Добірка наукової літератури з теми "Leloir pathway"

Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with 13C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in Glc-free media and switching Glc-grown GBM cells into Gal-enriched/Glc-free media produced elevated levels of Glut3 and/or Glut14 enzymes. The 13C NMR-based metabolic flux analysis demonstrated a fully functional Leloir pathway and elevated pentose phosphate pathway activity for efficient Gal metabolism in GBM cells. Conclusion: Expression of Glut3 and/or Glut14 together with the enzymes of the Leloir pathway allows GBM to transport and metabolize Gal at physiological glucose concentrations, providing GBM cells with an alternate energy source. The presence of this pathway in GBM and its selective targeting may provide new treatment strategies.

Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose–galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose–galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.

Galactosemias are a family of autosomal recessive genetic disorders resulting from impaired enzymes of the Leloir pathway of galactose metabolism including galactokinase, galactose uridyltransferase, and UDP-galactose 4-epimerase that are critical for conversion of galactose into glucose-6-phosphate. To better understand pathophysiological mechanisms involved in galactosemia and develop novel therapies to address the unmet need in patients, it is important to develop reliable assays to measure the activity of the Leloir pathway enzymes. Here we describe in-depth methods for indirectly measuring Galacose-1-Phosphate Uridyltransferase activity in cell culture and animal tissues.

ABSTRACTThe GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization inBifidobacterium longumas it is an energy-saving variant of the Leloir pathway. Both pathways are present inB. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1,galT2, andugpA) fromBifidobacterium bifidumwere cloned in anEscherichia colimutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates whenB. bifidumgrows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.

ABSTRACT The gal genes from the chromosome ofLactobacillus casei 64H were cloned by complementation of the galK2 mutation of Escherichia coli HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA HindIII fragment. Detailed studies with other E. coli K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of theL. casei gal operon is galKETRM. Because parts of the gene for the mutarotase as well as the promoter region upstream of galK were not cloned on pKBL1, the regions flanking theHindIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the gal genes constitute an operon that is transcribed from two promoters. The galKppromoter is inducible by galactose in the medium, whilegalEp constitutes a semiconstitutive promoter located ingalK.

Abstract BACKGROUND We have recently shown that GBM use D-galactose (Gal) as a substrate, in vitro and in vivo. Gal is imported via Glut3 and/or Glut14 and metabolized through the Leloir pathway. We investigated 4-deoxy-4-fluorogalactose (4DFG) as the lead compound in a family of galactose-based antimetabolites. 4DFG is a potent chemotherapeutic in monotherapy and can bolster existing therapies. METHODS We examined the alteration of glioma metabolism in vitro and in vivo induced by 4DFG. 1H/13C-NMR and optical probes were used to interrogate the effects of 4DFG on glycolysis and mitochondrial respiration in primary glioma cell cultures. Labeled lectins were used to assay for the disruption of glycan synthesis induced by 4DFG. An intracranial model of primary GBM was used to assess efficacy and toxicity in vivo. RESULTS NMR reveals that at physiological concentrations of glucose, low concentrations of 4DFG (5 μM) is able to inhibit glycolytic and mitochondrial flux by approximately 12%, p< 0.05. Analysis using lectins shows a collapse in general glycan synthesis, but most especially in the incorporation of both Gal and GalNAc sugars. In nude mice with intracranial primary GBM, six treatments of 4DFG increased survival from 23 to 50 days, p< 0.002. DISCUSSION The ability of GBM to scavenge galactose allows us to target the Glut3/14 import and Leloir metabolic pathway using galactose-based anti-metabolites. Our first-generation compound is highly effective as a monotherapy, inhibiting glucose metabolism and glycan synthesis.

ABSTRACT The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.

Streptococcus pneumoniae is a formidable human pathogen. Responsible for between 1 and 2 million deaths annually, the pneumococcus makes a major contribution to global morbidity and mortality. In order to cause disease, the pneumococcus must first colonise the human nasopharynx. This colonisation is typically asymptomatic and provides the ideal niche from which the pneumococcus can transmit itself to new hosts. However, in some cases, the pneumococcus will undergo a ‘switch’ from harmless coloniser to invasive pathogen, transiting to deeper, usually sterile niches in the body and causing invasive disease. A key determinant of successful colonisation of the nasopharynx is the ability to metabolise the different carbon sources that are available. While the pneumococcus typically prefers to metabolise glucose, this carbon source is actively eliminated from the human nasopharynx in an attempt to maintain airway sterility. In the absence of glucose, galactose is the predominant sugar in this niche. Galactose can be metabolised by two pathways in the pneumococcus, the Leloir pathway and the tagatose-6-phosphate pathway. A study by Trappetti et al., in 2017 was the first to show a link between carbohydrate metabolism and cell-to-cell signalling in the pneumococcus, demonstrating that the quorum sensing molecule Autoinducer 2 (AI-2) is likely phosphorylated during import into the cell. Phosphorylated AI-2 is then proposed to either directly or indirectly phosphorylate GalR, the regulator of the Leloir pathway, driving an increase in galactose metabolism and a subsequent hypervirulent phenotype. They propose that this phosphorylation occurs at the putative phosphorylation sites identified by Sun et al., in 2010: Serine 317, Threonine 319 and Threonine 323. To better understand the role of these putative phosphorylation sites, a series of amino acid substitution mutants were generated in which each of the sites were replaced, either singly or in combination, with either the non-phosphorylatable residue alanine (A) or the phosphomimetic aspartic acid (D) or glutamic acid (E). While the use of phosphomimetic residues proved somewhat challenging, the use of non-phosphorylatable alanine residues revealed that each of the three putative phosphorylation sites are required for growth in galactose, successful activation of the Leloir pathway and disease progression in a murine model of infection. What became clear during this study was that despite having two functional pathways encoded for galactose metabolism, there was an inability for one pathway to rescue the other during times of metabolic distress. This indicated that these pathways may not be as discreet as once thought. To further investigate this potential interplay, a series of mutants were generated, deleting key genes from either the Leloir or the T6P pathways. This approach revealed that deleting genes from either pathway resulted in an inability to metabolise galactose, as well as transcriptional changes indicating that there is indeed interplay between these two pathways, with GalR possibly playing a key role as the central regulator of both pathways. Additionally, we found that there is differential accumulation of metabolites intracellularly as a result of these mutations, which may hold the key to deciphering exactly how these two pathways are linked. Finally, using dual in vivo RNA sequencing, we have revealed that GalR and its putative phosphorylation sites play an important role in virulence, leading to skewing of the immune response during infection. Collectively, the findings of this thesis have significantly advanced our understanding of pneumococcal galactose metabolism, particularly in terms of its regulation via GalR. Additionally, we have shed light on the interplay between the Leloir and T6P pathways, showing for the first time that there is a definitive link that requires both pathways to be present and functional in order to survive in the presence of galactose, much like what is found in the human nasopharynx. We have also shown that the putative GalR phosphorylation sites play a key role in pneumococcal galactose metabolism, pneumococcal virulence and the host response to infection. This project provides the foundation for further investigation into the regulation of pneumococcal galactose metabolism, and the wide-reaching impacts this pathway has on pneumococcal virulence and disease.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2022