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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., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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 Mg 2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and β-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-β-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-β-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Classic galactosemia is a rare inborn error of metabolism that affects the metabolism of galactose, a sugar derived from milk and derivates. Classic galactosemia is caused by variants of the GALT gene, which lead to absent or misfolded forms of the ubiquitously present galactose-1-phosphate uridylyltransferase enzyme (GALT) driving galactose metabolites to accumulate, damaging cells from neurons to hepatocytes. The disease has different prevalence around the world due to different allele frequencies among populations and its symptoms range from cognitive and psychomotor impairment to hepatic, ophthalmological, and bone structural damage. The practice of newborn screening still varies among countries, dairy restriction treatment is a consensus despite advances in preclinical treatment strategies. Recent clinical studies in Duarte variant suggest dairy restriction could be reconsidered in these cases. Despite noteworthy advances in the classic galactosemia understanding, preclinical trials are still crucial to fully understand the pathophysiology of the disease and help propose new treatments. This review aims to report a comprehensive analysis of past studies and state of art research on galactosemia screening, its clinical and preclinical trials, and treatments with the goal of shedding light on this complex and multisystemic innate error of the metabolism., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Classic galactosemia is an autosomal recessive inherited liver disorder of carbohydrate metabolism caused by deficient activity of galactose-1-phosphate uridylyltransferase (GALT). While a galactose-restricted diet is lifesaving, most patients still develop long-term complications. In this study, we report on a two-week-old female patient who is a compound heterozygote for a known pathogenic variant (p.K285N) and a novel missense variant (p.A303D) in the GALT gene. Segregation analysis showed that the patient inherited the p.K285N pathogenic variant from her father and the p.A303D variant from her mother. A bioinformatics analysis to predict the impact of the p.A303D missense variant on the structure and stability of the GALT protein revealed that it may be pathogenic. Based on this finding, we performed a literature review of all GALT missense variants identified in homozygous and compound heterozygous galactosemia patients carrying the p.K285N pathogenic variant to explore their molecular effects on the clinical phenotype of the disease. Our analysis revealed that these missense variants are responsible for a wide range of molecular defects. This study expands the clinical and mutational spectrum in classic galactosemia and reinforces the importance of understanding the molecular consequences of genetic variants to incorporate genetic analysis into clinical care.

During our biochemical characterization of select bacterial phosphatases belonging to the haloacid dehalogenase superfamily of hydrolases, we discovered a strong bias of Salmonella YidA for glucose-1-phosphate (Glc-1-P) over galactose-1-phosphate (Gal-1-P). We sought to exploit this ability of YidA to discriminate these two sugar-phosphate epimers in a simple coupled assay that could be a substitute for current cumbersome alternatives. To this end, we focused on Gal-1-P uridylyltransferase (GalT) that is defective in individuals with classical galactosemia, an inborn disorder. GalT catalyzes the conversion of Gal-1-P and UDP-glucose to Glc-1-P and UDP-galactose. When recombinant YidA was coupled to GalT, the final orthophosphate product (generated from selective hydrolysis of Glc-1-P by YidA) could be easily measured using the inexpensive malachite green reagent. When this new YidA-based colorimetric assay was benchmarked using a recombinant Duarte GalT variant, it yielded kcat/Km values that are ~2.5-fold higher than the standard coupled assay that employs phosphoglucomutase and glucose-6-phosphate dehydrogenase. Although the simpler design of our new GalT coupled assay might find appeal in diagnostics, a testable expectation, we spotlight the GalT example to showcase the untapped potential of sugar-phosphate phosphatases with distinctive substrate-recognition properties for measuring the activity of various metabolic enzymes (e.g. trehalose-6-phosphate synthase, N-acetyl-glucosamine-6-phosphate deacetylase, phosphofructokinase)., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Primary ovarian insufficiency is characterized by accelerated loss of primordial follicles, which results in ovarian failure and concomitant menopause before age 40. About 1-3% of females in the general population are diagnosed with POI; however, greater than 80% of females with the inherited disease Classic Galactosemia will develop POI. Classic Galactosemia is caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase. While dietary restriction of galactose is lifesaving in the neonatal period, the development of complications including primary ovarian insufficiency is not mitigated. Additionally, the pattern(s) of follicle loss have not been completely characterized. The chronic accumulation of aberrant metabolites such as galactose-1-phosphate and galactitol are suspected culprits in the development of the sequelae, yet the mechanisms remain elusive.Our group uses a GalT gene-trapped mouse model to study the pathophysiology of primary ovarian insufficiency in Classic Galactosemia. We recently showed that differences in the Integrated Stress Response pathway occur in mutant ovaries that likely contribute to their primary ovarian insufficiency phenotype. Using immunofluorescent staining of histological sections of ovaries at progressive ages, we saw evidence of altered Integrated Stress Response activity in granulosa cells and primordial oocytes consistent with accelerated primordial follicle growth activation, aberrant DNA damage and/or repair, and increased cellular stress/death. Overall, our findings indicate that abnormal Integrated Stress Response in the Classic Galactosemia model ovary results in accelerated primordial follicle growth activation, sometimes referred to as "burnout." These aberrant early events help further clarify when/how the primary ovarian insufficiency phenotype arises under galactosemic conditions., (© 2022. The Author(s).)

Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Classic Galactosemia (CG) is a devastating inborn error of the metabolism caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase in galactose metabolism. Severe complications of CG include neurological impairments, growth restriction, cognitive delays, and, for most females, primary ovarian insufficiency. The absence of the GALT enzyme leads to an accumulation of aberrant galactose metabolites, which are assumed to be responsible for the sequelae. There is no treatment besides the restriction of dietary galactose, which does not halt the development of the complications; thus, additional treatments are sorely needed. Supplements have been used in other inborn errors of metabolism but are not part of the therapeutic regimen for CG. The goal of this study was to test two generally recognized as safe supplements (purple sweet potato color (PSPC) and myo -inositol (MI)) that may impact cellular pathways contributing to the complications in CG. Our group uses a GalT gene-trapped mouse model to study the pathophysiology in CG, which phenocopy many of the complications. Here we report the ability of PSPC to ameliorate dysregulation in the ovary, brain, and liver of our mutant mice as well as positive results of MI supplementation in the ovary and brain.

Messenger RNA (mRNA) has emerged as a novel therapeutic approach for inborn errors of metabolism. Classic galactosemia (CG) is an inborn error of galactose metabolism caused by a severe deficiency of galactose-1-phosphate:uridylyltransferase (GALT) activity leading to neonatal illness and chronic impairments affecting the brain and female gonads. In this proof of concept study, we used our zebrafish model for CG to evaluate the potential of human GALT mRNA (hGALT mRNA) packaged in two different lipid nanoparticles to restore GALT expression and activity at early stages of development. Both one cell-stage and intravenous single-dose injections resulted in hGALT protein expression and enzyme activity in the CG zebrafish (galt knockout) at 5 days post fertilization (dpf). Moreover, the levels of galactose-1-phosphate (Gal-1-P) and galactonate, metabolites that accumulate because of the deficiency, showed a decreasing trend. LNP-packaged mRNA was effectively translated and processed in the CG zebrafish without signs of toxicity. This study shows that mRNA therapy restores GALT protein and enzyme activity in the CG zebrafish model, and that the zebrafish is a suitable system to test this approach. Further studies are warranted to assess whether repeated injections safely mitigate the chronic impairments of this disease., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin - an inhibitor of the de novo sphingolipid synthesis pathway - inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose-1-phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno-associated virus (AAV)-mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV-mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFβRII protein levels and that these effectors of cellular homeostasis could be restored following AAV-mediated expression of GALT. Finally, we show initial in vivo proof-of-concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV-GALT., (© 2021 SSIEM.)

Classic galactosemia (CG) results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection by newborn screening and lifelong dietary restriction of galactose, most patients grow to experience a range of long-term complications. Recently, we developed and characterized a GALT-null rat model of CG and demonstrated that AAV9-hGALT, administered by tail vein injection to neonatal pups, dramatically improved plasma, liver, and brain galactose metabolites at 2 weeks posttreatment. Here we report a time-course study of GALT restoration in rats treated as neonates with scAAV9-hGALT and harvested at 8, 14, 30, and 60 days. Cohorts of rats in the two older groups were weaned to diets containing either 1% or 3% of calories from galactose. As expected, GALT activity in all treated animals peaked early and then diminished over time, most notably in liver, ostensibly due to dilution of the nonreplicating episomal vector as transduced cells divided. All treated rats showed dramatic metabolic rescue through 1 month, and those weaned to the lower galactose diet showed continued strong metabolic rescue into adulthood (2 months). Prepubertal growth delay and cataracts were both partially rescued by treatment. Finally, we found that UDP glucose pyrophosphorylase (UGP), which offers a metabolic bypass around missing GALT, was 3-fold more active in brain samples from adult rats than from young pups, offering a possible explanation for the improved ability of older GALT-null rats to metabolize galactose. Combined, these results document promising metabolic and phenotypic efficacy of neonatal GALT gene replacement in a rat model of classic galactosemia., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Classic galactosemia is an inborn error of metabolism associated with mutations that impair the activity and the stability of galactose-1-phosphate uridylyltransferase (GALT), catalyzing the third step in galactose metabolism. To date, no treatments (including dietary galactose deprivation) are able to prevent or alleviate the long-term complications affecting galactosemic patients. Evidence that arginine is able to improve the activity of the human enzyme expressed in a prokaryotic model of classic galactosemia has induced researchers to suppose that this amino acid could act as a pharmacochaperone, but no effects were detected in four galactosemic patients treated with this amino acid. Given that no molecular characterizations of the possible effects of arginine on GALT have been performed, and given that the samples of patients treated with arginine are extremely limited for drawing definitive conclusions at the clinical level, we performed computational simulations in order to predict the interactions (if any) between this amino acid and the enzyme. Our results do not support the possibility that arginine could function as a pharmacochaperone for GALT, but information obtained by this study could be useful for identifying, in the future, possible pharmacochaperones for this enzyme.

The third step of the catabolism of galactose in mammals is catalyzed by the enzyme galactose-1-phosphate uridylyltransferase (GALT), a homodimeric enzyme with two active sites located in the proximity of the intersubunit interface. Mutations of this enzyme are associated to the rare inborn error of metabolism known as classic galactosemia; in particular, the most common mutation, associated with the most severe phenotype, is the one that replaces Gln188 in the active site of the enzyme with Arg (p.Gln188Arg). In the past, and more recently, the structural effects of this mutation were deduced on the static structure of the wild-type human enzyme; however, we feel that a dynamic view of the proteins is necessary to deeply understand their behavior and obtain tips for possible therapeutic interventions. Thus, we performed molecular dynamics simulations of both wild-type and p.Gln188Arg GALT proteins in the absence or in the presence of the substrates in different conditions of temperature. Our results suggest the importance of the intersubunit interactions for a correct activity of this enzyme and can be used as a starting point for the search of drugs able to rescue the activity of this enzyme in galactosemic patients.

Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia., (© 2021 Wiley-VCH GmbH.)

The pathogenesis and molecular mechanisms of ovarian low malignant potential (LMP) tumors or borderline ovarian tumors (BOTs) have not been fully elucidated to date. Surgery remains the cornerstone of treatment for this disease, and diagnosis is mainly made by histopathology to date. However, there is no integrated analysis investigating the tumorigenesis of BOTs with open experimental data. Therefore, we first utilized a functionome-based speculative model from the aggregated obtainable datasets to explore the expression profiling data among all BOTs and two major subtypes of BOTs, serous BOTs (SBOTs) and mucinous BOTs (MBOTs), by analyzing the functional regularity patterns and clustering the separate gene sets. We next prospected and assembled the association between these targeted biomolecular functions and their related genes. Our research found that BOTs can be accurately recognized by gene expression profiles by means of integrative polygenic analytics among all BOTs, SBOTs, and MBOTs; the results exhibited the top 41 common dysregulated biomolecular functions, which were sorted into four major categories: immune and inflammatory response-related functions, cell membrane- and transporter-related functions, cell cycle- and signaling-related functions, and cell metabolism-related functions, which were the key elements involved in its pathogenesis. In contrast to previous research, we identified 19 representative genes from the above classified categories (IL6, CCR2 for immune and inflammatory response-related functions; IFNG, ATP1B1, GAS6, and PSEN1 for cell membrane- and transporter-related functions; CTNNB1, GATA3, and IL1B for cell cycle- and signaling-related functions; and AKT1, SIRT1, IL4, PDGFB, MAPK3, SRC, TWIST1, TGFB1, ADIPOQ, and PPARGC1A for cell metabolism-related functions) that were relevant in the cause and development of BOTs. We also noticed that a dysfunctional pathway of galactose catabolism had taken place among all BOTs, SBOTs, and MBOTs from the analyzed gene set databases of canonical pathways. With the help of immunostaining, we verified significantly higher performance of interleukin 6 (IL6) and galactose-1-phosphate uridylyltransferase (GALT) among BOTs than the controls. In conclusion, a bioinformatic platform of gene-set integrative molecular functionomes and biophysiological pathways was constructed in this study to interpret the complicated pathogenic pathways of BOTs, and these important findings demonstrated the dysregulated immunological functionome and dysfunctional metabolic pathway as potential roles during the tumorigenesis of BOTs and may be helpful for the diagnosis and therapy of BOTs in the future.

Background: Classical Galactosemia (CG) is an inherited disorder of galactose metabolism caused by a deficiency of the galactose-1-phosphate uridylyltransferase (GALT) enzyme resulting in neurocognitive complications. As in many Inborn Errors of Metabolism, the metabolic pathway of CG is well-defined, but the pathophysiology and high variability in clinical outcome are poorly understood. The aim of this study was to investigate structural changes of the brain of CG patients on MRI and their association with clinical outcome., Methods: In this prospective cohort study an MRI protocol was developed to evaluate gray matter (GM) and white matter (WM) volume of the cerebrum and cerebellum, WM hyperintensity volume, WM microstructure and myelin content with the use of conventional MRI techniques, diffusion tensor imaging (DTI) and quantitative T1 mapping. The association between several neuroimaging parameters and both neurological and intellectual outcome was investigated., Results: Twenty-one patients with CG (median age 22 years, range 8-47) and 24 controls (median age 30, range 16-52) were included. Compared to controls, the WM of CG patients was lower in volume and the microstructure of WM was impaired both in the whole brain and corticospinal tract (CST) and the lower R1 values of WM, GM and the CST were indicative of less myelin. The volume of WM lesions were comparable between patients and controls. The 9/16 patients with a poor neurological outcome (defined as the presence of a tremor and/or dystonia), demonstrated a lower WM volume, an impaired WM microstructure and lower R1 values of the WM indicative of less myelin content compared to 7/16 patients without movement disorders. In 15/21 patients with a poor intellectual outcome (defined as an IQ < 85) both GM and WM were affected with a lower cerebral and cerebellar WM and GM volume compared to 6/21 patients with an IQ ≥ 85. Both the severity of the tremor (as indicated by the Tremor Rating Scale) and IQ (as continuous measure) were associated with several neuroimaging parameters such as GM volume, WM volume, CSF volume, WM microstructure parameters and R1 values of GM and WM., Conclusion: In this explorative study performed in patients with Classical Galactosemia, not only WM but also GM pathology was found, with more severe brain abnormalities on MRI in patients with a poor neurological and intellectual outcome. The finding that structural changes of the brain were associated with the severity of long-term complications indicates that quantitative MRI techniques could be of use to explain neurological and cognitive dysfunction as part of the disease spectrum. Based on the clinical outcome of patients, the absence of widespread WM lesions and the finding that both GM and WM are affected, CG could be primarily a GM disease with secondary damage to the WM as a result of neuronal degeneration. To investigate this further the course of GM and WM should be evaluated in longitudinal research, which could also clarify if CG is a neurodegenerative disease., Competing Interests: Declaration of Competing Interest Mendy M. Welsink-Karssies, Anouk G.M. Schrantee, Mirian C.H. Janssen, Esmee Oussoren, Stefan D. Roosendaal, Marc Engelen and Maaike de Vries declare they have no conflict of interest. Matthan W.A. Caan is shareholder or Nico.lab Ltd. Carla E.M. Hollak is involved in premarketing studies with Sanofi, Protalix and Idorsia in the field of lysosomal storage disorders. She reports no conflicts of interest in relation to the current study. Annet M. Bosch was member of an advisory board of Biomarin., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nucleotide sugars (NS) are fundamental molecules in life and play a key role in glycosylation reactions and signal conduction. Several pathways are involved in the synthesis of NS. The Leloir pathway, the main pathway for galactose metabolism, is crucial for production of uridine diphosphate (UDP)-glucose and UDP-galactose. The most common metabolic disease affecting this pathway is galactose-1-phosphate uridylyltransferase (GALT) deficiency, that despite a lifelong galactose-restricted diet, often results in chronically debilitating complications. Alterations in the levels of UDP-sugars leading to galactosylation abnormalities have been hypothesized as a key pathogenic factor. However, UDP-sugar levels measured in patient cell lines have shown contradictory results. Other NS that might be affected, differences throughout development, as well as tissue specific profiles have not been investigated. Using recently established UHPLC-MS/MS technology, we studied the complete NS profiles in wildtype and galt knockout zebrafish (Danio rerio). Analyses of UDP-hexoses, UDP-hexosamines, CMP-sialic acids, GDP-fucose, UDP-glucuronic acid, UDP-xylose, CDP-ribitol, and ADP-ribose profiles at four developmental stages and in tissues (brain and gonads) in wildtype zebrafish revealed variation in NS levels throughout development and differences between examined tissues. More specifically, we found higher levels of CMP-N-acetylneuraminic acid, GDP-fucose, UDP-glucuronic acid, and UDP-xylose in brain and of CMP-N-glycolylneuraminic acid in gonads. Analysis of the same NS profiles in galt knockout zebrafish revealed no significant differences from wildtype. Our findings in galt knockout zebrafish, even when challenged with galactose, do not support a role for abnormalities in UDP-glucose or UDP-galactose as a key pathogenic factor in GALT deficiency, under the tested conditions., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

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., (© 2019 Wiley Periodicals, Inc.)

Classical galactosemia (CG) patients frequently develop long-term complications despite early dietary treatment. The highly variable clinical outcome is poorly understood and a lack of prognostic biomarkers hampers individual prognostication and treatment. The aim of this study was to investigate the association between residual galactose oxidation capacity and clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. The noninvasive 1- 13 C galactose breath test was used to assess whole body galactose oxidation capacity. Participants received a 7 mg/kg oral dose of 1- 13 C labelled galactose. The galactose oxidation capacity was determined by calculating the cumulative percentage dose of the administered galactose (CUMPCD) recovered as 13 CO 2 in exhaled air. Forty-one CG patients (5-47 years) and four adult controls were included. The median galactose oxidation capacity after 120 minutes (CUMPCDT120) of 34 classical patients (0.29; 0.08-7.51) was significantly lower when compared to two homozygous p.Ser135Leu patients (9.44; 8.66-10.22), one heterozygous p.Ser135Leu patient 18.59, four NBS detected variant patients (13.79; 12.73-14.87) and four controls (9.29; 8.94-10.02). There was a clear correlation between Gal-1-P levels and CUMPCDT120 (P < .0005). In the classical patients, the differences in CUMPCDT120 were small and did not distinguish between patients with poor and normal clinical outcomes. The galactose breath test distinguished classical patients from homo- and heterozygous p.Ser135Leu and NBS detected variant patients, but was not able to predict clinical outcomes in classical patients. Future studies are warranted to enable individualised prognostication and treatment, especially in NBS variants with galactose oxidation capacities in the control range., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

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., (© 2019 SSIEM.)

Since the first description of galactosemia in 1908 and despite decades of research, the pathophysiology is complex and not yet fully elucidated. Galactosemia is an inborn error of carbohydrate metabolism caused by deficient activity of any of the galactose metabolising enzymes. The current standard of care, a galactose-restricted diet, fails to prevent long-term complications. Studies in cellular and animal models in the past decades have led to an enormous progress and advancement of knowledge. Summarising current evidence in the pathophysiology underlying hereditary galactosemia may contribute to the identification of treatment targets for alternative therapies that may successfully prevent long-term complications. A systematic review of cellular and animal studies reporting on disease complications (clinical signs and/or biochemical findings) and/or treatment targets in hereditary galactosemia was performed. PubMed/MEDLINE, EMBASE, and Web of Science were searched, 46 original articles were included. Results revealed that Gal-1-P is not the sole pathophysiological agent responsible for the phenotype observed in galactosemia. Other currently described contributing factors include accumulation of galactose metabolites, uridine diphosphate (UDP)-hexose alterations and subsequent impaired glycosylation, endoplasmic reticulum (ER) stress, altered signalling pathways, and oxidative stress. galactokinase (GALK) inhibitors, UDP-glucose pyrophosphorylase (UGP) up-regulation, uridine supplementation, ER stress reducers, antioxidants and pharmacological chaperones have been studied, showing rescue of biochemical and/or clinical symptoms in galactosemia. Promising co-adjuvant therapies include antioxidant therapy and UGP up-regulation. This systematic review provides an overview of the scattered information resulting from animal and cellular studies performed in the past decades, summarising the complex pathophysiological mechanisms underlying hereditary galactosemia and providing insights on potential treatment targets., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Genetic defects of human galactose-1-phosphate uridyltransferase ( h GALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by h GALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant h GALT with a K I of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and r h MUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae . To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

Hereditary galactosemia is an inborn error of carbohydrate metabolism. Galactose is metabolized by Leloir pathway enzymes; galactokinase (GALK), galactose-1-phosphate uridylyltransferase (GALT) and UDP-galactose 4-epimerase (GALE). The defects in these enzymes cause galactosemia in an autosomal recessive manner. The severe GALT deficiency, or classic galactosemia, is life-threatening in the newborn period. The treatment for classic galactosemia is dietary restriction of lactose. Although implementation of lactose restricted diet is efficient in resolving the acute complications, it is not sufficient to prevent long-term complications affecting the brain and female gonads, the two main target organs of damage. Implementation of molecular genetics diagnostic tools and GALT enzyme assays are instrumental in distinguishing classic galactosemia from clinical and biochemical variant forms of GALT deficiency. Better understanding of mechanisms responsible for the phenotypic variation even within the same genotype is essential to provide appropriate counseling for families. Utilization of a lactose restricted diet is also recommended for GALK deficiency and some rare forms of GALE deficiency. Novel modes of therapies are being explored; they may be beneficial if access issues to the affected tissues are circumvented and optimum use of therapeutic window is achieved., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Classic galactosemia results from deficient activity of galactose-1-phosphate uridylyltransferase (GALT), a key enzyme of galactose metabolism. Despite early diagnosis and early postnatal therapeutic intervention, patients still develop neurologic and fertility impairments. Prenatal developmental toxicity has been hypothesized as a determinant factor of disease. In order to shed light on the importance of prenatal GALT activity, several studies have examined GALT activity throughout development. GALT was shown to increase with gestational age in 7-28 weeks human fetuses; later stages were not investigated. Prenatal studies in animals focused exclusively on brain and hepatic GALT activity. In this study, we aim to examine GALT specific activity in late prenatal and adult stages, using a sheep model. Galactosemia acute target-organs-liver, small intestine and kidney-had the highest late prenatal activity, whereas the chronic target-organs-brain and ovary-did not exhibit a noticeable pre- or postnatal different activity compared with nontarget organs. This is the first study on GALT specific activity in the late prenatal stage for a wide variety of organs. Our findings suggest that GALT activity cannot be the sole pathogenic factor accounting for galactosemia long-term complications, and that some organs/cells might have a greater susceptibility to galactose toxicity. Anat Rec, 300:1570-1575, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Introduction: Newborn screening (NBS) for classical galactosemia (CG) was introduced in the Netherlands in 2007. Multiple screening methods have been used since, and currently a two-tier system is used, with residual enzyme activity of galactose-1-phosphate-uridyltransferase (GALT) and total galactose concentration in dried blood spots as the primary and secondary markers. As it is essential to monitor effectiveness of NBS programs, we assessed the effectiveness of different screening methods used over time (primary aim), and aimed to identify and investigate patients identified through NBS with previously unreported clinical and biochemical phenotypes (secondary aim)., Methods: The effectiveness of different screening methods and their cut-off values (COVs), as used from 2007 through 2015, was determined, and the clinical and biochemical data of all identified patients were retrospectively collected., Results: All screening methods and COVs resulted in relatively high false-positive rates and low positive predictive values. Total galactose levels in dried blood spots were far above the COV for NBS in all true positive cases. A total of 31 galactosemia patients were identified, and when corrected for a family with three affected siblings, 14% had a previously unreported phenotype and genotype. These individuals did not demonstrate any symptoms at the time of diagnosis while still being exposed to galactose, had galactose-1-phosphate values below detection limit within months after the start of diet, and had previously unreported genotypes., Conclusion: Optimization of NBS for CG in the Netherlands is warranted because of the high false-positive rate, which may result in significant harm. Furthermore, a surprising 14% of newborns identified with CG by screening had previously unreported clinical and biochemical phenotypes and genotypes. For them, individualized prognostication and treatment are warranted, in order to avoid unnecessary stringent galactose restriction., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most lethal types of cancer worldwide, with poor prognosis and limited treatments. In order to identify novel therapeutic targets that will lead to development of effective therapies with manageable side effects, we tested the hypothesis that knocking-down galactokinase (GALK1) or galactose-1 phosphate uridylyltransferase (GALT) gene expression would control the growth of cultured hepatoma cells. Our results showed small interfering RNA (siRNA) against GALK1 or GALT inhibited the growth of HepG2 cells in culture. Western blot analysis revealed simultaneous down-regulation of multiple players of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) growth signaling pathway, as well as heat-shock protein 90 (HSP90) and poly ADP ribose polymerase (PARP). Reverse transcription-polymerase chain reaction (RT-PCR) data, however, showed no significant mRNA reduction of the encoded genes. Our study thus not only supports GALK1 and GALT as being possible novel targets for treating HCC, but also uncovers new post-transcriptional regulatory mechanisms that link the galactose metabolic pathway to protein expression of the PI3K/AKT pathway in hepatoma., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Galactosemia, an inborn error of galactose metabolism, was first described in the 1900s by von Ruess. The subsequent 100years has seen considerable progress in understanding the underlying genetics and biochemistry of this condition. Initial studies concentrated on increasing the understanding of the clinical manifestations of the disease. However, Leloir's discovery of the pathway of galactose catabolism in the 1940s and 1950s enabled other scientists, notably Kalckar, to link the disease to a specific enzymatic step in the pathway. Kalckar's work established that defects in galactose 1-phosphate uridylyltransferase (GALT) were responsible for the majority of cases of galactosemia. However, over the next three decades it became clear that there were two other forms of galactosemia: type II resulting from deficiencies in galactokinase (GALK1) and type III where the affected enzyme is UDP-galactose 4'-epimerase (GALE). From the 1970s, molecular biology approaches were applied to galactosemia. The chromosomal locations and DNA sequences of the three genes were determined. These studies enabled modern biochemical studies. Structures of the proteins have been determined and biochemical studies have shown that enzymatic impairment often results from misfolding and consequent protein instability. Cellular and model organism studies have demonstrated that reduced GALT or GALE activity results in increased oxidative stress. Thus, after a century of progress, it is possible to conceive of improved therapies including drugs to manipulate the pathway to reduce potentially toxic intermediates, antioxidants to reduce the oxidative stress of cells or use of "pharmacological chaperones" to stabilise the affected proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (-74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT(2) Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The personal glucose meter (PGM) was recently shown to be a general meter to detect many targets. Most studies, however, focus on transforming either target binding or enzymatic activity that cleaves an artificial substrate into the production of glucose. More importantly, almost all reports exhibit their methods by using artificial samples, such as buffers or serum samples spiked with the targets. To expand the technology to even broader targets and to validate its potential in authentic, more complex clinical samples, we herein report expansion of the PGM method by using alkaline phosphatase (ALP) that links the enzymatic activity of galactose-1-phosphate uridyltransferase to the production of glucose, which allows point-of-care galactosemia diagnosis in authentic human clinical samples. Given the presence of ALP in numerous enzymatic assays for clinical diagnostics, the methods demonstrated herein advance the field closer to point-of-care detection of a wide range of targets in real clinical samples., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection., (Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Galactosemia is an inborn error of galactose metabolism that occurs mainly as the outcome of galactose-1-phosphate uridyltransferase (GALT) deficiency. The ability to assess galactose oxidation following administration of a galactose-labeled isotope (1-(13)C-galactose) allows the determination of galactose metabolism in a practical manner. We aimed to assess the level of galactose oxidation in both healthy and galactosemic Brazilian children. Twenty-one healthy children and seven children with galactosemia ranging from 1 to 7 years of age were studied. A breath test was used to quantitate (13)CO2 enrichment in exhaled air before and at 30, 60, and 120 min after the oral administration of 7 mg/kg of an aqueous solution of 1-(13)C-galactose to all children. The molar ratios of (13)CO2 and (12)CO2 were quantified by the mass/charge ratio (m/z) of stable isotopes in each air sample by gas-isotope-ratio mass spectrometry. In sick children, the cumulative percentage of (13)C from labeled galactose (CUMPCD) in the exhaled air ranged from 0.03% at 30 min to 1.67% at 120 min. In contrast, healthy subjects showed a much broader range in CUMPCD, with values from 0.4% at 30 min to 5.58% at 120 min. The study found a significant difference in galactose oxidation between children with and without galactosemia, demonstrating that the breath test is useful in discriminating children with GALT deficiencies.

We explored the regulatory mechanism of Leloir pathway genes in Streptococcus pneumoniae D39. Here, we demonstrate that the expression of galKT is galactose dependent. By microarray analysis and quantitative RT-PCR, we further show the role of the transcriptional regulator GalR, present upstream of galKT, as a transcriptional activator of galKT in the presence of galactose. Moreover, we predict a 19-bp regulatory site (5'-GATAGTTTAGTAAAATTTT-3') for the transcriptional regulator GalR in the promoter region of galK, which is also highly conserved in other streptococci. Growth comparison of D39 ΔgalK with the D39 wild type grown in the presence of galactose shows that galK is required for the proper growth of S. pneumoniae on galactose., (© 2015 S. Karger AG, Basel.)

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.

Knowledge of the impact of variations on protein structure can enhance the comprehension of the mechanisms of genetic diseases related to that protein. Here, we present a new version of GALT Protein Database, a Web-accessible data repository for the storage and interrogation of structural effects of variations of the enzyme galactose-1-phosphate uridylyltransferase (GALT), the impairment of which leads to classic Galactosemia, a rare genetic disease. This new version of this database now contains the models of 201 missense variants of GALT enzyme, including heterozygous variants, and it allows users not only to retrieve information about the missense variations affecting this protein, but also to investigate their impact on substrate binding, intersubunit interactions, stability, and other structural features. In addition, it allows the interactive visualization of the models of variants collected into the database. We have developed additional tools to improve the use of the database by nonspecialized users. This Web-accessible database (http://bioinformatica.isa.cnr.it/GALT/GALT2.0) represents a model of tools potentially suitable for application to other proteins that are involved in human pathologies and that are subjected to genetic variations., (© 2014 WILEY PERIODICALS, INC.)

Classic galactosemia is an autosomal recessive disorder caused by deficient galactose-1-phosphate uridylyltransferase (GALT) activity. Patients develop symptoms in the neonatal period, which can be ameliorated by dietary restriction of galactose. Many patients develop long-term complications, with a broad range of clinical symptoms whose pathophysiology is poorly understood. The high allelic heterogeneity of GALT gene that characterizes this disorder is thought to play a determinant role in biochemical and clinical phenotypes. We aimed to characterize the mutational spectrum of GALT deficiency in Portugal and to assess potential genotype-phenotype correlations. Direct sequencing of the GALT gene and in silico analyses were employed to evaluate the impact of uncharacterized mutations upon GALT functionality. Molecular characterization of 42 galactosemic Portuguese patients revealed a mutational spectrum comprising 14 nucleotide substitutions: ten missense, two nonsense and two putative splicing mutations. Sixteen different genotypic combinations were detected, half of the patients being p.Q188R homozygotes. Notably, the second most frequent variation is a splicing mutation. In silico predictions complemented by a close-up on the mutations in the protein structure suggest that uncharacterized missense mutations have cumulative point effects on protein stability, oligomeric state, or substrate binding. One splicing mutation is predicted to cause an alternative splicing event. This study reinforces the difficulty in establishing a genotype-phenotype correlation in classic galactosemia, a monogenic disease whose complex pathogenesis and clinical features emphasize the need to expand the knowledge on this "cloudy" disorder.

The 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 in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. 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, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant 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 when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.

Classic galactosemia is a potentially lethal disorder that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. Although early diagnosis and rigorous dietary restriction of galactose prevent or resolve the potentially lethal acute symptoms, patients are at markedly increased risk of long-term complications including significant cognitive, speech, and behavioral difficulties, among other problems. The mechanisms that underlie these long-term complications remain unclear, as do the factors that modify their severity. Here we explored the scholastic and behavioral outcomes experienced by a cohort of 54 school age children with classic galactosemia. Data collected included survey responses from parents and teachers, school records including standardized test scores, and GALT genotype data used to estimate predicted residual GALT activity based on a yeast expression system. As expected, many but not all of the children in our study demonstrated speech, scholastic, and behavioral difficulties. Perhaps most striking, we found that predicted cryptic residual GALT activity, often below the threshold of detection of clinical assays, appeared to modify scholastic outcome. These data raise the intriguing possibility that cryptic GALT activity might also influence the severity of other long-term complications in classic galactosemia.

In connection with prospective (18)F-PET imaging studies, the potential for enzymatic synthesis of fluorine-labelled glycosides of small molecules was investigated. Approaches to the enzymatic synthesis of anomeric phosphates of d-gluco-configured fluorosugars proved ineffective. In contrast, starting in the d-galacto series and relying on the consecutive action of Escherichia coli galactokinase (GalK), galactose-1-phosphate uridylyltransferase (GalPUT), uridine-5'-diphosphogalactose 4-epimerase (GalE) and oat root glucosyltransferase (SAD10), a quick and effective synthesis of 6-deoxy-6-fluoro-d-glucosyl N-methylanthranilate ester was achieved., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Context: Classic galactosemia is a potentially lethal genetic disorder resulting from profound impairment of galactose-1P uridylyltransferase (GALT). More than 80% of girls and women with classic galactosemia experience primary or premature ovarian insufficiency despite neonatal diagnosis and rigorous lifelong dietary galactose restriction., Objective: The goal of this study was to test the relationship between markers of ovarian reserve, cryptic residual GALT activity, and spontaneous pubertal development in girls with classic galactosemia., Design and Setting: This was a cross-sectional study with some longitudinal follow-up in a university research environment., Patients: Patients included girls and women with classic galactosemia and unaffected controls, <1 month to 30 years old., Main Outcome Measures: We evaluated plasma anti-Müllerian hormone (AMH) and FSH levels, antral follicle counts ascertained by ultrasound, and ovarian function as indicated by spontaneous vs assisted menarche., Results: More than 73% of the pre- and postpubertal girls and women with classic galactosemia in this study, ages >3 months to 30 years, demonstrated AMH levels below the 95% confidence interval for AMH among controls of the same age, and both pre- and postpubertal girls and women with classic galactosemia also demonstrated abnormally low antral follicle counts relative to age-matched controls. Predicted residual GALT activity ≥ 0.4% significantly increased the likelihood that a girl with classic galactosemia would demonstrate an AMH level ≥ 0.1 ng/mL., Conclusions: A majority of girls with classic galactosemia demonstrate evidence of diminished ovarian reserve by 3 months of age, and predicted cryptic residual GALT activity is a modifier of ovarian function in galactosemic girls and women.

Classical Galactosemia is an autosomal recessive disorder of galactose metabolism caused by severe reduction or absence of the galactose-1-phosphate uridyl transferase (GALT) enzyme. Till date, no reports are available on clinical and molecular spectrum of galactosemia from Indian population. The characterization of underlying GALT gene lesions was performed in 55 unrelated galactosemia patients. The GALT mutational spectrum comprised 16 distinct mutations including 10 previously unreported mutations. N314D was the most common mutation with a frequency of 40% followed by Q188R at 2.7%. The novel GALT gene mutations included 6 missense mutations viz. Y89H, Q103R, P166A, S181F, K285R, R333L; one nonsense mutation, S307X and 3 silent mutations--Q103Q, K210K and H319H. The functional significance of the novel GALT missense mutations was investigated using SNPs&GO and SIFT tools. Further, modeling studies using 3D models of mutant and wild type GALT proteins revealed mutations to exert their effects at the molecular level by altering H-bonds, salt bridges, secondary structure or surface features. The study highlighted the heterogeneity of classical galactosemia in the Indian population and also emphasizes the importance of GALT gene analysis in diagnosis of galactosemia. It also revealed that the Indian GALT mutational profile differs significantly from other populations studied., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Classic galactosemia is a potentially lethal metabolic disorder that results from profound impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT); despite decades of research, the underlying mechanism of pathophysiology remains unclear. Previous studies of plasma and tissue samples from patients with classic galactosemia have revealed defects of protein and lipid glycosylation, however, the underlying bases for these defects and their clinical significance, if any, has remained unclear. As a step toward addressing these questions we characterized both the N- and O-linked glycomes of plasma proteins from neonates, infants, children, and adults with galactosemia using mass spectrometry and asked (1) whether similar or disparate defects exist for N-linked and O-linked modifications, (2) what factors correlate with the severity of these defects in different patients, and perhaps most important, (3) whether there is any apparent relationship between chronic glycosylation defects and long-term outcome in patients. We found that some but not all of the galactosemic neonates tested exhibited abnormal N- and O-linked glycosylation of plasma proteins. The types of abnormalities seen were similar between N- and O-linked moieties, but the extent of the defects varied between patients. Age, gender, GALT genotype, and predicted residual GALT activity all failed to explain the extent of the glycosylation defect in the samples studied. Dietary galactose restriction markedly normalized both the N- and O-linked glycosylation patterns for all infants tested; however, any remaining glycosylation defects evident in the plasma of older children or adults on galactose-restricted diets showed no correlation with clinical outcome. These data cannot rule out the possibility that subtle or localized glycosylation defects, not detectable by our methods or not reflected in plasma, may contribute to acute or long-term outcome severity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Wiley Periodicals, Inc.)

Classic galactosemia is a potentially lethal disorder that results from profound impairment of galactose-1-phosphate uridylyltransferase (GALT). Despite decades of research, the underlying pathophysiology of classic galactosemia remains unclear, in part owing to the lack of an appropriate animal model. Here, we report the establishment of a Drosophila melanogaster model of classic galactosemia; this is the first whole-animal genetic model to mimic aspects of the patient phenotype. Analogous to humans, GALT-deficient D. melanogaster survive under conditions of galactose restriction, but accumulate elevated levels of galactose-1-phosphate and succumb during larval development following galactose exposure. As in patients, the potentially lethal damage is reversible if dietary galactose restriction is initiated early in life. GALT-deficient Drosophila also exhibit locomotor complications despite dietary galactose restriction, and both the acute and long-term complications can be rescued by transgenic expression of human GALT. Using this new Drosophila model, we have begun to dissect the timing, extent and mechanism(s) of galactose sensitivity in the absence of GALT activity.

Background: Galactose-1-phosphate:uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal). Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes. The most commonly used assays require radio labelled substrates or indirect coupled assays., Methods: GALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc. UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection. Clinical validity was assessed using blood samples from galactosaemic patients., Results: UDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 42.5 nmol haemoglobin. Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was <1.4% and <2.4%, respectively. Mean GALT activity in 33 individuals was 601+/-79 nmol UDP-Gal/(micromol Hb.h) (range 492-697). Patients with classical galactosaemia were easily detected by their extremely low activity., Conclusions: We have developed a reliable and convenient direct method to measure GALT activity in human erythrocytes using HPLC with UV detection., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)., Methods: [(13)C6]-galactose, [(13)C2]-galactose-1-phosphate, and UDP-glucose were used as substrates for 3 galactose-metabolizing enzymes. The end products from the combined reaction mixtures, [(13)C6]-galactose-1-phosphate, UDP-[(13)C2]-galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Linearity, imprecision, ion suppression, and the effects of substrate were evaluated to determine assay performance. Enzyme activities from 35 healthy individuals, 8 patients with enzyme deficiency, and 18 mutant cells were analyzed., Results: Substrates, products, and internal standards from the mixture of 3 enzyme reactions were clearly separated by using UPLC-MS/MS, with an injection cycle time of 10 min. Ion suppression was 0.1%-2.5%, the interassay imprecision of UPLC-MS/MS was 3.3%-10.6% CV, and the linearity of each system was good (R(2) = 0.994-0.999). Patient samples and mutated cells showed consistently low enzyme activities compared with those of normal individuals and wild-type cells., Conclusions: This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.