Exploring the roles of UDP-galactose 4'-epimerase in Drosophila melanogaster development and homeostasis Open Access

Abstract

In both humans and Drosophila melanogaster, UDP-galactose 4'-epimerase (GALE) catalyzes two distinct reactions. It catalyzes the interconversion of UDP-galactose (UDP-Gal) and UDP-glucose (UDP-Glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverts UDP-N-acetylgalactosamine (UDP-GalNAc) and UDP-N-acetylglucosamine (UDP-GlcNAc). All four of these UDP-sugars serve as critical substrates in the synthesis of glycoproteins and glycolipids. Loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia and profound loss is potentially lethal.

Here we present work describing the first whole-animal model of GALE deficiency. Our studies reveal that loss of GALE is incompatible with life in Drosophila, and demonstrate that GALE is required throughout development. We further show that GALE is necessary and sufficient when expressed in specific tissues, and demonstrate that partial loss of GALE results in galactose-induced lethality.

Whether the outcomes observed in human patients result from loss of one GALE activity, the other, or both is unknown. To address this question we have uncoupled the activities of dGALE in our Drosophila model. By replacing the endogenous dGALE with either of two prokaryotic genes, Escherichia coli galE which efficiently interconverts only UDP-Gal/UDP-Glc, and Plesiomonas shigelloides wbgU which efficiently interconverts only UDP-GalNAc/UDP-GlcNAc, we were able to generate animals with only one or the other GALE activity. Using this system we demonstrate that activity with respect to both UDP-Gal and UDP-GalNAc is required for Drosophila survival. Furthermore, distinct roles for each activity were observed during specific windows of development and in response to dietary galactose. Combined, these data reinforce the unique and essential roles of both GALE activities in Drosophila, and may provide insight into the pathophysiology of human GALE deficiency.

Table of Contents

Table of Contents

Page

Chapter 1 Introduction 1

1.1 Galactose Metabolism 2

1.2 Galactosemia 6

1.3 The Pathophysiology of Galactosemia 15

1.4 UDP-galactose 4'-Epimerase 17

1.5 Model Systems for Studying Galactosemia 20

1.6 Summary 24

1.7 References 26

Chapter 2 UDP-galactose 4' epimerase (GALE) is essential 42

for development of Drosophila melanogaster

2.1 Abstract 43

2.2 Introduction 44

2.3 Materials and Methods 48

2.4 Results 57

2.5 Discussion 69

2.6 References 76

Chapter 3 UDP-galactose 4'-epimerase activities toward 83

UDP-Gal and UDP-GalNAc play different roles in

the development of Drosophila melanogaster

3.1 Abstract 84

3.2 Author Summary 85

3.3 Introduction 86

3.4 Results 90

3.5 Discussion 103

3.6 Materials and Methods 109

3.7 References 115

Chapter 4 Conclusions and Future Directions 119

4.1 Summary 120

4.2 Significance and Future Directions 123

4.3 References 129

Appendix Development of an Assay to Permit High-throughput 131

Screening for Modulators of UGP Activity

A.1 Introduction 132

A.2 Materials and Methods 137

A.3 Results 140

A.4 Discussion 150

A.5 References 152

List of Figures

Page

1.1 The Leloir Pathway of Galactose Metabolism 4

1.2 Expanded Diagram of Galactose Metabolism 7

2.1 The Leloir Pathway of Galactose Metabolism 46

2.2 Schematic of dGALE and Alleles 60

2.3 Viability of dGALE Mutants 62

2.4 dGALE Is Required Throughout Development 68

2.5 Hypomorphic dGALE Mutants Are Sensitive To 70

Dietary Galactose

3.1 The Leloir Pathway of Galactose Metabolism 87

3.2 Enzyme activities of flies expressing different 91

GALE transgenes

3.3 Differentially impaired fecundity of flies lacking 98

different GALE activities

3.4 Flies lacking GALE activity toward UDP-Gal/UDP-Glc 101

have a shortened life span when exposed to galactose

as adults

3.5 Metabolite profiles of Drosophila exposed to galactose 104

A.1 The Leloir Pathway of Galactose Metabolism 133

A.2 UGP Forms a Bypass Pathway Around a Deficient 135

GALT Enzyme

A.3 Schematic of Coupled UGP Assay 141

A.4 Performance of the Coupled UGP Assay 143

A.5 hUGP KM Determination Toward Gal-1P 145

A.6 hUGP KM Detmination Toward Glc-1P 146

A.7 DMSO Increases hUGP Activity Toward Gal-1P 148

A.8 DMSO Displays Opposite Effects on UGP Activity 149

Toward Gal-1P versus Glc-1P

List of Tables

Page

2.1 Leloir Pathway Enzyme Activities 58

3.1 Crosses to test rescue of wbgU and eGALE transgenes 95

individually and in combination

3.2 D. melanogaster stocks and alleles used in this study 112

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Chemistry, Biochemistry
Keyword	Drosophila GALE deficiency Galactosemia
Committee Chair / Thesis Advisor	Fridovich-Keil, Judith, Emory University
Committee Members	Sanyal, Subhabrata, Emory University Cubells, Joseph F, Emory University Moberg, Kenneth H, Emory University Kelly, William G, Emory University

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