Galactose-1-phosphate inhibits cytochrome c oxidase and causes mitochondrial dysfunction in classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.

Heterologous expression of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-ß-1,4-GlcNAc-X.

Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and ß-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-ß-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-ß-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides.

Hand fine motor control in classic galactosemia.

Classic galactosemia (CG) is a rare inborn error of metabolism that results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection and rapid and lifelong dietary restriction of galactose, which is the current standard of care, most patients grow to experience a broad range of complications that can include motor difficulties. The goal of this study was to characterize hand fine motor control deficit among children and adults with classic galactosemia (CG). Specifically, we used Neuroglyphics software to collect digital Archimedes spiral drawings on a touch screen from 57 volunteers with CG (cases) and 80 controls. Hand fine motor control was scored as root mean square (RMS) of spirals drawn relative to an idealized template. Presence of tremor was defined as a peak in periodicity of changes in drawing speed or direction in the 4-8 Hz range. We observed a highly significant difference (P < .001) in RMS scores between cases and controls, with almost 51% of cases showing at least 1 of 4 spirals scoring outside the 95th percentile for controls. The corresponding prevalence for controls was 10%. Similarly, more than 35% of cases, and almost 14% of controls, showed at least 1 of 4 spirals with a tremor amplitude above the 95th % cutoff for controls. Our results both confirm and extend what is known about hand fine motor control deficit among children and adults with CG and establish digital assessment as a useful approach to quantify this outcome.

Receptor-mediated attenuation of insulin-like growth factor-1 activity by galactose-1-phosphate in neonate skin fibroblast cultures: Galactosemia pathogenesis.

BACKGROUND: The pathogenesis of classical galactosemia, a rare metabolic disorder associated with developmental complications in neonates and children due to inherited deficiency of galactose-1-phosphate (Gal-1-P) uridylyltransferase (GALT), is known to be mediated by elevated Gal-1-P levels and involves a cascade of cytokines, reactive oxygen species (ROS) and growth factors. OBJECTIVES: To examine ex vivo the effect of Gal-1-P on the mitogenic activity of different growth factors, particularly insulin-like growth factor-1 (IGF-1), known to regulate growth and development from the fetal stage to adulthood. MATERIAL AND METHODS: Fibroblasts derived from the foreskin of 3-8-day-old healthy neonates were cultured for 1-14 days with 0-20 mM galactose or 0-10 mM Gal-1-P and then stimulated with 5% fetal bovine serum (FBS) or 50 ng/mL of platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF) or IGF-1 for 24 h. DNA synthesis was measured and protein expression of PDGFR, FGFR and IGF-1R was assessed with western blotting. RESULTS: Supra-physiological concentrations of galactose significantly decreased FBSand IGF-1-induced BrdU incorporation. The presence of Gal-1-P (5-10 mM) in culture medium for 7-14 days significantly (p < 0.01) decreased IGF-1-, PDGFand FBS-stimulated DNA synthesis. While treatment with Gal-1-P selectively and significantly (p < 0.01) reduced the protein expression of IGF-1 receptor, galactose treatment did not have any marked effect on examined growth factor receptors. CONCLUSIONS: This study demonstrates that Gal-1-P impairs IGF-1 activity through IGF-1-receptor impairment, thereby providing a new insight into the molecular mechanisms of galactosemia pathogenesis.

The yeast protein Ubx4p contributes to mitochondrial respiration and lithium-galactose-mediated activation of the unfolded protein response.

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

BACKGROUND: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. METHODS: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. RESULTS: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001). CONCLUSIONS: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.

Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.

Galactose 1-phosphate accumulates to high levels in galactose-treated cells due to low GALT activity and absence of product inhibition of GALK.

Classic Galactosaemia is a genetic disorder, characterised by galactose intolerance in newborns. It occurs due to recessive mutations in the galactose-1-phosphate uridylyltransferase (GALT) gene. One of the main alterations caused by GALT deficiency is the accumulation of galactose 1-phosphate (Gal-1P) in cells. Studies have suggested that Gal-1P exerts cellular toxicity, possibly by inhibiting cellular metabolism. However, the exact significance of Gal-1P in disease pathogenesis remains unclear. In this study, we tested the hypothesis that Gal-1P inhibits cellular glucose utilisation by competing with substrates in the glycolytic pathway. We also investigated the metabolism of both galactose and glucose in GALT-expressing HEK293T and 143B cells to identify critical reactions steps contributing to the metabolic toxicity of galactose. Notably, we found that galactose-treated HEK293T and 143B cells, which express endogenous GALT, accumulate markedly high intracellular Gal-1P concentrations. Despite very high intracellular Gal-1P concentrations, no inhibition of cellular glucose uptake and no significant changes in the intracellular concentrations of glycolytic metabolites were observed. This indicates that Gal-1P does not exert an inhibitory effect on glycolysis in cells and rules out one potential hypothesis for cellular Gal-1P toxicity. We also investigated the mechanism responsible for the observed Gal-1P accumulation. Our results suggest that Gal-1P accumulation is a result of both low GALT activity and the absence of product inhibition by Gal-1P on galactokinase (GALK1), the enzyme responsible for phosphorylating galactose to Gal-1P. These findings provide a better understanding of the disease mechanisms underlying Classic Galactoaemia.

A galactose-1-phosphate uridylyltransferase-null rat model of classic galactosemia mimics relevant patient outcomes and reveals tissue-specific and longitudinal differences in galactose metabolism.

Classic galactosemia (CG) is a potentially lethal inborn error of metabolism, if untreated, that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the middle enzyme of the Leloir pathway of galactose metabolism. While newborn screening and rapid dietary restriction of galactose prevent or resolve the potentially lethal acute symptoms of CG, by mid-childhood, most treated patients experience significant complications. The mechanisms underlying these long-term deficits remain unclear. Here we introduce a new GALT-null rat model of CG and demonstrate that these rats display cataracts, cognitive, motor, and growth phenotypes reminiscent of patients outcomes. We further apply the GALT-null rats to test how well blood biomarkers, typically followed in patients, reflect metabolic perturbations in other, more relevant tissues. Our results document that the relative levels of galactose metabolites seen in GALT deficiency differ widely by tissue and age, and that red blood cell Gal-1P, the marker most commonly followed in patients, shows no significant association with Gal-1P in other tissues. The work reported here establishes our outbred GALT-null rats as an effective model for at least four complications characteristic of CG, and sets the stage for future studies addressing mechanism and testing the efficacy of novel candidate interventions.

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24 h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT-/- newborn pup.

Profiling of intracellular metabolites produced from galactose and its potential for galactosemia research.

BACKGROUND: Clinical outcome of patients with a classical presentation of galactosemia (classical patients) varies substantially, even between patients with the same genotype. With current biomarkers, it is not possible to predict clinical outcome early in life. The aim of this study was to develop a method to provide more insight into galactose metabolism, which allows quantitative assessment of residual galactose metabolism in galactosemia patients. We therefore developed a method for galactose metabolite profiling (GMP) in fibroblasts using [U-13C]-labeled galactose. METHODS: GMP analysis was performed in fibroblasts of three classical patients, three variant patients and three healthy controls. The following metabolites were analyzed: [U13C]-galactose, [U13C]-galactose-1-phosphate (Gal-1-P) and [13C6]- uridine diphosphate(UDP)-galactose. The ratio of [U13C]-Gal-1-P/ [13C6]-UDP-galactose was defined as the galactose index (GI). RESULTS: All patient cell lines could be distinguished from the control cell lines and there was a clear difference between variant and classical patients. Variant patients had lower levels of [U13C]-galactose and [U13C]-Gal-1-P than classical patients (though substantially higher than healthy controls) and higher levels of [13C6]-UDP-galactose than classical patients (though substantially lower than healthy controls) resulting in a different GI in all groups. CONCLUSIONS: GMP in fibroblasts is a sensitive method to determine residual galactose metabolism capacity, which can discriminate between patients with a classical presentation of galactosemia, patients with a variant presentation and healthy controls. GMP may be a useful method for early prognostication after further validation in a larger cohort of patients representing the full phenotypic spectrum of galactosemia.

Galactose metabolism and toxicity in Ustilago maydis.

In most organisms, galactose is metabolized via the Leloir pathway, which is conserved from bacteria to mammals. Utilization of galactose requires a close interplay of the metabolic enzymes, as misregulation or malfunction of individual components can lead to the accumulation of toxic intermediate compounds. For the phytopathogenic basidiomycete Ustilago maydis, galactose is toxic for wildtype strains, i.e. leads to growth repression despite the presence of favorable carbon sources as sucrose. The galactose sensitivity can be relieved by two independent modifications: (1) by disruption of Hxt1, which we identify as the major transporter for galactose, and (2) by a point mutation in the gene encoding the galactokinase Gal1, the first enzyme of the Leloir pathway. The mutation in gal1(Y67F) leads to reduced enzymatic activity of Gal1 and thus may limit the formation of putatively toxic galactose-1-phosphate. However, systematic deletions and double deletions of different genes involved in galactose metabolism point to a minor role of galactose-1-phosphate in galactose toxicity. Our results show that molecular triggers for galactose toxicity in U. maydis differ from yeast and mammals.

Common and divergent features of galactose-1-phosphate and fructose-1-phosphate toxicity in yeast.

Metabolic dysregulation leading to sugar-phosphate accumulation is toxic in organisms ranging from bacteria to humans. By comparing two models of sugar-phosphate toxicity in Saccharomyces cerevisiae, we demonstrate that toxicity occurs, at least in part, through multiple, isomer-specific mechanisms, rather than a single general mechanism.

Human NUDT22 Is a UDP-Glucose/Galactose Hydrolase Exhibiting a Unique Structural Fold.

Human NUDT22 belongs to the diverse NUDIX family of proteins, but has, until now, remained uncharacterized. Here we show that human NUDT22 is a Mg2+-dependent UDP-glucose and UDP-galactose hydrolase, producing UMP and glucose 1-phosphate or galactose 1-phosphate. We present the structure of human NUDT22 alone and in a complex with the substrate UDP-glucose. These structures reveal a partially conserved NUDIX fold domain preceded by a unique N-terminal domain responsible for UDP moiety binding and recognition. The NUDIX domain of NUDT22 contains a modified NUDIX box identified using structural analysis and confirmed through functional analysis of mutants. Human NUDT22's distinct structure and function as a UDP-carbohydrate hydrolase establish a unique NUDIX protein subfamily.

Structural basis for enzyme bifunctionality - the case of Gan1D from Geobacillus stearothermophilus.

6-phospho-ß-glucosidases and 6-phospho-ß-galactosidases are enzymes that hydrolyze the ß-glycosidic bond between a terminal non-reducing glucose-6-phosphate (Glc6P) or galactose-6-phosphate (Gal6P), respectively, and other organic molecules. Gan1D, a glycoside hydrolase (GH) belonging to the GH1 family, has recently been identified in a newly characterized galactan-utilization gene cluster in the bacterium Geobacillus stearothermophilus T-1. Gan1D has been shown to exhibit bifunctional activity, possessing both 6-phospho-ß-galactosidase and 6-phospho-ß-glucosidase activities. We report herein the complete 3D crystal structure of Gan1D, together with its acid/base catalytic mutant Gan1D-E170Q. The tertiary structure of Gan1D conforms well to the (ß/α)8 TIM-barrel fold commonly observed in GH enzymes, and its quaternary structure adopts a dimeric assembly, confirmed by gel-filtration and small-angle X-ray scattering results. We present also the structures of Gan1D in complex with the putative substrate cellobiose-6-phosphate (Cell6P) and the degradation products Glc6P and Gal6P. These complexes reveal the specific enzyme-substrate and enzyme-product binding interactions of Gan1D, and the residues involved in its glycone, aglycone, and phosphate binding sites. We show that the different ligands trapped in the active sites adopt different binding modes to the protein, providing a structural basis for the dual galactosidase/glucosidase activity observed for this enzyme. Based on this information, specific mutations were performed on one of the active site residues (W433), shifting the enzyme specificity from dual activity to a significant preference toward 6-phospho-ß-glucosidase activity. These data and their comparison with structural data of related glucosidases and galactosidases are used for a more general discussion on the structure-function relationships in this sub-group of GH1 enzymes. DATABASES: Atomic coordinates of Gan1D-wild-type (WT)-P1, Gan1D-WT-C2, Gan1D-E170Q, Gan1D-WT-Gal6P, Gan1D-WT-Glc6P, and Gan1D-E170Q-Cell6P have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, under accession codes 5OKB, 5OKJ/5OKH, 5OKA/5OK7, 5OKQ/5OKK, 5OKS/5OKR, and 5OKG/5OKE, respectively.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

Galactose and its Metabolites Deteriorate Metaphase II Mouse Oocyte Quality and Subsequent Embryo Development by Disrupting the Spindle Structure.

Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare spontaneous pregnancies have been reported. Here, we evaluate the effect of D-galactose and its metabolites, galactitol and galactose 1-phosphate, on oocyte quality as well as embryo development to elucidate the mechanism through which these compounds mediate oocyte deterioration. Metaphase II mouse oocytes (n = 240), with and without cumulus cells (CCs), were exposed for 4 hours to D-galactose (2 µM), galactitol (11 µM) and galactose 1-phosphate (0.1 mM), (corresponding to plasma concentrations in patients on galactose-restricted diet) and compared to controls. The treated oocytes showed decreased quality as a function of significant enhancement in production of reactive oxygen species (ROS) when compared to controls. The presence of CCs offered no protection, as elevated ROS was accompanied by increased apoptosis of CCs. Our results suggested that D-galactose and its metabolites disturbed the spindle structure and chromosomal alignment, which was associated with significant decline in oocyte cleavage and blastocyst development after in-vitro fertilization. The results provide insight into prevention and treatment strategies that may be used to extend the window of fertility in these patients.

Acute and long-term outcomes in a Drosophila melanogaster model of classic galactosemia occur independently of galactose-1-phosphate accumulation.

Classic galactosemia (CG) is a potentially lethal inborn error of metabolism that results from the profound loss of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. Neonatal detection and dietary restriction of galactose minimizes or resolves the acute sequelae of CG, but fails to prevent the long-term complications experienced by a majority of patients. One of the substrates of GALT, galactose-1-phosphate (Gal-1P), accumulates to high levels in affected infants, especially following milk exposure, and has been proposed as the key mediator of acute and long-term pathophysiology in CG. However, studies of treated patients demonstrate no association between red blood cell Gal-1P level and long-term outcome severity. Here, we used genetic, epigenetic and environmental manipulations of a Drosophila melanogaster model of CG to test the role of Gal-1P as a candidate mediator of outcome in GALT deficiency. Specifically, we both deleted and knocked down the gene encoding galactokinase (GALK) in control and GALT-null Drosophila, and assessed the acute and long-term outcomes of the resulting animals in the presence and absence of dietary galactose. GALK is the first enzyme in the Leloir pathway of galactose metabolism and is responsible for generating Gal-1P in humans and Drosophila Our data confirmed that, as expected, loss of GALK lowered or eliminated Gal-1P accumulation in GALT-null animals. However, we saw no concomitant rescue of larval survival or adult climbing or fecundity phenotypes. Instead, we saw that loss of GALK itself was not benign and in some cases phenocopied or exacerbated the outcome seen in GALT-null animals. These findings strongly contradict the long-standing hypothesis that Gal-1P alone underlies pathophysiology of acute and long-term outcomes in GALT-null Drosophila and suggests that other metabolite(s) of galactose, and/or other pathogenic factors, might be involved.

Biochemical characterization of a novel bifunctional glycosyl-1-phosphate transferase involved in the exopolysaccharide biosynthesis.

Ebosin produced by Streptomyces sp. 139 is a novel exopolysaccharide with anti-rheumatic arthritis activity in vivo and its biosynthesis gene cluster (ste) has been previously identified. In our previous research, ste5 gene has been identified as priming glycosyltransferase involved in Ebosin biosynthesis. However, it remains unclear how ste5 initiated Ebosin biosynthesis in molecular level. Here we show that Ebosin derivative produced by ste5 mutant lost the antagonist activities for IL-1R and Overexpression of ste5 in mutant dramatically enhanced the antagonist activities for IL-1R. For biochemical characterization of Ste5, the ste5 gene was cloned and expressed in Escherichia coli BL21. We identified that the recombinant Ste5 can transfer galactose-1-Phosphate (Gal-1-P) or glucose-1-Phosphate (Glc-1-P) from UDP-galactose and UDP-glucose to the lipid carrier located in the cytoplasmic membrane of Streptomyces sp. 139 (ste5(-)) with a continuous coupled spectrophotometric assay. 12.6 µM of Km was for UDP-galactose and 23.9 µM for UDP-glucose respectively. Our results indicate that Ste5 is bifunctional Gal-1-P and Glc-1-P transferase to initiate Ebosin biosynthesis and may be further applied in remoulding carbohydrate compounds.

Genetic and functional studies reveal a novel noncoding variant in GALT associated with a false positive newborn screening result for galactosemia.

BACKGROUND: Classic galactosemia (CG) is a potentially lethal genetic disorder that results from profound loss of galactose-1-phosphate uridylyltransferase (GALT). CG is detected by newborn screening (NBS) in many countries; however, conclusive diagnosis can be complex due to broad and overlapping ranges of GALT activity. Molecular studies can also be complex due to allelic heterogeneity at the GALT locus. METHODS: We conducted both biochemical and molecular follow-up studies for an infant flagged by NBS for possible galactosemia. To clarify the diagnosis we also conducted biochemical and RNA studies of lymphoblasts prepared from the child and one parent. RESULTS: We identified a novel noncoding GALT variant, c.377+17C>T, that was homozygous in the child and heterozygous in both parents. The child and both parents also showed diminished GALT activity in red blood cells, and transformed lymphoblasts from the child and one parent further showed diminished GALT activity. However, qRT-PCR studies demonstrated apparently normal GALT mRNA levels in lymphoblasts, and Gal-1P values measured in the child following galactose exposure in infancy and at 1 year were normal. CONCLUSIONS: These results highlight the existence of rare but apparently benign variants in GALT and underscore the need for functional studies to distinguish pathogenic from benign variants.