UDP-Galactose 4′-Epimerase Activities toward UDP-Gal and UDP-GalNAc Play Different Roles in the Development of Drosophila melanogaster

In both humans and Drosophila melanogaster, UDP-galactose 4′-epimerase (GALE) catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc). All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE) efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU) efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans

Differentially impaired fecundity of flies lacking different GALE activities.

<p>(A) Diagram of the method used to achieve expression of different <i>GALE</i> transgenes in the background of <i>dGALE</i> knockdown animals. The timing of knockdown and concurrent transgene expression was controlled by switching flies from the permissive temperature (18°C) to the restrictive temperature (28–29°C), as indicated. (B) Knockdown efficiency in male and female animals switched to the restrictive temperature as early to mid-stage pupa and harvested for biochemical analysis as newly eclosed adults or three days after eclosion. Of note, GALT activity was completely normal in all samples tested and apparently unaffected by the <i>dGALE</i> knockdown (data not shown). (C) Each box represents the outcome of flies switched from 18°C to 28°C at the stage indicated in the column on the left. The number of days the flies developed at 18°C to reach each stage is shown in parentheses.</p

Enzyme activities of flies expressing different <i>GALE</i>

<p><b>transgenes.</b> Assays for all genotypes were performed on flies with <i>dGALE</i> knockdown (KD) driven by the <i>Act5C-GAL4</i> driver with the exception of flies labeled “no knockdown”; those flies carried the same <i>UAS-RNAi^dGALE</i> and <i>GAL80^ts</i> alleles, but were balanced over TSTL, and thus lacked the driver. In addition to RNAⁱ knockdown of <i>dGALE</i>, <i>Act5C-GAL4</i> also drives expression of the specified transgenes in these animals. Panel A: GALE activity using UDP-gal as substrate. Panel B: GALE activity using UDP-galNAc as substrate.</p

Flies lacking GALE activity toward UDP-gal/UDP-glc have a shortened life span when exposed to galactose as adults.

<p>The life spans of male (A and B) and female (C and D) flies reared on molasses food and then tapped as newly eclosed adults to food containing either 555 mM glucose only (A and C), or 555 mM glucose plus 175 mM galactose (B and D), is illustrated. As indicated by the key, these cohorts of flies included controls expressing endogenous <i>dGALE</i> as well as animals that expressed endogenous <i>dGALE</i> early in development but then were subjected late in development to <i>dGALE</i> knockdown coupled with induced expression of either no <i>GALE</i> transgene, or <i>wbgU</i>, <i>eGALE</i>, <i>hGALE</i>, or both <i>wbgU</i> and <i>eGALE</i> in combination. Based on Log rank and Wilcoxon tests for significance, the life spans of knockdown animals expressing either no transgene or expressing only <i>wbgU</i> were significantly decreased on food containing galactose compared with food containing only glucose (p<0.0001).</p

The Leloir pathway of galactose metabolism.

<p>UDP-galactose 4′-epimerase, the third enzyme in the pathway, also interconverts UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc) in humans, <i>Drosophila</i>, and other metazoans tested.</p

Metabolite profiles of <i>Drosophila</i> exposed to galactose.

<p>Metabolites were extracted from cohorts of larvae raised on food containing either 555 mM glucose or 555 mM glucose+175 mM galactose. Animals were shifted from the permissive temperature (18°C) to the restrictive temperature (28°C) as first instar larvae and allowed to develop for four days before harvest. Accumulated metabolite values for gal-1P (A), UDP-gal (B), and UDP-galNAc (C) are shown on food containing glucose and glucose+galactose. To demonstrate the impact of diet on metabolite levels, values for gal-1P (D), UDP-gal (E), and UDP-galNAc (F) are shown as ratios of the amount of each metabolite accumulated by animals on food containing galactose over that accumulated by animals of the same genotype on food containing only glucose. Error bars show the 95% confidence interval for each ratio.</p

Crosses to test rescue of <i>wbgU</i> and <i>eGALE</i> transgenes individually and in combination.

<p>Crosses to test rescue of <i>wbgU</i> and <i>eGALE</i> transgenes individually and in combination.</p

Acute and long-term outcomes in a Drosophila melanogaster model of classic galactosemia occur independently of galactose-1-phosphate accumulation

Classic galactosemia (CG) is a potentially lethal inborn error of metabolism that results from the profound loss of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. Neonatal detection and dietary restriction of galactose minimizes or resolves the acute sequelae of CG, but fails to prevent the long-term complications experienced by a majority of patients. One of the substrates of GALT, galactose-1-phosphate (Gal-1P), accumulates to high levels in affected infants, especially following milk exposure, and has been proposed as the key mediator of acute and long-term pathophysiology in CG. However, studies of treated patients demonstrate no association between red blood cell Gal-1P level and long-term outcome severity. Here, we used genetic, epigenetic and environmental manipulations of a Drosophila melanogaster model of CG to test the role of Gal-1P as a candidate mediator of outcome in GALT deficiency. Specifically, we both deleted and knocked down the gene encoding galactokinase (GALK) in control and GALT-null Drosophila, and assessed the acute and long-term outcomes of the resulting animals in the presence and absence of dietary galactose. GALK is the first enzyme in the Leloir pathway of galactose metabolism and is responsible for generating Gal-1P in humans and Drosophila. Our data confirmed that, as expected, loss of GALK lowered or eliminated Gal-1P accumulation in GALT-null animals. However, we saw no concomitant rescue of larval survival or adult climbing or fecundity phenotypes. Instead, we saw that loss of GALK itself was not benign and in some cases phenocopied or exacerbated the outcome seen in GALT-null animals. These findings strongly contradict the long-standing hypothesis that Gal-1P alone underlies pathophysiology of acute and long-term outcomes in GALT-null Drosophila and suggests that other metabolite(s) of galactose, and/or other pathogenic factors, might be involved