UNIVERSITY OF CALIFORNIA Los Angeles From Vitamin C to Metabolite Repair: The Role of Novel Sugar Nucleotide Phosphorylases A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biochemistry and Molecular Biology by Lital Noah Adler 2012 © Lital Noah Adler 2012 2 ABSTRACT OF THE DISSERTATION From Vitamin C to Metabolite Repair: The Role of Novel Sugar Nucleotide Phosphorylases by Lital Noah Adler Doctor of Philosophy in Biochemistry and Molecular Biology University of California, Los Angeles, 2012 Professor Steven G. Clarke, Chair The pcm-1 gene in the soil nematode Caenorhabditis elegans that encodes the L- isoaspartyl methyltransferase has roles in protein repair as well as in the worm’s insulin-like signaling pathway. Interestingly, the pcm-1 gene displays overlapping exon sequences with a previously undescribed gene (mcp-1, C10F3.4) that is transcribed from the opposite strand. This rare topology in the C. elegans genome (less than 0.05% of all genes) led us to ask whether the expression of the pcm-1 and mcp-1 genes may be co-regulated or whether their products may have a related function. PSI-BLAST analyses revealed that MCP-1 has homologs in plants, vertebrates and invertebrates and that all the homologs share a conserved HIT (histidine triad) motif. Mutations in the Arabidopsis thaliana homolog, vtc2, lead to vitamin C deficiency in these plants. ii In this thesis, I have characterized the enzymatic activity of VTC2 from A. thaliana as a GDP-L-galactose phosphorylase, the last missing step in the Smirnoff-Wheeler pathway for vitamin C biosynthesis in plants. Additional work has been done to show that the green alga Chlamydomonas reinhardtii shares homologs with all of the genes in the Smirnoff-Wheeler pathway as well as the specific activity of GDP-L-galactose phosphorylase. While vitamin C is an apparent important antioxidant and enzymatic cofactor in C. elegans, no reports had been made as to the ability of this nematode to produce it. In my work I found out that C. elegans extracts contain low levels of vitamin C. Nevertheless, as levels of vitamin C in the mcp-1 deletion strain are not different from the values in the control animals and as C. elegans lack key homologs in the biosynthesis pathway, I concluded that mcp-1 was not involved in vitamin C synthesis in C. elegans. Enzymatic analysis revealed that C. elegans’ mcp-1 and its mammalian homologs encode GDP-D-glucose phosphorylases. mcp-1 mutant worms that lacked this activity accumulated GDP-D-glucose. This unique metabolite, which shares high similarity with GDP-D-mannose, does not have a reported role in invertebrates or mammals. With my collaborators, I suggested that GDP-D-glucose could compete with GDP-D-mannose for binding to glycosyltransferases in the first steps of protein N-glycosylation in the cell, thus potentially replacing mannose residues with glucose residues in a step that could lead to N-glycosylation defect. Thus, MCP-1 may serve as a metabolite repair enzyme that eliminates adventitiously formed GDP-D-glucose from the cytosol to ensure correct protein N-glycosylation. I have tested this hypothesis by observing morphological changes in mcp-1 mutant worms and by directly comparing the N-glycan profiles of mcp-1 and control animals. iii The dissertation of Lital Noah Adler is approved. _____________________________ Catherine F. Clarke _____________________________ Stephen G. Young _____________________________ Wayne W. Grody _____________________________ Robert T. Clubb _____________________________ Steven G. Clarke, Committee Chair University of California, Los Angeles 2012 iv I would like to dedicate this work to my family. To my best friend and companion, Tahg K Adler, thank you for being such a supportive partner in this journey. Thank you for pushing me and encouraging me all these years to pursue my ambition. I love you and could not have done this without you. To my amazing boys Shai, Ilai and Itai, I am so blessed to have you in my life. To my beloved grandmother, our family matriarch, Klara Grinfeld, whose inspiring life story and love made me believe that any obstacle can be overcome. To my parents, Tzvi and Nusha Levidor, for their endless love and support. To my sisters and their families Rinat, Orit, Aviad, Lavie, Avinoam and Elkana. To all of my aunts and uncles and their families in Israel – The Kuttners; Rimona, Avigdor, Yifat, Elad, Chaya, Namma and Neta The Waksmans; Haim, Ruti, Yaron, Tamar, Vered and Nurit. To my father in-law Dr. Norman Adler, probably the only one in my family who actually understands this work. To my mother in-law Shelley Adler for always believing in me. I love you all and I am looking forward to starting a new chapter in my life together with all my family. v TABLE OF CONTENTS Abstract of the Dissertation.............................................................................................................ii List of Figures...............................................................................................................................viii List of Tables..................................................................................................................................xi Acknowledgements......................................................................................................................xiii Vita...............................................................................................................................................xvi CHAPTER 1 Plan of the Dissertation............................................................................................1 References................................................................................................................9 CHAPTER 2 Arabidopsis VTC2 Encodes a GDP-L-Galactose Phosphorylase, the Last Unknown Enzyme in the Smirnoff-Wheeler Pathway to Ascorbic Acid in Plants…..................................................................................................................13 References..............................................................................................................20 CHAPTER 3 A Second GDP-L-Galactose Phosphorylase in Arabidopsis en Route to Vitamin C. Covalent Intermediate and Substrate Requirements for the Conserved Reaction. .............................................................................................21 References..............................................................................................................31 CHAPTER 4 Impact of Oxidative Stress on Ascorbate Biosynthesis in Chlamydomonas via Regulation of the VTC2 Gene Encoding a GDP-L-Galactose Phosphorylase........................................................................................................32 References..............................................................................................................42 CHAPTER 5 Ascorbate is Present in Caenorhabditis elegans but its Origin is Unclear............53 References..............................................................................................................79 vi CHAPTER 6 A Novel GDP-D-Glucose Phosphorylase Involved in Quality Control of the Nucleoside Diphosphate Sugar Pool in Caenorhabditis elegans and Mammals.............................................................................................................84 References...........................................................................................................96 CHAPTER 7 The Search for Phenotypes in Caenorhabditis elegans Mutants that Lack the GDP-D-Glucose Phosphorylase Activity Encoded by the mcp-1 Gene...........105 References.........................................................................................................123 CHAPTER 8 Future Goals and Perspectives..........................................................................127 References.........................................................................................................137 APPENDIX I Glycan Analysis of Wild Type N2 and mcp-1 Mutant C. elegans....................141 References.........................................................................................................155 vii LIST OF FIGURES 2-1 Amino acid alignment of VTC2 homologs......................................................................15 2-2 Dependence of VTC2 activity on GME and Pi. ..............................................................16 2-3 VTC2-catalyzed phosphorolysis of GDP-L-Gal and formation of GDP.........................17 2-4 Smirnoff-Wheeler pathway from D-glucose to L-ascorbic acid......................................19 3-1 SDS-PAGE analysis of recombinant VTC5 (VTC2 homolog) and wild-type and mutant VTC2 proteins ......................................................................................................24 3-2 Identification of a guanylylated enzyme intermediate formed during the catalytic cycle of VTC2 that is dependent upon His-238……………....………………….……...25 3-3 Recombinant VTC2 is highly specific for Pi as the guanylyl acceptor used during conversion of GDP-L-Gal to L-Gal-1-P……………................………………….….......27 3-4 Effect of Pi, D-Glc-1-P, and D-Man-1-P concentrations on the activity of recombinant VTC2……………................................................…………………….…...28 3-5 Effect of Pi, D-Glc-1-P, and D-Man-1-P concentrations on the activity of recombinant VTC5………………………………….………....…………………….…..28 3-6 Effect of Pi, D-Glc-1-P, and D-Man-1-P