Studies of the V94M‐substituted human UDPgalactose‐4‐epimerase enzyme associated with generalized epimerase‐deficiency galactosaemia

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.The enzyme galactose-1-phosphate uridylyltransferase (GALT) 1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose and galactose 1-phosphate (gal-1-P) to glucose 1-phosphate and UDP-galactose (UDP-gal) (1, 2). Impairment of human GALT (hGALT) results in the potentially lethal disorder classic galactosemia (2, 3).Currently, most infants with classic galactosemia born in industrialized nations are detected in the neonatal period by mandated newborn screening procedures. Dietary restriction of galactose initiated early and maintained throughout life for these patients prevents the potentially lethal sequelae of the disorder. Unfortunately, despite treatment, the long term outcome for these patients is mixed; 85% of girls with galactosemia experience primary ovarian failure, and 30 -50% of patients of both genders demonstrate learning disabilities and speech and/or motor dysfunction, among other complications (4). Although aberrant accumulation or depletion of key galactose metabolites, including gal-1-P, UDP-gal, galactitol, and others are hypothesized as underlying the observed complications (reviewed in Refs. 2 and 3), the biochemical mechanism of pathophysiology in galactosemia remains unknown.

Section: Methodsmentioning

confidence: 99%

Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

Riehman

Crews

Fridovich‐Keil³

2001

“…These results demonstrate that GALE-null yeast are not only Gal Ϫ or unable to metabolize galactose fully, they are also galactose-sensitive (15,18,19). Prior and ongoing studies have helped to identify potential factors mediating galactose sensitivity in GALE-null yeast (15,18,19); nonetheless, how these results relate to galactose metabolism and sensitivity in GALE-deficient mammalian cells has remained unknown.…”

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confidence: 88%

“…As one approach toward addressing this question, both we and others have developed and applied yeast model systems in which GALE (GAL10) is either deleted or impaired (11,14,15,18,19). GALE-deficient yeast are not only unable to grow on media containing galactose as the sole carbon source; they also fail to grow on media containing alternate carbon sources, such as glycerol/ethanol or raffinose, if even trace amounts of galactose are added (e.g.…”

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confidence: 99%

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

Schulz

Ross

Malmström

et al. 2005

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, UDPGalNAc 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.

“…Role of Cys 307 in hGALE-The issue of substrate specificity of hGALE, and the impact of naturally occurring mutations on that specificity, have been questions of both basic science and clinical interest for many years, in part because it remains unknown which activity of hGALE mediates severity of outcome for patients with epimerase-deficiency galactosemia (13,14). Structural and/or mutational studies from E. coli (20,23), human (8,23), Yersinia enterocolitica (35), and P. aeruginosa (24) all suggest that too small an active site cleft volume can prevent an otherwise functional epimerase from acting upon larger substrates (UDP-GalNAc/UDP-GlcNAc), although these same reports also demonstrate that a large active site cleft may or may not pose challenges to the epimerization of smaller substrates (UDP-Gal/UDP-Glc).…”

Section: Expression Of Wild-type and Substituted Human Gale Enzymes Imentioning

confidence: 99%

“…G90E) impact interconversion of UDP-Gal/UDP-Glc and UDP-GalNAc/UDP-GlcNAc equally, others do not. For example, as compared with the wild-type enzyme, V94M-hGALE, associated with severe epimerase-deficiency galactosemia, retains only 5% activity with regard to UDP-Gal/UDP-Glc but 25% activity with regard to UDPGalNAc/UDP-GlcNAc (13,14). The role of impaired activity with regard to each substrate in defining patient outcome remains unknown.…”

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confidence: 99%

Determinants of Function and Substrate Specificity in Human UDP-galactose 4′-Epimerase

Schulz

Watson

Sanders

et al. 2004

UDP-galactose 4-epimerase (GALE) interconvertsUDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.