RESUMO
The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fucosiltransferases/metabolismo , Guanosina Difosfato Fucose/metabolismo , Guanosina Difosfato Manose/metabolismo , Receptores Notch/metabolismo , Asas de Animais/metabolismo , Alelos , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/metabolismo , Endocitose/genética , Fucosiltransferases/genética , Guanosina Difosfato Fucose/genética , Guanosina Difosfato Manose/genética , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Receptores Notch/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Asas de Animais/anatomia & histologiaRESUMO
The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.
Assuntos
Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fucosiltransferases/metabolismo , Guanosina Difosfato Fucose/metabolismo , Guanosina Difosfato Manose/metabolismo , Receptores Notch/metabolismo , Asas de Animais/metabolismo , Alelos , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/metabolismo , Endocitose/genética , Fucosiltransferases/genética , Guanosina Difosfato Fucose/genética , Guanosina Difosfato Manose/genética , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Notch/genética , Transdução de Sinais , Asas de Animais/anatomia & histologiaRESUMO
In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies. The null mutants attach avidly to mouse macrophages, but unlike the wild type organisms, they do not bind to the complement receptor 3 and are slow to induce phagocytosis. Once internalised, they localise to the phagolysosome, but in contrast to wild type organisms which transform into the intracellular amastigote and establish in the macrophage, they are cleared by 24 h in culture and by 5 h in vivo. The null mutants are hypersensitive to human but not mouse complement and to temperature and acidic pH. Surprisingly, in view of the lack of several known host-protective antigens, injection of the mutant parasites into BALB/c mice confers significant and long lasting protection against infection, suggesting that these temperature sensitive mutants are an attractive candidate for a live attenuated vaccine.