty acid methyl esters are potential pro-toxins metabolized by sEH to more toxic diols. Furthermore, our results suggest that the endogenous compounds, leukotoxin methyl ester, 9,10(Z)-epoxyoctadec-12(Z)-enoic acid methyl ester, and isoleukotoxin methyl ester, 12, 13(Z)-epoxyoctadec-9(Z)-enoic acid methyl ester, are structurally optimized to elicit the observed effect.
Asunto(s)
Epóxido Hidrolasas/fisiología , Compuestos Epoxi/toxicidad , Exotoxinas/toxicidad , Ácidos Grasos/toxicidad , Humanos , Relación Estructura-ActividadRESUMEN
Leukotoxin (ltx) and isoleukotoxin (iltx) methyl esters, are metabolites of methyl linoleic acid, an essential fatty acid. They have been associated with acute respiratory distress syndrome. The observed toxicity of ltx and iltx is, in fact, due to the metabolism of the epoxides to their corresponding diols by soluble epoxide hydrolase (sEH). Herein, we demonstrate that ltx/iltx are toxic in a time-dependent manner to human sEH expressing cells with a LT(50) of 10.6 +/- 0.8 h and that ltx and iltx have K(M) of 6.15 +/- 1.0 and 5. 17 +/- 0.56 microM, respectively, and V(max) of 2.67 +/- 0.04 and 1. 86 +/- 0.06 micromol/min/mg, respectively, which can be inhibited by sEH inhibitors. We show that four major metabolites of ltx/iltx are formed in our system, including ltx/iltx free acid, ltxd/iltxd, free acid, and phosphotidylcholine and phosphotidylethanolamine containing the carboxylic acid forms of both ltx/iltx and ltxd/iltxd, but that the only metabolite associated with toxicity is the carboxylic acid form of ltxd/iltxd, suggesting the involvement of cellular esterases. We demonstrate that a serine esterase inhibitor provides some protection from the toxicity of epoxy fatty esters to sEH expressing cells as do intercellular free sulfhydryls, but that this protection is not due to glutathione conjugation. With these data, we have proposed an extension of the metabolic pathway for ltx/iltx in eukaryotic cells.
Asunto(s)
Compuestos Epoxi/metabolismo , Compuestos Epoxi/toxicidad , Ácidos Linoleicos/metabolismo , Ácidos Linoleicos/toxicidad , Animales , Ácidos Carboxílicos/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Esterasas/antagonistas & inhibidores , Esterasas/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Glutatión/farmacología , Glicerofosfolípidos/metabolismo , Humanos , Inactivación Metabólica , Cinética , Modelos Biológicos , Fluoruro de Fenilmetilsulfonilo/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Compuestos de Sulfhidrilo/antagonistas & inhibidores , Compuestos de Sulfhidrilo/metabolismoRESUMEN
During Plasmodium falciparum gametogenesis, proteolysis of Pfs230, a 360 kDa gametocyte surface protein, generates two large polypeptides, 307 and 300 kDa, that remain associated with the surface of the newly formed gamete. Using peptide specific antibodies, the amino termini of the 307 and 300 kDa forms have been mapped to between aa 477-487 and aa 523-555, respectively, which is the region between the glutamate rich repeats and the cysteine motif domains. Concomitantly, two peptides, 47 and 35 kDa, corresponding to the region upstream from the cleavage site are released into the medium. The membrane permeant cysteine protease inhibitor, E64d, blocks production of the 300 and 35 kDa forms of Pfs230, but does not alter the formation of the 307 or 47 kDa forms. In contrast, E64, which has been shown to inhibit the development of P. falciparum trophozoites, does not block proteolytic processing of Pfs230. Production of both the 307 and 300 kDa forms was reduced by a metallo-protease inhibitor, 1,10-phenanthroline, whereas the rest of the protease inhibitors tested had no effect on Pfs230 processing. This is the first study of proteolysis during gametogenesis and it demonstrates that the two large forms of Pfs230 produced are generated by proteases with different specificities. The data also suggest that Pfs230 undergoes proteolytic processing prior to emergence from the red blood cell.
Asunto(s)
Antígenos de Protozoos/análisis , Antígenos de Superficie/análisis , Gametogénesis/inmunología , Plasmodium falciparum/inmunología , Animales , Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Cisteína Endopeptidasas/farmacología , Dimetilsulfóxido/farmacología , Células Germinativas/inmunología , Immunoblotting , Pepstatinas/farmacología , Fenantrolinas/farmacología , Fluoruro de Fenilmetilsulfonilo/farmacología , Inhibidores de Proteasas/farmacología , XanturenatosRESUMEN
Proteolytically processed 310 kDa form of Plasmodium falciparum gamete surface antigen, Pfs230, is the target of malaria transmission-blocking monoclonal antibodies. To design a recombinant malaria transmission-blocking subunit vaccine, the amino terminus of the 310 kDa surface-exposed form of Pfs230 was mapped to amino acids (aa) 522 and 584 using a series of peptides and recombinant proteins encoding distinct regions of Pfs230. Antiserum generated against an Escherichia coli-produced recombinant protein, spanning the Pfs230 processing site and extending into the cysteine domains, r230/MBP.C (aa 443-1132), reduced parasite infectivity by 71.2-89.8%. To determine if the region spanning the cleavage site blocked malaria transmission when produced as a secreted protein by Saccharomyces cerevisiae, y230.CA14 (aa 467-584) was generated, purified, emulsified in adjuvant and used to vaccinate mice. In contrast to E. coli-produced r230/MBP.C, the immune response generated against y230. CA14 was very weak. To enhance the response, y230.CA14 was mixed with tetanus toxoid, chemically crosslinked, repurifed, and its immunogenicty compared with unconjugated y230.CA14. Conjugated-y230. CA14/TT required fewer booster injections to induce an immune response against Pfs230 and the antibodies generated reacted with the surface of intact gametes and immunoprecipitated radiolabelled Pfs230 extracted from 125I surface-labelled gametes to a greater extent. After seven injections, all y230.CA14 vaccinated mice developed anti-Pfs230 antibodies and the isotype profile was the same. In addition to enhancing the initial immune response generated against y230.CA14, conjugation focuses the immune response toward epitopes within the region of Pfs230 present on the surface of the gamete.
Asunto(s)
Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Toxoide Tetánico , Animales , Anticuerpos Antiprotozoarios/sangre , Especificidad de Anticuerpos , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Ratones , Plasmodium falciparum/química , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Toxoide Tetánico/inmunología , Transformación Bacteriana , Vacunas Conjugadas/inmunologíaRESUMEN
Leukotoxin is a linoleic acic oxide produced by leukocytes and has been associated with the multiple organ failure and adult respiratory distress syndrome seen in some severe burn patients. Leukotoxin has been reported to be toxic when injected into animals intravenously. Herein, we report that this lipid is not directly cytotoxic in at least two in vitro systems. Using a baculovirus expression system we demonstrate that leukotoxin is only cytotoxic in the presence of epoxide hydrolases. In addition, it is the diol metabolite that proves toxic to pulmonary alveolar epithelial cells, suggesting a critical role for the diol in leukotoxin-associated respiratory disease. In vivo data also support the toxicity of leukotoxin diol. For the first time we demonstrate that soluble epoxide hydrolase can bioactivate epoxides to diols that are apparently cytotoxic. Thus leukotoxin should be regarded as a protoxin corresponding to the more toxic diol. This clearly has implications for designing new clinical interventions.
Asunto(s)
Citotoxinas/toxicidad , Epóxido Hidrolasas/metabolismo , Ácidos Linoleicos/toxicidad , Animales , Baculoviridae , Línea Celular , Permeabilidad de la Membrana Celular , Células Cultivadas , Citotoxinas/metabolismo , Conductividad Eléctrica , Células Epiteliales , Epitelio/fisiología , Humanos , Uniones Intercelulares , Transporte Iónico , Ácidos Linoleicos/metabolismo , Masculino , Ratones , Alveolos Pulmonares/citología , Alveolos Pulmonares/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , SpodopteraRESUMEN
During Plasmodium falciparum gametocytogenesis, Pfs230, a malaria transmission-blocking vaccine candidate, is expressed as a 360 kDa protein localized to the parasitophorous vacuole/parasite plasma membrane. When gametocytes emerge from red blood cells, as they do when taken up in a blood meal by a mosquito, Pfs230 is processed from a 360 kDa form to a 310 kDa form the latter of which is exposed on the exterior surface of gametes. The 50 kDa portion of Pfs230, removed from the amino-terminus of the 360 kDa form, contains 25 contiguous glutamates and an EEVG16 repeat. Analogous to other P. falciparum proteins, the repeat region appears to be immunodominant. As the gamete emerges from the red blood cell and is exposed to the antibodies in the blood meal, cleavage of the immunodominant region of Pfs230 may contribute to an immune evasion strategy by the parasite.
Asunto(s)
Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Humanos , Vacunas contra la Malaria/química , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Malaria Falciparum/transmisión , Microscopía Inmunoelectrónica , Peso Molecular , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/química , Secuencias Repetitivas de Ácidos Nucleicos , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Six regions of malaria transmission-blocking target antigen, Pfs230, encoding 80% of the 363-kDa protein, were expressed as recombinant proteins in E. coli as fusions with maltose-binding protein (MBP). Antisera generated against amylose-purified recombinant Pfs230/MBP fusion proteins (r230/MBP.A-r230/MBP.F) all recognized the 360-kDa form of parasite-produced Pfs230 by immunoblot. However, only antisera against the four carboxy regions (C-F) of Pfs230 and not the two amino regions (A and B) recognized the 310-kDa form of Pfs230, the form expressed on the surface of gametes. The data suggest that the 310-kDa form of Pfs230 arises from the cleavage of 50 kDa from the amino terminus of the 360-kDa form. Furthermore, antisera against r230/MBP.C bound to the surface of intact gametes and significantly reduced (by 71.2-89.8% (rank sum analysis, P < 0.01)) the infectivity of P. falciparum parasites to mosquitoes. This is the first report of a recombinant form of a P. falciparum gametocyte protein capable of inducing antisera that reduce malaria parasite infectivity to mosquitoes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Culicidae/parasitología , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Complejo Antígeno-Anticuerpo , Antígenos de Protozoos/biosíntesis , Proteínas Portadoras/biosíntesis , Clonación Molecular , Cisteína , Escherichia coli , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas de Unión a Maltosa , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Plasmodium falciparum/patogenicidad , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Homología de Secuencia de AminoácidoRESUMEN
The 230 kD gametocyte/gamete-specific surface protein of Plasmodium falciparum, Pfs230, is a target of antibodies which inhibit the development of the parasite inside the mosquito vector. A transmission blocking vaccine based on Pfs230 may be a powerful tool for malaria control. As a first step, Pfs230 has been expressed in E. coli as a series of recombinant proteins, fused to maltose binding protein. We have used the fusion proteins to assess cellular and humoral immune responses to Pfs230 in malaria-immune adult Gambian blood donors; responses to the fusion proteins have been compared with responses to native Pfs230. The tetrapeptide repeat region of the molecule appears to be immunodominant for both antibody-producing cells and peripheral blood T cells. We postulate that this may represent a mechanism for immune evasion since the N-terminal repeat region of the molecule is cleaved from the mature protein and shed into the plasma. Responses to fusion proteins representing the seven-cysteine motifs were correlated within individual donors, suggesting that cross-reactive epitopes occur within the motifs. Antibody responses to recombinant proteins were poorly correlated with responses to native Pfs230 suggesting that dominant epitopes of the native protein are not adequately represented in the recombinant proteins. Although prokaryotic expression products may be suitable for induction of cellular immune responses to Pfs230, alternative expression systems may be needed for creation of appropriate B cell epitopes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antígenos de Protozoos/inmunología , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antiprotozoarios/sangre , Proteínas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Estudios de Casos y Controles , Escherichia coli/inmunología , Humanos , Epítopos Inmunodominantes , Interferón gamma/sangre , Activación de Linfocitos , Proteínas de Unión a Maltosa , Persona de Mediana Edad , Proteínas Protozoarias/biosíntesis , Proteínas Recombinantes/biosíntesisAsunto(s)
Antígenos de Protozoos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Polimorfismo Genético , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Malaria Falciparum/prevención & control , Datos de Secuencia Molecular , Vacunas Antiprotozoos/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la EspecieAsunto(s)
Antígenos de Protozoos/genética , Genes Protozoarios , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunologíaRESUMEN
Immunoaffinity chromatography and electroelution of protein from solid-phase matrices are two powerful tools often used to purify proteins. In this study, we combined these two techniques and found that antigen was effectively recovered from immunoaffinity resins by electroelution. Yields ranged from 90.5% to 62.8%, with a mean of 74.0 +/- 7.4% (mean +/- standard deviation). A major portion of the eluate, 79.4 +/- 13.1%, was concentrated in a volume of 100 microliters and 94.0 +/- 2.0% was recovered in 200 microliters, even when 1 ml of resin was used. Electroelution had no major effect on the electrophoretic mobility of the antigen. Two distinct antigens, a relatively hydrophilic 230-kDa protein and a hydrophobic 28-kDa protein were successfully electroeluted. In addition two types of immunoaffinity resins, monoclonal antibody covalently linked to CNBr-activated Sepharose or immobilized protein A, were found to be compatible with this method.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Protozoos/aislamiento & purificación , Cromatografía de Afinidad/métodos , Plasmodium falciparum/química , Proteínas Protozoarias/aislamiento & purificación , Animales , Antígenos de Protozoos/biosíntesis , Autorradiografía/métodos , Electroquímica/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Radioisótopos de Yodo , Peso Molecular , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/biosíntesisRESUMEN
Mono-ADP-ribosylation is a reversible modification of proteins with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyzing the forward and reverse reactions, respectively. Hydrolase activities were present in a variety of animal species, with the highest specific activities found in rat and mouse brain, spleen, and testis. Rat and mouse hydrolases were dithiothreitol- and Mg(2+)-dependent, whereas the bovine and guinea pig enzymes were dithiothreitol-independent. A rat brain hydrolase was purified approximately 20,000-fold and represented the major approximately 39-kDa protein on denaturing gels. Immunoaffinity-purified rabbit polyclonal antibodies reacted with 39-kDa proteins from turkey erythrocytes and rat, mouse, and calf brains. A rat brain cDNA library was screened using oligonucleotide and polymerase chain reaction-generated cDNA probes. Inserts from two overlapping clones yielded a composite sequence that included a 1086-base pair open reading frame, which contained amino acid sequences found in the purified hydrolase. A hydrolase fusion protein, synthesized in Escherichia coli, reacted with anti-39-kDa polyclonal antibodies and exhibited Mg(2+)- and dithiothreitol-dependent hydrolase activity. A coding region cDNA hybridized readily to a 1.7-kilobase band in rat and mouse poly(A)+ RNA, but poorly to bovine, chicken, rabbit, and human poly(A)+ RNA. The immunological and molecular biological data are consistent with partial conservation of hydrolase structure across animal species.
Asunto(s)
Glicósido Hidrolasas , Hidrolasas/genética , N-Glicosil Hidrolasas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Encéfalo/enzimología , Cationes Bivalentes , Bovinos , ADN/genética , Ditiotreitol/metabolismo , Cobayas , Hidrolasas/inmunología , Magnesio/metabolismo , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN/genética , Ratas , Especificidad de la Especie , Distribución Tisular , PavosRESUMEN
Two C3 ADP-ribosyltransferase substrates with different characteristics were isolated from bovine brain cytosol. Amino acid sequences of tryptic peptides from the two substrates were identical to rhoA and rhoB; hence, the purified proteins are referred to as rhoA* and rhoB*, respectively. Soluble rhoA* exhibits properties different from those previously reported for rho proteins. In contrast to other C3 substrates, rhoA* behaved as a 77-80-kDa protein on gel filtration, although on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the ADP-ribosylated moiety had a mobility consistent with a 21.5-kDa protein. Furthermore, C3-catalyzed ADP-ribosylation of rhoA* was dependent on guanine nucleotides in the presence of 1 mM Mg2+ or 1 mM EDTA (0.19 microM free Mg2+). Half-maximal stimulation by GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), guanylyl-imidodiphosphate (Gpp(NH)p), and GDP was observed at 16, 20, 220, and 380 nM, respectively; guanosine 5'-O-(2-thiodiphosphate), GMP, and adenine nucleotides were ineffective. In the presence of GTP gamma S, the rate and extent of ADP-ribosylation was enhanced by dimyristoylphosphatidylcholine and/or cholate. This increase in ADP-ribosylation was specific for rhoA*; it was not observed with rhoB* and has not been reported for other C3 substrates. These distinct properties suggest that rhoA* is a newly recognized type of C3 substrate, differing from the rhoA-like proteins previously reported. rhoB*, on the other hand, has properties similar to those reported for membrane-associated rhoB and its ADP-ribosylation was independent of guanine nucleotides in the presence of 1 mM Mg2+ and not affected by dimyristoylphosphatidylcholine and/or cholate.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Toxinas Botulínicas , Clostridium botulinum/enzimología , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Ácido Cólico , Ácidos Cólicos/farmacología , Cromatografía , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Dimiristoilfosfatidilcolina/farmacología , Durapatita , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/aislamiento & purificación , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Hidroxiapatitas , Cinética , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Proteína de Unión al GTP rhoBAsunto(s)
ADP Ribosa Transferasas/metabolismo , Toxinas Botulínicas , Clostridium botulinum/enzimología , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Bovinos , Proteínas de Unión al GTP/genética , Guanosina Trifosfato/metabolismo , Técnicas In Vitro , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fosfolípidos/metabolismo , Homología de Secuencia de Ácido Nucleico , Especificidad por Sustrato , Proteína de Unión al GTP rhoA , Proteína de Unión al GTP rhoBRESUMEN
Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on the partial amino acid sequence of one of the soluble ARFs from bovine brain. Comparison of the deduced amino acid sequence of lambda ARF2B with sequences of peptides from the ARF protein (total of 60 amino acids) revealed only two differences. Whether these are cloning artifacts or reflect the existence of more than one ARF protein remains to be determined. Deduced amino acid sequences of ARF, Go alpha (the alpha subunit of a G protein that may be involved in regulation of ion fluxes), and c-Ha-ras gene product p21 show similarities in regions believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF apparently lacks a site analogous to that ADP-ribosylated by choleragen in G-protein alpha subunits. Although both the ARF proteins and the alpha subunits bind guanine nucleotides and serve as choleragen substrates, they must interact with the toxin A1 peptide in different ways. In addition to serving as an ADP-ribose acceptor, ARF interacts with the toxin in a manner that modifies its catalytic properties.
Asunto(s)
ADN/genética , Proteínas de Unión al GTP/genética , Proteínas de la Membrana/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Ribosilacion-ADP , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Toxina del Cólera/metabolismo , Clonación Molecular , Proteínas de Unión al GTP/metabolismo , Datos de Secuencia MolecularRESUMEN
The formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) dependent Ca2+ uptake by human neutrophils consists of at least two components, one of which is sensitive to dihydropyridine derivatives. Inhibition by dihydropyridine derivatives showed the rank order of nisoldepine greater than nitrendipine greater than nimodepine greater than/Bay K 8644. The nisoldepine-sensitive calcium uptake exhibited an ID50 of 1.5 microM and maximal inhibition were observed at 5 microM. Neither calcium efflux or [3H]fMet-Leu-Phe binding was affected by nisoldepine up to 10 microM. The inhibition of nisoldepine was inversely proportional to the extracellular calcium concentration. Unlabeled nisoldepine and other dihydropyridine derivatives displaced the specific binding of [3H]PN 200-110 to human neutrophils. Our data suggest a relationship between dihydropyridine binding and the inhibition of fMet-Leu-Phe-dependent Ca2+ uptake.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Neutrófilos/metabolismo , Nifedipino/análogos & derivados , Receptores Inmunológicos/fisiología , Adulto , Transporte Biológico/efectos de los fármacos , Humanos , Técnicas In Vitro , Isradipino , Neutrófilos/efectos de los fármacos , Nifedipino/farmacología , Nisoldipino , Oxadiazoles/metabolismo , Receptores de Formil PéptidoRESUMEN
A novel G protein which appears to couple chemotactic peptide receptors to a polyphosphoinositide phospholipase C has been purified from rabbit neutrophils. Neutrophil membranes were solubilized with sodium cholate and fractionated by successive anion exchange, gel filtration and hydrophobic chromatography. Guanosine-5'-(3-O-thio)triphosphate binding activity was purified 170-fold from the soluble extract. The alpha-subunit of the purified G protein was identified by pertussis toxin-catalyzed ADP-ribosylation, and found to have an Mr of 40,000. The beta-subunit (Mr 36,000) comigrated on SDS-polyacrylamide gel electrophoresis with the beta-subunits of bovine brain Gi and Go. The neutrophil pertussis toxin substrate is highly unstable in cholate solution unless 30% ethylene glycol is added. Structural and functional analysis of this novel G protein will advance our understanding of the molecular mechanisms of coupling of receptors to phospholipase C.