GALT deficiency causes UDP-hexose deficit in human galactosemic cells

Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.     

Relationship between genotype, activity, and galactose sensitivity in yeast expressing patient alleles of human galactose-1-phosphate uridylyltransferase

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.     

Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosemia

Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.     

Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes

Classic galactosemia, an inborn error of human galactose metabolism, is characterized by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The current model for the pathophysiology of this disease ascribes most of its symptoms to the toxicity of intracellular galactose-1-phosphate (Gal-1-P), one of the substrates of GALT which accumulates in the untreated disease state. Recently, a reduction in the intracellular concentration of UDP-Gal (uridine diphosphogalactose), one of the products of GALT, has been described in treated galactosemic patients. We investigated whether galactosemic patients might also have reduced amounts of those macromolecules that depend on UDP-Gal for their biosynthesis. We report a reduction in glycolipids that contain either galactose or its derivative N-acetylgalactosamine and an accumulation of the precursors to these compounds in the brain of a neonate with galactosemia. We also found an imbalance in glycolipids in galactosemic lymphoblasts. This novel biochemical abnormality observed in galactosemic patients is not addressed by dietary galactose-restriction therapy and could explain some of the chronic neurologic and other complications of galactosemia.     

The structural and molecular biology of type I galactosemia: Enzymology of galactose 1-phosphate uridylyltransferase

Reduced galactose 1-phosphate uridylyltransferase (GALT) activity is associated with the genetic disease type I galactosemia. This results in an increase in the cellular concentration of galactose 1-phosphate. The accumulation of this toxic metabolite, combined with aberrant glycoprotein and glycolipid biosynthesis, is likely to be the major factor in molecular pathology. The mechanism of GALT was established through classical enzymological methods to be a substituted enzyme in which the reaction with UDP-glucose results in the formation of a covalent, UMP-histidine adduct in the active site. The uridylated enzyme can then react with galactose 1-phosphate to form UDP-galactose. The structure of the enzyme from Escherichia coli reveals a homodimer containing one zinc (II) and one iron (II) ion per subunit. This enzymological and structural knowledge provides the basis for understanding the biochemistry of this critical step in the Leloir pathway. However, a high-resolution crystal structure of human GALT is required to assist greater understanding of the effects of disease-associated mutations.     

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.     

Reversion from deficiency of galactose-1-phosphate uridylytransferase (GALT) in an SV40-transformed human fibroblast line

Control SV40-transformed human fibroblasts can be readily adapted to growth on medium containing galactose as sole hexose source (galactose-MEH). However, most cells from a line of SV40-transformed skin fibroblasts from a patient with galactosemia (galactose-1-phosphate uridylyltransferase (GALT) deficiency) died in galactose-MEM. Surviving cells of this line either grew in completely sugar-free media or had acquired significant amounts of GALT activity. Two presumptive revertant cell lines with GALT activity were characterized in detail. The expression of GALT in these two lines was stable in nonselective conditions. Each had different reaction maximum velocities with respect to uridine diphosphoglucose (UDPg) concentration as compared to residual activity in the parental cell strain or control cells. Both appeared to demonstrate heat-inactivation profiles for GALT than differed from the parental cells or controls. UDPG concentration was found to significantly alter the thermostability of GALT. A competitive radioimmunoassay for GALT showed that these two lines had amounts of the GALT protein comparable to that of the parental cell strain or control cells. The electrophoretic mobility of GALT from the two presumptive revertants was found to differ from control cells. It was concluded that structural gene changes were probably responsible for the apparent reversion in these lines.     

The roles of galactitol, galactose-1-phosphate, and phosphoglucomutase in galactose-induced toxicity in Saccharomyces cerevisiae

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.     

Defective galactosylation of serum transferrin in galactosemia

The glycosylation of serum transferrin from galactosemic patients with a deficiency of galactose-1-phosphate uridyl transferase (EC 2. 7.7 12) is abnormal but becomes normal after treatment with a galactose-free diet. To understand the structural and biochemical basis of the abnormal glycosylation, transferrin was purified from the serum of untreated and treated galactosemic patients and normal controls and the N-linked glycans analyzed by HPLC. The glycans from normal transferrin consisted predominantly (86%) of the disialylated biantennary complex type. The glycans from untreated galactosemic patients were more heterogeneous and contained four major truncated glycans in addition to a smaller amount (13%) of the disialylated biantennary complex type. The truncated glycans were deficient in galactose and sialic acid and their structures were consistent with a decrease in galactosyltransferase activity in hepatocytes, the probable cells of origin of the transferrin. This is postulated to be due to direct inhibition of the galactosyltransferase activity by the accumulated galactose-1-phosphate or to an effect on the formation of UDP- galactose, the donor substrate in the reaction. After treatment the proportion of the truncated glycans decreased and the proportion of the disialylated biantennary complex type increased, returning almost but never completely to normal, even after prolonged treatment in some cases. There was no clear relationship between the length of treatment and the normalization of glycosylation and the level of galactose-1- phosphate in red blood cells, the usual parameter for monitoring the treatment of galactosemics. It is suggested that the persistence of abnormally glycosylated proteins may contribute to the long-term complications in galactosemia.      

N-Glycosylation of total cellular glycoproteins from the human ovarian carcinoma SKOV3 cell line and of recombinantly expressed human erythropoietin

Ovarian carcinoma is the leading cause of death from gynecological cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation and consequently in ovarian cancer. In this study, a detailed structure analysis of the N-linked glycans from total glycoproteins from the SKOV3 ovarian carcinoma cell line and from a recombinantly expressed secretory glycoprotein, erythropoietin (EPO), produced from the same cells has been performed using high-performance anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total cellular N-glycans contained high-mannose type and proximally fucosylated complex type partially agalactosylated structures. On the other hand, the recombinant human EPO secreted from SKOV3 cells contained predominantly core-fucosylated tetraantennary structures, which were partially lacking one or two galactose residues, and partially contained the LacdiNAc motif. Only minor amounts of di- and triantennary complex-type glycans were found, and high-mannose-type glycans were not present in the secreted EPO protein. A large amount of N-acetylneuraminic acid in α2,3-linkage was detected as well. Endogenous glycoproteins were also found to contain the LacdiNAc motif in N-linked glycans. This work contributes to the knowledge of the glycosylation of a human ovarian cancer cell line. It also establishes the basis to further explore high-mannose-type glycans, and the LacdiNAc motif as possible markers of ovarian carcinoma.     

Effect of uridine on hepatic galactose-1-phosphate uridyltransferase

Uridine sugar nucleotides are important intermediates in galactose metabolism and may play a role in the long-term galactose toxicity in human galactose-1-phosphate uridyltransferase deficiency galactosemia. Since administration of uridine, a precursor of uridine nucleotides, has been considered as a therapeutic measure, we have investigated the effects of this compound on the activity of rat hepatic transferase. Uridine has been found to be an inhibitor of the enzyme in in vitro studies and to cause an increase in galactose-1-phosphate in liver perfused with galactose which is consistent with physiologic inhibition of the enzyme. Uridine is a partial linear competitive inhibitor of UDPglucose and an uncompetitive inhibitor of galactose-1-phosphate. These findings suggest caution should be applied in giving the compound to subjects with genetically limited transferase activity because of the possibility of inhibiting the small amount of residual enzyme.     

The binding of decomposition products of UDP-galactose to the microsomes and polyribosomes isolated from rat liver

UDP-D-[U-14C]galactose is decomposed to [U-14C]galactose-1-phosphate and [U-14C]galactose by rat liver microsomal and crude polyribosomal fractions, under conditions commonly used to assay of glycosyltransferase activities. UDP-D-[U-14C]galactose, at neutral pH, is also chemically degraded to the [U-14C]galactose-1,2-cyclic phosphate. The 1,2-cyclic phosphate derivative of galactose also exists in the commercial UDP-D-[U-14C]galactose. It is a very important finding that products of the UDP-D-[U-14C]galactose decomposition are tightly, although nonenzymatically, bound to tested subcellular fractions and may create a false impression of protein glycosylation. The application of controls containing all radioactive substances present in suitable samples is recommended in order to avoid incorrect interpretations of the results.     

On the molecular nature of the Duarte variant of galactose-1-phosphate uridyl transferase (GALT)

Galactosemia is an inborn error of galactose metabolism secondary to deficiency of galactose-1-phosphate uridyl transferase (GALT). GALT is a polymorphic enzyme and Duarte (D) is the most common enzyme variant. This variant is characterized by faster electrophoretic mobility and reduced activity. Duarte/galactosemia compound heterozygotes (D/G) are commonly identified in galactosemia newborn screening programs. However, these patients do not generally require treatment. By using a candidate mutation approach to define the molecular basis of the Duarte variant of GALT, a close association between the previously reported N314D polymorphism and the Duarte variant of GALT was found. We suggest that N314D encodes the D variant of GALT and that molecular testing for N314D might be useful to confirm a biochemical diagnosis of Duarte variant of GALT.     

Galactose tolerance studies of individuals with reduced galactose pathway activity.

The galactose tolerance of individuals with mutant genotypes affecting the activities of galactokinase (GALK) and galactose-1-phosphate uridylyltransferase (GALT) was examined. Genotypes studied were heterozygotes for the GALK and GALT forms of galactosemia, the Duarte-variant GALT, and Philadelphia-variant GALK alleles. The measurements used were urinary concentration of galactose during pregnancy in adults and in infants from the newborn period through the first 5 months of life; the rate of elimination of an intravenous infusion of galactose; and slit-lamp examination of the lens for evidence of cataracts. No unusual urinary excretions of galactose were noted in any of the age groups studied. Intravenous galactose tolerance tests were normal in all but two women, a mother and daughter heterozygous for the GALK-deficient form of galactosemia (GALKG/GALKA). Six other GALKG/GALKA subjects had normal tolerance studies. The intrafamilial consistency and interfamilial differences in the galactose tolerance of GALKG/GALKA individuals suggest heterogeneity of the genes responsible for the GALK-deficient form of galactosemia. Although subclinical cataracts were observed in several individuals, their significance relative to the mutant genotype cannot be resolved with the available data.     

The human galactose-1-phosphate uridyltransferase gene.

Classical galactosemia is an inborn error of metabolism caused by a deficiency of galactose-1-phosphate uridyltransferase (GALT). Standard treatment with dietary galactose restriction will reverse the potentially lethal symptoms of the disease that are manifest in the newborn period. However, the long-term prognosis for these patients is variable. As a first step toward investigating the molecular basis for phenotypic variation in galactosemia, we have cloned and sequenced the entire gene for human galactose-1-phosphate uridyltransferase. This gene is organized into 11 exons spanning 4 kb. In exons 6, 9, and a portion of 10, there is a high degree of amino acid sequence conservation among Escherichia coli, yeast, mouse, and human. We have identified a number of nucleotide changes in the GALT genes of galactosemic patients that alter conserved amino acids. The most common of these is an A to G transition at nucleotide position 1470, converting a glutamine to an arginine at amino acid codon position 188 (Q188R).(ABSTRACT TRUNCATED AT 250 WORDS)     

The unfolded protein response has a protective role in yeast models of classic galactosemia

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.KEY WORDS: Yeast, Galactosemia, UPR, Lithium, Galactose     

Distinct galactose phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus lactis.

Lactose-negative (Lac-) mutants were isolated from a variant of Streptococcus lactis C2 in which the lactose plasmid had become integrated into the chromosome. These mutants retained their parental growth characteristics on galactose (Lac- Gal+). This is in contrast to the Lac- variants obtained when the lactose plasmid is lost from S. lactis, which results in a slower growth rate on galactose (Lac- Gal+). The Lac- Gal+ mutants were defective in [14C]thiomethyl-beta-D-galactopyranoside accumulation, suggesting a defect in the lactose phosphoenolpyruvate-dependent phosphotransferase system, but still possessed the ability to form galactose-1-phosphate and galactose-6-phosphate from galactose in a ratio similar to that observed from the parental strain. The Lac- Gald variant formed only galactose-1-phosphate. The results imply that galactose is not translocated via the lactose phosphoenolpyruvate-dependent phosphotransferase system, but rather by a specific galactose phosphoenolpyruvate-dependent phosphotransferase system for which the genetic locus is also found on the lactose plasmid in S. lactis.     

Overelaborated synaptic architecture and reduced synaptomatrix glycosylation in a Drosophila classic galactosemia disease model

Classic galactosemia (CG) is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT), which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP)-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP–N-acetylgalactosamine and UDP–N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG), showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT) greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ). Dietary galactose and mutation of galactokinase (dGALK) or UDP-glucose dehydrogenase (sugarless) genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP) moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG) co-receptor and Wnt ligand levels, which are also corrected by dGALK co-removal and sugarless overexpression. These results reveal synaptomatrix glycosylation losses, altered trans-synaptic signaling pathway components, defective synaptogenesis and impaired coordinated movement in a CG neurological disease model.KEY WORDS: Congenital disorder of glycosylation (CDG), sugarless, Galactokinase, Synaptogenesis, Trans-synaptic signaling, WNT, HSPG, Neuromuscular junction     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.     

Polymorphism of human red cell galactose-1-phosphate uridyl transferase in Serbia, Yugoslavia

Phenotypes of human red cell galactose-1-phosphate uridyl transferase (GALT) were determined in 283 unrelated adults from Serbia (Yugoslavia). The gene frequencies were 0.959 for GALT N, 0.018 for GALT D and 0.023 for GALT N.