GALE Expression Research Articles

GALE Promotes the Proliferation and Migration of Glioblastoma Cells and Is Regulated by miR-let-7i-5p.

PurposeGlioma is the most common and lethal type of brain tumor. While GALE (UDP-galactose-4-epimerase) has been shown to be overexpressed in some kinds of cancers, its expression in gliomas has not been reported. MicroRNAs (miRNAs) function as tumor suppressors in many cancers, and online datasets can be used to predict whether GALE is regulated by miR-let-7i-5p. In this investigation, we explored the effect and regulatory mechanisms of GALE on glioblastoma growth via miR-let-7i-5p.MethodsWe used a Cox proportional hazards model and publicly available datasets to examine the relationship between GALE and the survival rates of glioma patients. Bioinformatics predicted the targeting of GALE by miR-let-7i-5p. The proliferation, migration, cell cycle and apoptosis of human glioblastoma cells were assessed by relevant assays. We also demonstrated the effect of GALE on glioblastoma multiforme [GBM] tumor growth using an in vivo orthotopic xenograft model.ResultsGALE was upregulated in human gliomas, especially in high-grade gliomas (e.g., GBM). An obvious decline in GALE expression was observed in human glioblastoma cell lines (U87 and U251) following treatment with a small interfering RNA (siRNA) targeting GALE or miR-let-7i-5p mimics. Knockdown of GALE or overexpression of miR-let-7i-5p (with miR-let-7i-5p mimics) inhibited U87 and U251 cell growth. miR-let-7i-5p significantly restrained the migration ability of human glioblastoma cells in vascular mimic (VM), wound healing and transwell assays, and GALE promoted glioblastoma growth in vivo.ConclusionOur findings confirm that GALE plays an important role in promoting the development of human glioma and that GALE can be regulated by miR-let-7i-5p to inhibit human glioblastoma growth.Implications for cancer survivorsOur data show that cancer survivors have low GALE expression, which indicates that GALE may be a diagnostic biomarker and a promising therapeutic target in glioblastoma.

Overexpression of UDP-Glucose 4-Epimerase Is Associated with Differentiation Grade of Gastric Cancer.

The UDP-glucose 4-epimerase (GALE) is a glycosyltransferase, which acts on protein and lipid glycosylation in normal and neoplastic cells. This study is aimed at investigating the differential tissue expression of GALE and its possible association with clinical-pathological parameters and the outcome of gastric adenocarcinoma patients. Seventy-one patients were evaluated in relation to GALE expression by immunohistochemistry. Our results showed that 48 (67.6%) patients were GALE positive and 23 (32.4%) negative. Positive staining was present on well-differentiated and moderate-differentiated histological grade of gastric adenocarcinomas (p < 0.0001). There was no significant association with outcome parameters (p > 0.05). Besides that, our results corroborated with the validation cohort analysis, where the expression of GALE mRNA was also associated with the histological grade (p < 0.001). These results suggest a possible use of this enzyme as a biomarker for well and moderately differentiated tumors.

The critical role of UDP-galactose-4-epimerase in osteoarthritis: Modulating proteoglycans synthesis of the articular chondrocytes

UDP-galactose-4-epimerase (GALE) is a key enzyme catalyzing the interconversion of UDP-glucose and UDP-galactose, as well as UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine, which are all precursors for the proteoglycans (PGs) synthesis. However, whether GALE is essential in cartilage homeostasis remains unknown. Therefore, we investigated the role of GALE in PGs synthesis of human articular chondrocytes, the GALE expression in OA, and the regulation of GALE expression by interleukin-1beta (IL-1β). Silencing GALE gene with specific siRNAs resulted in a markedly inhibition of PGs synthesis in human articular chondrocytes. GALE protein levels were also decreased in both human and rat OA cartilage, thus leading to losses of PGs contents. Moreover, GALE mRNA expression was stimulated by IL-1β in early phase, but suppressed in late phase, while the suppression of GALE expression induced by IL-1β was mainly mediated by stress-activated protein kinase/c-Jun N-terminal kinase pathway. These data indicated a critical role of GALE in maintaining cartilage homeostasis, and suggested that GALE inhibition might contribute to OA progress.

TRANSLATIONAL IMPACT

UDP-galactose 4’ epimerase (GALE) plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose when exogenous sources are lacking, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. GALE catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway of galactose metabolism and, in humans, also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. No patients completely lacking GALE activity have been found, suggesting that loss of GALE must be lethal. However, partial loss of GALE can be frequent, at least in some populations, and results in epimerase deficiency galactosemia. This spectrum disorder ranges in severity from benign to potentially lethal, depending, among other factors, on the degree and tissue-specificity of GALE impairment. The prognosis and treatment for patients with epimerase deficiency galactosemia are currently ill-defined, partly because affected individuals are often either missed or misdiagnosed, but also owing to the lack of an appropriate animal model.This paper reports the generation and initial characterization of the first whole-animal model of GALE deficiency, using the fruit fly Drosophila melanogaster. Drosophila lacking GALE died as embryos, confirming that GALE function is essential in developing animals. Larvae in which Drosophila GALE (dGALE) expression was conditionally inhibited by dGALE-specific RNA interference (RNAi) also died within days of GALE loss. Both types of affected embryos were rescued by expression of a human GALE (hGALE) transgene, showing the specificity of the mutation. To examine possible tissue specificity of the lethal GALE phenotype, conditional knockdown and hGALE transgene expression were used to show that GALE expression in the gut primordium and Malpighian tubules was both necessary and sufficient for Drosophila survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with a partial loss of GALE activity survived in the absence of galactose, but succumbed during development if exposed to dietary galactose.These data establish the utility of the fly model of generalized GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

UDP-galactose 4′ epimerase (GALE) is essential for development ofDrosophila melanogaster

UDP-galactose 4' epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway; human GALE (hGALE) also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. GALE therefore plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. Partial impairment of hGALE results in the potentially lethal disorder epimerase-deficiency galactosemia. We report here the generation and initial characterization of a first whole-animal model of GALE deficiency using the fruit fly Drosophila melanogaster. Our results confirm that GALE function is essential in developing animals; Drosophila lacking GALE die as embryos but are rescued by the expression of a human GALE transgene. Larvae in which GALE has been conditionally knocked down die within days of GALE loss. Conditional knockdown and transgene expression studies further demonstrate that GALE expression in the gut primordium and Malpighian tubules is both necessary and sufficient for survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with partial GALE loss survive in the absence of galactose but succumb in development if exposed to dietary galactose. These data establish the utility of the fly model of GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

Relationship between UDP-Galactose 4′-Epimerase Activity and Galactose Sensitivity in Yeast

UDP-galactose 4'-epimerase (GALE) catalyzes the final step of the highly conserved Leloir pathway of galactose metabolism. Loss of GALE in humans results in a variant form of the metabolic disorder, galactosemia. Loss of GALE in yeast results in galactose-dependent growth arrest. Although the role of GALE in galactose metabolism has been recognized for decades, the precise relationship between GALE activity and galactose sensitivity has remained unclear. Here we have explored this relationship by asking the following. 1) Is GALE rate-limiting for galactose metabolism in yeast? 2) What is the relationship between GALE activity and galactose-dependent growth arrest in yeast? 3) What is the relationship between GALE activity and the abnormal accumulation of galactose metabolites in yeast? To answer these questions we engineered a strain of yeast in which GALE was doxycycline-repressible and studied these cells under conditions of intermediate GALE expression. Our results demonstrated a smooth linear relationship between galactose metabolism and GALE activity over a range from 0 to approximately 5% but a steep threshold relationship between growth rate in galactose and GALE activity over the same range. The relationship between abnormal accumulation of metabolites and GALE activity was also linear over the range from 0 to approximately 5%, suggesting that if the abnormal accumulation of metabolites underlies galactose-dependent growth-arrest in GALE-impaired yeast, either the impact of individual metabolites must be synergistic and/or the threshold of sensitivity must be very steep. Together these data reveal important points of similarity and contrast between the roles of GALE and galactose-1-phosphate uridylyltransferase in galactose metabolism in yeast and provide a framework for future studies in mammalian systems.

Mediators of Galactose Sensitivity in UDP-Galactose 4′-Epimerase-impaired Mammalian Cells

UDP-galactose 4'-epimerase (GALE) catalyzes the final step in the Leloir pathway of galactose metabolism, interconverting UDP-galactose and UDP-glucose. Unlike its Escherichia coli counterpart, mammalian GALE also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. Considering the key roles played by all four of these UDP-sugars in glycosylation, human GALE therefore not only contributes to the Leloir pathway, but also functions as a gatekeeper overseeing the ratios of important substrate pools required for the synthesis of glycosylated macromolecules. Defects in human GALE result in the disorder epimerase-deficiency galactosemia. To explore the relationship among GALE activity, substrate specificity, metabolic balance, and galactose sensitivity in mammalian cells, we employed a previously described GALE-null line of Chinese hamster ovary cells, ldlD. Using a transfection protocol, we generated ldlD derivative cell lines that expressed different levels of wild-type human GALE or E. coli GALE and compared the phenotypes and metabolic profiles of these lines cultured in the presence versus absence of galactose. We found that GALE-null cells accumulated abnormally high levels of Gal-1-P and UDP-Gal and abnormally low levels of UDP-Glc and UDP-GlcNAc in the presence of galactose and that human GALE expression corrected each of these defects. Comparing the human GALE- and E. coli GALE-expressing cells, we found that although GALE activity toward both substrates was required to restore metabolic balance, UDP-GalNAc activity was not required for cell proliferation in the presence of otherwise cytostatic concentrations of galactose. Finally, we found that uridine supplementation, which essentially corrected UDP-Glc and, to a lesser extent UDP-GlcNAc depletion, enabled ldlD cells to proliferate in the presence of galactose despite the continued accumulation of Gal-1-P and UDP-Gal. These data offer important insights into the mechanism of galactose sensitivity in epimerase-impaired cells and suggest a potential novel therapy for patients with epimerase-deficiency galactosemia.

Molecular cloning and characterization of a UDP-glucose-4-epimerase gene ( galE) and its expression in pea tissues

We cloned a cDNA encoding for the enzyme UDP-glucose-4-epimerase (EC 5.1.3.2; UDP-galactose-4-epimerase or ‘galactowaldenase’) in pea ( Pisum sativum L. cv. Wando). This enzyme functions in the interconversion of UDP-glucose and UDP-galactose. The open reading frame within this cDNA contains 350 amino acid residues and the polypeptide product has a predicted molecular mass of 38 994 Da. The pea enzyme exhibits extensive homology with the deduced sequences of other prokaryotic and eukaryotic UDP-glucose-4-epimerases. Northern analysis of expression of the UDP-glucose-4-epimerase gene ( galE) in pea tissues revealed 3- and 5-fold higher mRNA levels in the leaves and roots, respectively, of 3-week old seedlings, as compared with 4-day old seedlings. GalE transcript levels did not differ significantly in the roots and leaves of light-grown versus etiolated 4-day old seedlings. Increased galE expression in the roots of older seedlings may be correlated with the copious secretion of mucigel at the root tip, as increased availability of the UDP-galactose precursor may be required to support the synthesis of this pectinaceous extracellular matrix.

Differential UDP-galactose-4′-epimerase (GALE) enzymatic activity and mRNA expression in the rat mammary gland during lactation

We have investigated the UDP-galactose-4′-epimerase (GALE) enzymatic activity and mRNA expression in the rat mammary gland during lactation. We report a dramatic increase in the GALE enzymatic activity correlated with an increase in the mRNA transcript expression. These results indicate a transcriptional regulation of the enzyme during lactation in the rat mammary gland. Our data are of double interest for further investigation: first, the mammary gland provides a suitable model for the characterisation of the transcriptional regulation elements of GALE which are still unknown in mammals; second, GALE expression could help to compensate UDP-galactose deficiency in classic galactosaemia.

Point mutations that affect translation initiation in the Escherichia coli galE gene

This paper describes the selection and characterization of several mutations in the Escherichia coli galactose operon that affect translation initiation of the galE gene but are located outside of the Shine-Dalgarno sequence and the initiator codon. One mutation lies in the gal promoter region and shifts transcription initiation from the galP1 to the galP2 promoter. This results in a gal messenger that is five nucleotides longer and that is translated threefold more efficiently in vivo. This accords with previous observations from in vitro experiments which showed that the longer gal messenger was better translated (Queen & Rosenberg, 1981). The other mutations that affect galE translation are located in the coding sequence immediately downstream from the initiator codon. In contrast to the promoter mutation, these cause alterations in galE expression only when the gene carries a mutated initiator codon or Shine-Dalgarno sequence and have no effect on the wild-type galE gene. These findings are discussed with respect to our present knowledge of translation initiation mechanisms.

Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation

Using hydroxylamine mutagenesis in vitro, mutations were introduced into a short DNA fragment containing the two overlapping promoters of the Escherichia coli galactose operon and the start of the first gal gene, galE. The mutagenised fragment was inserted into a lac expression plasmid. In such a vector, lac expression is controlled by the gal promoter region. Amongst eighteen candidates in which expression was reduced due to mutations in the gal fragment, twelve contained promoter mutations and six carried mutations that reduce the initiation of galE translation. The candidates in which promoter activity was reduced contained mutations affecting the promoter PI, which is dependent on the cyclic AMP-receptor protein complex (cAMP-CRP) for activation. All carried mutations in the sequence 5'GTGA3' at the CRP binding site. One of the twelve also contained a second mutation affecting the second promoter, P2, which normally functions in the absence of cAMP-CRP. Amongst the six candidates affecting galE translation, two contained a mutation that changes the initiator codon from AUG to AUA and almost completely suppresses galE expression. The mutations in the other four candidates affect the ribosome binding sequence, 5'GGAG3'. However, multiple mutations that abolish this sequence do not totally suppress galE expression, showing that there must be another way to guide ribosomes to the correct initiation site.

Escherichia coli gal operon proteins made after prophage lambda induction.

Expression of the EScherichia coli gal operon under the control of the prophage lambda promoter pL leads to gross discoordinacy of gal expression. Expression of the most promoter-distal cistron galK is much greater than expression of the promoter-proximal cistron galE. We had previously shown that transcription of the gal operon is coordinate after prophage induction. A survey of protein synthesized after prophage induction indicated that lack of expression of galE is due to a failure of translation of the galE sequence in the pL-gal transcript. This failure of translation of the galE sequence may be due to extensive dyad symmetry present in the vicinity of the gal promoter region of the pL-gal transcript. This symmetry could result in a ribonucleic acid stem-loop structure, blocking the attachment of ribosomes at the Shine-Dalgarno sequence of galE. To test this model, strains bearing the IS1 or IS2 insertion, deletion, or new promoter mutation within the symmetrical region were constructed. The restoration of some galE expression after such disruptions of the symmetrical region indicated that the ribonucleic acid stem-loop structure did play a role in the discoordinate expression of gal from pL. However, failure to obtain galE expression coordinated with high levels of galK expression suggested that other components were involved, perhaps other symmetries between galE and the pL transcript.