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
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
We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP+ as a cofactor and, like its E. coli counterpart, is inhibited by GDP-fucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.