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
We describe the first biochemical characterization of the gumI gene product, an essential protein for xanthan polysaccharide synthesis. Cellular fractionation experiments reveal the presence of a protein associated with the membrane fraction, even in the absence of the other proteins responsible for the synthesis of glycolipid intermediates and the proteins involved in the polymerization and transport of the xanthan chains. By alkaline buffer extraction and detergent phase partitioning, GumI was categorized as a monotopic membrane protein. GumI was overexpressed in Escherichia coli, solubilized and purified in an active and stable form using a simple and reproducible two-step procedure. The purified recombinant GumI is a nonprocessive ß-mannosyltransferase that uses GDP-Man as a donor substrate and glucuronic acid-ß-1,2-mannose-α-1,3-glucose-ß-1,4-glucose-PP-polyisoprenyl as an acceptor. We also established the optimal biochemical conditions for GumI enzymatic activity. Sequence analysis revealed the presence of a conserved domain for glycosyltransferases (GTs) of the GT-B superfamily and homologous proteins in several prokaryote organisms. On the basis of this biochemical characterization, GumI may represent the founding member of a new GT family in the Carbohydrate-Active EnZymes classification.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Guanosina Difosfato Manose/metabolismo , Manosiltransferases/isolamento & purificação , Manosiltransferases/metabolismo , Polissacarídeos Bacterianos/metabolismo , Xanthomonas campestris/enzimologia , Proteínas de Bactérias/genética , Teste de Complementação Genética , Manosiltransferases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Frações SubcelularesRESUMO
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
Leptospira interrogans synthesizes a range of mannose-containing glycoconjugates relevant for its virulence. A prerequisite in the synthesis is the availability of the GDP-mannose, produced from mannose-1-phosphate and GTP in a reaction catalyzed by GDP-mannose pyrophosphorylase. The gene coding for a putative enzyme in L. interrogans was expressed in Escherichia coli BL21(DE3). The identity of this enzyme was confirmed by electrospray-mass spectroscopy, Edman sequencing and immunological assays. Gel filtration chromatography showed that the dimeric form of the enzyme is catalytically active and stable. The recombinant protein was characterized as a mannose-1-phosphate guanylyltransferase. S (0.5) for the substrates were determined both in GDP-mannose pyrophosphorolysis: 0.20 mM (GDP-mannose), 0.089 mM (PPi), and 0.47 mM; and in GDP-mannose synthesis: 0.24 mM (GTP), 0.063 mM (mannose-1-phosphate), and 0.45 mM (Mg(2+)). The enzyme was able to produce GDP-mannose, IDP-mannose, UDP-mannose and ADP-glucose. We obtained a structural model of the enzyme using as a template the crystal structure of mannose-1-phosphate guanylyltransferase from Thermus thermophilus HB8. Binding of substrates and cofactor in the model agree with the pyrophosphorylases reaction mechanism. Our studies provide insights into the structure of a novel molecular target, which could be useful for detection of leptospirosis and for the development of anti-leptospiral drugs.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Leptospira interrogans/enzimologia , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Adenosina Difosfato Glucose/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Western Blotting , Cromatografia em Gel , Guanosina Difosfato Manose/metabolismo , Manosefosfatos/metabolismo , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Açúcares de Uridina Difosfato/metabolismoAssuntos
Alinhamento de Sequência , DNA Bacteriano/isolamento & purificação , Dados de Sequência Molecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Fosfatidilinositóis/metabolismo , Guanosina Difosfato Manose/metabolismo , Lipopolissacarídeos/metabolismo , Manosiltransferases/genética , Manosiltransferases/isolamento & purificação , Modalidades de Fisioterapia , Mycobacterium bovis/genética , Mycobacterium smegmatis , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Sequência de AminoácidosAssuntos
Alinhamento de Sequência , DNA Bacteriano/isolamento & purificação , Dados de Sequência Molecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Fosfatidilinositóis/metabolismo , Guanosina Difosfato Manose/metabolismo , Lipopolissacarídeos/metabolismo , Manosiltransferases/genética , Manosiltransferases/isolamento & purificação , Mycobacterium bovis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Sequência de AminoácidosRESUMO
Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (DeltaPMM and DeltaDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicana DeltaDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana DeltaPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.
Assuntos
Leishmania mexicana/enzimologia , Leishmania mexicana/patogenicidade , Manosiltransferases/genética , Fosfotransferases (Fosfomutases)/genética , Virulência/genética , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Clonagem Molecular , Dolicol Monofosfato Manose/metabolismo , Regulação para Baixo , Citometria de Fluxo , Deleção de Genes , Marcação de Genes , Glicoconjugados/metabolismo , Glicosilação , Guanosina Difosfato Manose/metabolismo , Leishmania mexicana/genética , Macrófagos/parasitologia , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
The biosynthesis of the extracellular polysaccharide xanthan in Xanthomonas campestris pv. campestris is directed by a cluster of 12 genes, gumB-gumM. Several xanthan-deficient mutants of the wild-type strain 8004 have previously been described which carry Tn5 insertions in this region of the chromosome. Here it is shown that the transposon insertion in one of these mutants, strain 8397, is located 15 bp upstream of the translational start site of the gumB gene. EDTA-treated cells of strain 8397 were able to synthesize the lipid-linked pentasaccharide repeating unit of xanthan from the three nucleotide sugar donors (UDP-glucose, GDP-mannose and UDP-glucuronic acid) but were unable to polymerize the pentasaccharide into mature xanthan. A subclone of the gum gene cluster carrying gumB and gumC restored xanthan production to strain 8397 to levels approximately 28% of the wild-type. In contrast, subclones carrying gumB or gumC alone were not effective. These results are discussed with reference to previous speculations, based on computer analysis, that gumB and gumC are both involved in the translocation of xanthan across the bacterial membranes.
Assuntos
Proteínas de Bactérias/metabolismo , Genes Bacterianos/fisiologia , Polissacarídeos Bacterianos/metabolismo , Xanthomonas campestris/enzimologia , Cromatografia DEAE-Celulose , Cromatografia por Troca Iônica , Elementos de DNA Transponíveis , Guanosina Difosfato Manose/metabolismo , Reação em Cadeia da Polimerase , Radioquímica , Uridina Difosfato Glucose/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo , Xanthomonas campestris/genéticaRESUMO
Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a trisaccharide side chain.