RESUMO
The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Glicosilação , Humanos , Dados de Sequência Molecular , Açúcares de Nucleosídeo Difosfato/química , Açúcares de Nucleosídeo Difosfato/genética , Proteínas de Transporte de Nucleotídeos/química , Proteínas de Transporte de Nucleotídeos/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.
Assuntos
Humanos , Animais , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Glicosilação , Dados de Sequência Molecular , Açúcares de Nucleosídeo Difosfato/síntese química , Açúcares de Nucleosídeo Difosfato/genética , Proteínas de Transporte de Nucleotídeos/química , Proteínas de Transporte de Nucleotídeos/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-L: -rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.
Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Carboxílicos/metabolismo , Escherichia coli/metabolismo , Glicolipídeos/biossíntese , Hexosiltransferases/metabolismo , Pseudomonas aeruginosa/enzimologia , Tensoativos/metabolismo , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Clonagem Molecular , Meios de Cultura , Escherichia coli/genética , Expressão Gênica , Genes Reporter , Glicolipídeos/genética , Hexosiltransferases/genética , Açúcares de Nucleosídeo Difosfato/metabolismo , Óperon/genética , Pseudomonas aeruginosa/genética , Nucleotídeos de Timina/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
It is accepted that glycosyltransferase-generated nucleoside diphosphates are converted to monophosphates in the secretory pathway by nucleoside diphosphatases (NDPases) to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the lumen is coupled to exit of nucleoside monophosphates. Working with Saccharomyces cerevisiae mutants affected in anterograde and/or retrograde endoplasmic reticulum (ER)-Golgi vesicular traffic and/or defective in one or both secretory pathway (Golgi) NDPases, we show that UDP-Glc: glycoprotein glucosyltransferase-mediated glucosylation is not dependent on the presence of NDPases or on ER-Golgi vesicular traffic and that GDP-Man-dependent N- and O-mannosylations are reduced but not abolished in the absence of NDPases in the secretory pathway. Further, the absence of the main Man-1-P transferase (a Golgi GMP-generating enzyme) does not modify the limited mannosylation observed in the absence of NDPases. Based on these results and on available additional information, we suggest that in the absence of NDPases, the already characterized nucleotide sugar transporters allow entrance of nucleotide sugars into the luminal compartments and that resulting nucleoside diphosphates exit to the cytosol by a still unknown mechanism. Further, an unexpected side result suggests that formation of Ser/Thr-Man(2) may occur in the ER and not exclusively in the Golgi.
Assuntos
Hidrolases Anidrido Ácido/fisiologia , Glicosilação/efeitos dos fármacos , Hexosiltransferases/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Retículo Endoplasmático/metabolismo , Expressão Gênica , Complexo de Golgi/metabolismo , Hexosaminas/metabolismo , Hexosiltransferases/genética , Hidrólise , Mutação , Açúcares de Nucleosídeo Difosfato/metabolismo , Nucleotídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Schizosaccharomyces/enzimologia , Transfecção , Uridina Difosfato Glucose/metabolismoRESUMO
Changes of energy metabolism in leucocytes have been studied longitudinally as well as cross-sectionally in three groups of children born: 1.- at term, with normal weight; 2.- with intra-uterine malnutrition; 3.- prematurely. The longitudinal study showed that, at birth, the activity of adenylate kinase as well as concentrations of ATP, ADP AND AMP were significantly lower in the malnourished than in the normal child. At one year of age, the values of enzyme activity are approximately equal in both groups, while the concentrations, of ATP and ADP are higher in malnourished children. Finally, at two years of age all values found, with the exception of AMP concentration, were higher in malnourished than in normal children born at term. In the data of the cross-sectional study this accelerated biochemical maturation is also shown.