RESUMO
A giant protein with an apparent molecular mass of 2,300-kDa was identified in the Triton X-100 soluble fraction of Crithidia luciliae thermophila. Polyclonal antibody raised against this protein reacted by immunoblot analysis with proteins of similar molecular mass in Crithidia fasciculata and Crithidia oncopelti. In addition, the antibody immunoprecipitates the protein either after in vivo phosphorylation with [32P]orthophosphoric acid or after metabolically labeling with [35S]methionine. Indirect immunofluorescence microscopy analysis performed either with fixed or with live parasites showed a single fluorescent spot at the level of the flagellar pocket region. Immunogold electron microscopy of thin sections of the parasite revealed that the antigen is localized at a restricted area of the spongiome, between the contractile vacuole and the flagellar pocket. Furthermore, Triton X-114 phase separation of whole cell membrane proteins, metabolically labeled with [35S]methionine, demonstrated that the giant protein remains in the aqueous phase. These results indicate that this phosphoprotein behaves as a peripheral membrane protein localized at the spongiome region, suggesting that it might be involved in the osmoregulatory process.
Assuntos
Crithidia/química , Fosfoproteínas/análise , Proteínas de Protozoários/análise , Vacúolos/química , Animais , Antígenos de Protozoários/análise , Crithidia/ultraestrutura , Epitopos , Técnica Indireta de Fluorescência para Anticorpo , Glicosilação , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Peso Molecular , Fosfoproteínas/química , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , SolubilidadeRESUMO
Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200), respectively. Polyclonal antibodies to Lt 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. In addition to reacting with giant polypeptides of the Leptomonas species, anti-Ls 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500, Ls 2500, and Lt 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas Lt 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. In addition, there is also evidence that Ps 3500 and Ls 2500, in contrast to Lt 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Leishmania/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosomatina/metabolismo , Animais , Antígenos de Protozoários/análise , Técnicas de Cultura de Células , Proteínas do Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Immunoblotting , Leishmania/imunologia , Leishmania/ultraestrutura , Microscopia Confocal , Microscopia Imunoeletrônica , Peso Molecular , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/imunologia , Trypanosomatina/imunologia , Trypanosomatina/ultraestruturaRESUMO
We have purified and characterized a novel high molecular mass glycoprotein of P. chabaudi chabaudi (Pc550gp) that is transported to the erythrocyte membrane during the intraerythrocytic cycle. Immuno fluorescence assays with polyclonal monospecific antibodies against Pc550gp show that the protein to be localized in the periphery of young trophozoite stages i.e., on the plasma membrane or parasitophorous vacuole membrane. However, in late trophozoites and schizonts the antigen is distributed in both parasite and host cell membranes. These results were confirmed by immunoblotting of isolated parasites and infected host cell membranes at different stages of parasite development. Moreover, alkali extraction of purified infected erythrocyte membranes at mature stages of parasite development does not solubilize Pc550gp, suggesting that it is an integral membrane protein. In addition proteinase K digestion of intact infected host cells induced the disappearance of Pc550gp. Further indicating its transmembrane nature and that it presents extracellular domains susceptible to proteolysis. Brefeldin A or low temperature (15 degrees C) treatment did not affect the translocation of Pc550gp from the parasite compartments to the erythrocyte membrane, indicating that the secretion of Pc550gp does not follow the classical transport pathway described in most eukaryotic cells.
Assuntos
Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Plasmodium chabaudi/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico , Brefeldina A/farmacologia , Carbonatos , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Feminino , Glicosilação , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Peptídeos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , TemperaturaAssuntos
Antígenos de Protozoários/metabolismo , Glicoproteínas/metabolismo , Malária/parasitologia , Plasmodium chabaudi/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Eritrócitos/parasitologia , Técnica Indireta de Fluorescência para Anticorpo , Glicoproteínas/biossíntese , Glicoproteínas/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Plasmodium chabaudi/crescimento & desenvolvimento , Plasmodium chabaudi/imunologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , SolubilidadeRESUMO
A megadalton protein was found to be a cytoskeleton component of the promastigote forms of the flagellate Phytomonas serpens. This protein migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as a doublet of polypeptides with a molecular mass similar to muscle beta-connectin (titin) 2500-3000 kDa. A polyclonal antibody raised against this protein reacts, by immunoblot analysis, with Phytomonas serpens and two others Phytomonas species. In addition, the Phytomonas serpens protein was immunoprecipitated after being metabolically labeled with [35S]methionine. This antibody did not cross-react with the cytoskeletal proteins of Trypanosoma cruzi, Crithidia luciliae thermophila, Crithidia fasciculata and Leptomonas samueli or with beta-connectin (titin). Indirect immunofluorescence microscopy analysis revealed a punctate fluorescence staining at the anterior region of the parasite's body skeleton. Moreover, immunogold electron microscopy of cytoskeletal preparations and of thin sections of whole cells indicates that the giant protein appears to cap the anterior end of the cell body microtubules at the level of the junctional complex. We suggest that this giant protein may serve as a linker between the cell body skeleton and the flagellum membrane.
Assuntos
Citoesqueleto/química , Trypanosomatina/química , Animais , Western Blotting , Citoesqueleto/ultraestrutura , Imunofluorescência , Imuno-Histoquímica , Microscopia Imunoeletrônica , Peso Molecular , Testes de Precipitina , Proteínas de Protozoários/análise , Proteínas de Protozoários/isolamento & purificação , Trypanosomatina/citologia , Trypanosomatina/ultraestruturaRESUMO
Cytoskeletal preparations of Trypanosoma cruzi trypomastigotes and epimastigotes contain a protein recognized by a monoclonal antibody (2G4) which is connected to the flagellar attachment zone of both stages of the parasite. Western blot analysis revealed that the antibody was able to recognize protein bands of molecular masses higher than 700 kDa up to 2500 kDa. These giant proteins do not seem to share sequences with beta-connectin since an anti-beta-connectin antibody did not recognize the T. cruzi proteins nor did the 2G4 monoclonal antibody recognize authentic beta-connectin. Immunofluorescence and immunogold electron microscopy provided evidence that this protein is located inside the cell body of the parasite, closely related to a corset of four microtubules known as subpellicular microtubule quartet. Immunogold labeling shows that the protein accompanies the flagellar attachment zone as long as the flagellum adheres to the cell body. It is proposed that these microtubule-associated proteins recognized by the 2G4 monoclonal antibody exist only in trypanosomatid forms having a junctional complex between the flagellum and the cell body and may act as transmembrane elements connecting the subpellicular microtubular quartet with the flagellum at the desmosome region.