Galactose-1-phosphate Uridylyltransferase Enzyme Research Articles

Novel mRNA therapy restores GALT protein and enzyme activity in a zebrafish model of classic galactosemia.

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.

Simulation of the Interactions of Arginine with Wild-Type GALT Enzyme and the Classic Galactosemia-Related Mutant p.Q188R by a Computational Approach.

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.

Analysis of the Structure-Function-Dynamics Relationships of GALT Enzyme and of Its Pathogenic Mutant p.Q188R: A Molecular Dynamics Simulation Study in Different Experimental Conditions.

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.

Класична галактоземія: особливості діагностики та лікування

Актуальність. Галактоземія І типу, або класична галактоземія (КГ), — найбільш тяжка форма спадкових порушень обміну вуглеводів, зокрема галактози. Галактоземія не включена до програми масового неонатального скринінгу в Україні. КГ характеризується неспецифічністю дебютних симптомів, супроводжується високими показниками смертності у неонатальному періоді. Актуальними є питання ранньої діагностики та лікування КГ за відсутності програми масового неонатального скринінгу в Україні. Мета. Аналіз клініко-лабораторних, молекулярно-генетичних особливостей КГ; визначення комплексу клінічних симптомів щодо формування селективних груп пацієнтів для призначення дієтотерапії та проведення лабораторної діагностики; розробка поетапної схеми ефективної ранньої діагностики та лікування пацієнтів при КГ за відсутності масового неонатального скринінгу з метою запобігання інвалідизації хворих, зниження показника дитячої смертності. Матеріали та методи. Впродовж 2014–2017 рр. у Центрі орфанних захворювань обстежено 317 пацієнтів із діагнозом «гепатит невизначеного генезу». Вік хворих — від 3 днів до 16 років. Хлопчиків було 145 (45 %), дівчаток — 172 (55 %). У всіх пацієнтів відзначена полісиндромна клінічна картина з переважним ураженням печінки. В усіх пацієнтів визначали активність ферменту GАLT в еритроцитах периферичної крові, проводили ДНК-дослідження з метою виявлення мажорних мутацій у гені GАLT. Всі хворі були обстежені на наявність вірусних гепатитів. За результатами біохімічного дослідження визначали рівень печінкових трансаміназ, лужної фосфатази, білірубіну, показники згортання крові. Також проводили тандемну мас-спектрометрію з метою виявлення порушень обміну амінокислот, органічних кислот і мітохондріального бета-окислення жирних кислот. У 5 пацієнтів за наявності припущення про глікогеноз проведене кількісне визначення глікогену у біоптаті печінки. Результати. Діагноз КГ підтверджений у 15 (4,7 %) з 317 пацієнтів: у 9 хлопчиків і 6 дівчаток віком від 14 діб до 15 років. У 13 (86,7 %) дітей клінічний діагноз КГ встановлений у середньому через 47 ± 15 діб життя. У 2 пацієнтів КГ діагностована у віці 2 і 15 років. У всіх пацієнтів активність ферменту GALT в еритроцитах була знижена і становила 0,08–5,8 Од/г Hb, у середньому 3,3 ± 1,6 Од/г Hb (в нормі > 10 Од/г Hb). Молекулярно-генетичний аналіз проведений у 14 (93,3 %) пацієнтів. У 5 хворих виявлений гомозиготний генотип за Q188R, в одного — за K285N, у 6 — гетерозиготний генотип за двома мажорними мутаціями (Q188R/K285N), в одного пацієнта виявлений генотип був р.Y209S/р.K285N, ще в одного — виявлена мутація р.K285N тільки в одній алелі. Отже, у 14 пацієнтів досліджені 28 алелей у гені GALT, ідентифіковані 27 мутацій: Q188R — у 57 % спостережень, K285N — у 36 %, р.Y209S — у 3 % (виявлена в одного пацієнта в одній алелі). В цілому мутації Q188R і K285N виявляли з частотою 93 %, що свідчить про високий рівень їх мажорності в українській популяції. Жовтяниця виявлена у 93 % хворих, гепатомегалія — у 80 %, диспептичний синдром — у 87 %. Катаракта була діагностована у 6 (40 %) пацієнтів, в усіх спостереженнях поєднана з неврологічними симптомами. Ураження нервової системи (спастичний тетрапарез, дистонія, тремор, когнітивна недостатність, дизартрія, затримка мовленнєвого розвитку) відзначені у 7 (21,8 %) хворих при КГ. Померли 4 (26,7 %) з 15 пацієнтів віком від 20 днів до 2,5 місяця. За даними клініко-генеалогічного аналізу у родинах хворих на КГ виявлено часті випадки ранньої смерті дітей (віком до 14 тижнів) унаслідок печінкової недостатності. У неонатальному періоді померли 4 дітей із трьох родин з обтяженим анамнезом. У цілому в родинах наших пацієнтів померли 8 (42,1 %) із 19 дітей з аналогічними клінічними симптомами. Лікування всім пацієнтам призначали з суттєвим запізненням, у середньому на 44-й день. Запропоновано схему діагностично-лікувальних заходів у пацієнтів селективної групи. Висновки. КГ — загрозливе для життя захворювання, що характеризується ураженням клітин печінки, диспептичним синдромом, сепсисом і спричиняє високий рівень смертності немовлят у неонатальному періоді. В цілому мутації Q188R і K285N виявлені у 93 % спостережень, що свідчить про високий рівень їх мажорності в українській популяції. Всі немовлята з жовтяницею, зумовленою гіпербілірубінемією, за наявності гепатомегалії, диспепсії, порушень згортання крові, високого рівня трансаміназ мають бути обстежені щодо КГ і негайно переведені на безлактозну дієту до отримання остаточних результатів лабораторної діагностики.

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

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

Functional analysis of GALT variants found in classic galactosemia patients using a novel cell-free translation method.

ABSTRACTClassic galactosemia is an autosomal recessive disorder caused by deleterious variants in the galactose‐1‐phosphate uridylyltransferase (GALT) gene. GALT enzyme deficiency leads to an increase in the levels of galactose and its metabolites in the blood causing neurodevelopmental and other clinical complications in affected individuals. Two GALT variants NM_000155.3:c.347T>C (p.Leu116Pro) and NM_000155.3:c.533T>G (p.Met178Arg) were previously detected in Filipino patients. Here, we determine their functional effects on the GALT enzyme through in silico analysis and a novel experimental approach using a HeLa‐based cell‐free protein expression system. Enzyme activity was not detected for the p.Leu116Pro protein variant, while only 4.5% of wild‐type activity was detected for the p.Met178Arg protein variant. Computational analysis of the variants revealed destabilizing structural effects and suggested protein misfolding as the potential mechanism of enzymological impairment. Biochemical and computational data support the classification of p.Leu116Pro and p.Met178Arg variants as pathogenic. Moreover, the protein expression method developed has utility for future studies of GALT variants.

The natural history of classic galactosemia: lessons from the GalNet registry

BackgroundClassic galactosemia is a rare inborn error of carbohydrate metabolism, caused by a severe deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). A galactose-restricted diet has proven to be very effective to treat the neonatal life-threatening manifestations and has been the cornerstone of treatment for this severe disease. However, burdensome complications occur despite a lifelong diet. For rare diseases, a patient disease specific registry is fundamental to monitor the lifespan pathology and to evaluate the safety and efficacy of potential therapies. In 2014, the international Galactosemias Network (GalNet) developed a web-based patient registry for this disease, the GalNet Registry. The aim was to delineate the natural history of classic galactosemia based on a large dataset of patients.MethodsObservational data derived from 15 countries and 32 centers including 509 patients were acquired between December 2014 and July 2018.ResultsMost affected patients experienced neonatal manifestations (79.8%) and despite following a diet developed brain impairments (85.0%), primary ovarian insufficiency (79.7%) and a diminished bone mineral density (26.5%). Newborn screening, age at onset of dietary treatment, strictness of the galactose-restricted diet, p.Gln188Arg mutation and GALT enzyme activity influenced the clinical picture. Detection by newborn screening and commencement of diet in the first week of life were associated with a more favorable outcome. A homozygous p.Gln188Arg mutation, GALT enzyme activity of ≤ 1% and strict galactose restriction were associated with a less favorable outcome.ConclusionThis study describes the natural history of classic galactosemia based on the hitherto largest data set.

The ability of an LC-MS/MS-based erythrocyte GALT enzyme assay to predict the phenotype in subjects with GALT deficiency

BackgroundGALT deficiency is a rare genetic disorder of carbohydrate metabolism. Due to the decreased activity or absence of the enzyme galactose-1-phosphate uridylyltransferase (GALT), cells from affected individuals are unable to metabolize galactose normally. Lactose consumption in the newborn period could potentially lead to a lethal disease process with multi-organ involvement. In contrast to the newborn-stage disease, however, a galactose-restricted diet does not prevent long-term complications such as central nervous system (CNS) dysfunction with speech defects, learning disability and neurological disease in addition to hypergonadotropic hypogonadism or primary ovarian insufficiency (POI) in females. As the literature suggests an association between GALT enzyme activity and the long-term complications, it is of importance to have a highly sensitive assay to quantify the GALT enzyme activity. To that end, we had developed a sensitive and accurate LC-MS/MS method to measure GALT enzyme activity. Its ability to predict outcome is the subject of this report. Materials and methodsThe GALT enzyme activity in erythrocytes from 160 individuals, in which 135 with classic, clinical variant or biochemical variant galactosemia, was quantified by LC-MS/MS. Individuals with GALT deficiency were evaluated for the long-term complications of speech defects, dysarthria, ataxia, dystonia, tremor, POI, as well as intellectual functioning (full scale IQ). The LC-MS/MS results were compared to a variety of assays: radioactive, [14C]-galactose-1-phosphate, paper chromatography with scintillation counting, enzyme-coupled assays with spectrophotometric or fluorometric readout or high-pressure liquid chromatography with UV detection of UDP-galactose. ResultsThe LC-MS/MS method measured GALT activity as low as 0.2%, whereas other methods showed no detectable activity. Largely due to GALT activities that were over 1%, the LC-MS/MS measurements were not significantly different than values obtained in other laboratories using other methodologies. Severe long-term complications were less frequently noted in subjects with >1% activity. Patients with a p.Q188R/p.Q188R genotype have no residual enzyme activity in erythrocytes. ConclusionOur LC-MS/MS assay may be necessary to accurately quantify residual GALT activities below 5%. The data suggest that patients with >1% residual activity are less likely to develop diet-independent long-term complications. However, much larger sample sizes are needed to properly assess the clinical phenotype in patients with residual enzyme activities between 0.1 and 5%.

False-Positive Newborn Screen Using the Beutler Spot Assay for Galactosemia in Glucose-6-Phosphate Dehydrogenase Deficiency.

Classical galactosemia is detected through newborn screening by measuring galactose-1-phosphate uridylyltransferase (GALT) in the USA primarily via the Beutler spot assay. We report on an 18-month-old patient with glucose-6-phosphate dehydrogenase (G6PD) deficiency that was originally diagnosed with classical galactosemia. The patient presented with elevated liver function enzymes and bilirubinemia and was immediately treated with soy-based formula. Confirmatory tests revealed deficiency of the GALT enzyme, however, full-sequencing of GALT was normal, suggestive of a different ideology. The Beutler spot assay uses three other enzymatic steps in addition to GALT. A deficiency in either of these enzymes can result in suspected decreased GALT activity when using the Beutler assay. Congenital Disorders of Glycosylation screening for phosphoglucomutase-1 deficiency was negative. Quantitative analysis of G6PD enzyme in red blood cells showed a severe deficiency and a deletion in G6PD. Soy-formula, the standard treatment for galactosemia, has been reported to trigger hemolysis in G6PD deficient patients. G6PD and phosphoglucomutase-1 deficiencies should be considered when confirmatory tests are negative for pathogenic variants in GALT and galactose-1-phosphate level is normal.

GALT protein database: querying structural and functional features of GALT enzyme.

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.

Correlation assessment among clinical phenotypes, expression analysis and molecular modeling of 14 novel variations in the human galactose-1-phosphate uridylyltransferase gene

Galactose-1-phosphate uridylyltransferase (GALT) catalyzes the conversion of galactose-1-phosphate to UDP-galactose, a key step in the galactose metabolism. Deficiency of GALT activity in humans caused by deleterious variations in the GALT gene can cause a potentially lethal disease called classic galactosemia. In this study, we selected 14 novel nucleotide sequence changes in the GALT genes found in galactosemic patients for expression analysis and molecular modeling. Several variants showed decreased levels of expression and decreased abundance in the soluble fraction of the Escherichia coli cell extracts, suggesting altered stability and solubility. Only six variant GALT enzymes had detectable enzymatic activities. Kinetic studies showed that their V(max) decreased significantly. To further characterize the variants at molecular level, we performed static and dynamic molecular modeling studies. Effects of variations on local and/or global structural features of the enzyme were anticipated for the majority of variants. In-depth studies with molecular dynamic simulations on selected variants predicted the alteration of the protein structure even though static models apparently did not highlight any perturbation. Overall, these studies offered new insights on the molecular properties of GALT enzyme, with the aim of correlating them with the clinical outcome. Hum Mutat 33:1107-1115, 2012. © 2012 Wiley Periodicals, Inc.

Application of computational methods for structural and functional characterization of mutants of GALT enzyme

Motivations. Galactose-1-phosphate uridylyltransferase (GALT) catalyses the conversion of galactose-1-phosphate to UDP-galactose, a key step in the galactose metabolism. Deficiency of GALT activity in humans caused by mutations in the GALT gene are associated to a rare genetic disease called Classic Galactosemia. To date, more than 180 mutations are known on GALT gene, most of which are missense mutations. A previous study applied computational methods to predict the effect of known mutations [1] and results were stored in a publicly available database: http://bioinformatica.isa.cnr.it/GALT [2]. In this work, we have characterized the effects on structure and function of GALT enzyme of 14 novel missense mutations, applying not only static modelling, but also molecular dynamics simulations. Results were compared to the biochemical characterization of these mutants, expressed and purified from bacteria. Methods. Starting from the 3D model of human GALT enzyme [3] (the crystallographic structure of the human enzyme is not yet available), homodimeric mutans were created using Modeller 9v8 [4]. Each resulting mutant was analysed for variations in structural feature such as intersubunit interactions, secondary structures, solvent accessibility, H-bond and salt bridge patterns, and for predicted stability of the protein. Molecular dynamics simulations were applied to those selected mutants, for which the static modelling of mutations did not allow to highlight any variation in these properties, using GROMACS program [5]. Results were compared with those obtained on wild type molecule and on the most characterized mutant, Q188R. The human homodimeric mutant proteins were expressed in E. coli, purified and assayed for their activity and kinetic properties (Vmax and KM). Results. Our static modelling simulations predicted for most of the mutant GALT enzyme alterations at structural level: in two cases the alteration of intersubunit interactions, in other two cases the involvement of residues indirectly affecting the active site, and in most cases the alteration of the network of H-bonds and salt bridges, with either local or global effects. In addition, in most cases these mutants have predicted stability problems In two cases, however, static modelling was not able to predict any structural effect. In these cases, the results of molecular dynamics simulations were able to suggest possible effects that impair the correct activity and the stability of the enzyme, too. The comparison with simulations made on mutant Q188R allowed also to confirm the deleterious effects of this mutation at structural level. The biochemical characterization of these mutants showed results that are well explained by the simulations. In particular, those mutations that were predicted to have a large impact on protein structure are also those with minimal or no activity, whereas those mutants showing a residual activity, also show localized effects on protein structure. Acknowledgments This work was supported by a Parents of Galactosemic Children Research Grant (to A.M.), and by project FIRB MIUR ITALBIONET (RBPR05ZK2Z and RBIN064YAT_003) (to A.M., L.M). This work has been made in the frame of the Flagship Project InterOmics and of the project CNR-Bioinformatics. Research grant support to K.L. includes U.S. NIH grants 5R01 HD054744-04 and 3R01 HD054744-04S1.

Covalent Heterogeneity of the Human Enzyme Galactose-1-phosphate Uridylyltransferase

Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.

Biochemical characterization of the S135L allele of galactose‐1‐phosphate uridylyltransferase associated with galactosaemia

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosaemia. The S135L mutation, which accounts for almost 50% of the GALT alleles in galactosaemia patients of African-American descent, has been associated with activities ranging from null to wild-type by different investigators examining cell lysates representing different tissues or model systems. Because of the crude nature of the lysates examined, however, and the absence of quantitative measures concerning GALT abundance in most of those lysates, the available data do not distinguish between differences in GALT enzyme expression/abundance, specific activity, or kinetic constants in these different tissues or systems. In an effort to overcome this uncertainty and investigate the biochemical impact of the S135L substitution on human GALT function under defined conditions, we have overexpressed both wild-type and S135L-mutant GALT sequences in a null-background yeast expression system, and purified both proteins to near homogeneity. Abundance of the wild-type and mutant proteins in crude yeast lysates differed by approximately 2-fold. Kinetic studies of the purified proteins, however, demonstrated that although K(m) values differed by < 2-fold, specific activities differed by 10-fold. Temperature-activity profiles revealed no significant differences, and coprecipitation studies demonstrated that S135L-hGALT subunits remained competent to self-associate in vivo. We conclude that the S135L substitution causes either steric or electrochemical changes sufficiently close to the active site in human GALT to result in partial impairment of the transferase reaction.