A Chemoenzymatic Strategy toward Understanding O-GlcNAc Glycosylation in the Brain Thesis by Nelly Khidekel In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2007 (Defended July 28 2006) ii © 2007 Nelly Khidekel All Rights Reserved iii …for my parents Raisa and Roman… iv Acknowledgments I would like to thank Linda Hsieh-Wilson for her intellectual guidance and support over the last six years. Working in a young lab was scientifically challenging and invigorating and Linda had a fantastic ability to bring in the “30,000 ft” perspective when it was needed most. I would also like to thank my committee, Dennis Dougherty, Doug Rees, and Erin Schuman. Dennis and the entire Dougherty group consistently gave of their time for the young students of the Hsieh-Wilson lab, and I will always be grateful for their support and encouragement. I did not quite make it to a rotation in Doug Rees’ lab, but I had the opportunity to TA for him and it was a real joy to be able to think about biophysics, in light of my very mathless life at Caltech. I always appreciated Erin’s neuroscience insight during our meetings and I had the opportunity to learn from and interact with several of her students, which was likewise terrific. I owe so much of my scientific development and the success of our work to Eric Peters and Scott Ficarro at GNF. Working with them was not only scientifically rewarding, but also a heck of a lot of fun. They were my second mentors and I will always be indebted to them for their boundless wit and wisdom. Of course, I am tremendously indebted to all the amazing members of the Hsieh- Wilson lab over the years, who always gave of their time and energy and who supported me on the best days and were there for me on the worst. In particular, I must thank Nathan Lamarre-Vincent who has been my cohort on the O-GlcNAc project from the very beginning. We had some colorful discussions about science and life and I hope to have many more as we move onward…and eastward. I would also like to thank Bruce Tai for the collaborative effort in the development of our methodology and Sabine Arndt for the synthesis of our probe molecule. I owe much to Katherine Poulin-Kerstien, who was my scientific partner in the beginning, and a tremendous friend throughout and to Peter Clark, who has been a great collaborator in the last couple of years. I am also indebted to Helen Cheng, with whom I shared many lunchtime conversations about the meaning of O-GlcNAc and with whom I became fast friends. I owe my brief foray into synthetic chemistry to Sarah Tully and Claude Rogers and have appreciated Sarah’s generosity and camaraderie from the very beginning. v Ultimately, this thesis would not have been possible without the companionship and support of my wonderful friends at Caltech, Sarah Miller, Katherine Poulin-Kerstien, Sarah Tully, Adam Poulin-Kerstien, Nathan Lamarre-Vincent, Ray Doss, Nathan Lundblad and many others. In particular, I must thank Sherry Tsai—our conversations were bottomless, our analyses endless, and her friendship deeply meaningful to me. I hope it will stay bright for many years to come. Finally, I thank my family Roman, Raisa, Ann…and Mr. Fluff, the fattest cat the world has ever known. My parents are some of the most thoughtful, courageous, passionate people imaginable and they have been my life-long mentors and friends. They taught Ann and me the real meaning of what it is to be a ‘scientist’ from the time we were young, when they supported our every inquiry and discovery. Ann’s spirit, wit and kindness have only bettered with age, and I thank her for her friendship and love. Finally, to Ian Swanson—my best friend and the most indelibly charming man I have ever met. People of his caliber are rare and I am deeply grateful to be able to share my life with him. So here’s to Club 202 and Pre-Prufrock, sugars and ‘juicy’ mass spectra, Wendy’s runs and book club, Dim Sum and Vegas, and all the gifted, warm and wonderful people who have made the Caltech experience nothing short of magical. vi Abstract Posttranslational modification to proteins represents a fundamental mechanism by which protein function is extended and elaborated. In the brain, modifications such as phosphorylation play critical roles in mediating neuronal communication and development. Unique among carbohydrate modifications is the addition of a single monosaccharide, N-acetyl-D-glucosamine, to serine and threonine residues of proteins (O-GlcNAc glycosylation). The modification shares intriguing features with phosphorylation, including its intracellular and dynamic nature. The enzyme responsible for adding the modification to proteins is necessary for life at the single cell level and O- GlcNAc glycosylation has been linked to nutrient sensing, gene expression, and in the brain, to neurodegeneration. Despite tantalizing evidence for the modification’s importance, understanding O-GlcNAc glycosylation has been hampered by insufficient strategies to study it at single-protein level as well as across the proteome. Here we describe the development of a new, chemoenzymatic strategy to facilitate the discovery of O-GlcNAc proteins, as well as the first studies aimed at understanding O-GlcNAc proteome-wide, in the brain. Our approach capitalizes on an engineered enzyme and synthetic unnatural substrate to specifically ‘tag’ O-GlcNAc-modified proteins for rapid and sensitive detection. We applied the methodology to the discovery of low-abundance, endogenous O-GlcNAc proteins from cells. We also combined the approach with mass spectrometry for the isolation of O-GlcNAc peptides and the mapping of glycosylation sites, the first step toward functional analysis of the modification. Overall, our efforts led to the identification of nearly fifty new O-GlcNAc proteins, several of which serve as targets for mechanistic study. Many of the proteins function in the control of transcription and translation, highlighting the proposed role for O-GlcNAc in regulating gene expression. Additionally, we provide evidence that O-GlcNAc glycosylation is particularly prevalent on proteins at the nerve terminal, or synaptosome, where it may function to control vesicle cycling and neurotransmitter release. Finally, our work has also led to the first bioanalytical, quantitative assays for O-GlcNAc dynamics in both cells and tissue. We vii have shown that O-GlcNAc is reversible in neuronal tissue and can respond rapidly and robustly to neuronal stimulation in vivo. viii Table of Contents Acknowledgments iv Abstract vi Table of Contents viii List of Figures ix List of Schemes xii List of Tables xii Chapter 1 Introduction: O-GlcNAc Glycosylation in the Brain 1 Chapter 2 Development of a New Chemoenzymatic Strategy to Study 30 O-GlcNAc Glycosylation Chapter 3 Discovery of O-GlcNAc-Modified Proteins from Cell Lysates and61 and Identification of O-GlcNAc Glycosylated Sites via Mass Spectrometry Chapter 4 Exploring the O-GlcNAc Proteome: 95 Direct Identification of O-GlcNAc-Modified Proteins from the Brain Chapter 5 Probing the Dynamics of O-GlcNAc Glycosylation Using the 127 Chemoenzymatic Strategy and Quantitative Proteomics Appendix I Exploring O-GlcNAc Dynamics from Neuron Culture and 174 the Brain Appendix II Identification of O-GlcNAc Glycosylation Sites: Application 187 to Endogenous Proteins and Exploration of Fluorous Enrichment Strategies ix Figures Chapter 1 Page Figure 1.1 Protein phosphorylation regulates numerous facets of neuronal 3 communication Figure 1.2 Diverse molecules serve to modify proteins posttranslationally 4 Figure 1.3 O-GlcNAc glycosylation is a unique carbohydrate modification 7 to proteins Figure 1.4 O-GlcNAc glycosylation plays a role in many aspects of cell 11 function including transcriptional repression and nutrient sensing Figure 1.5 Mass spectrometry (MS)-based approaches for quantitative 20 proteomics Chapter 2 Figure 2.1 Strategy for detection of O-GlcNAc glycosylated proteins 34 Figure 2.2 Crystal Structure of the β1,4-galactosyltransferase complexed 35 with UDP-GalNAc Figure 2.3 Reverse phase LC-MS analysis of O-GlcNAc labeling reactions 36 with wildtype GalT Figure 2.4 Reverse phase LC-MS analysis of O-GlcNAc peptide labeling 37 reactions with Y289L GalT Figure 2.5 Electrospray ionization mass spectra of the LC-MS peaks 38 in figure 2.4 Figure 2.6 The chemonenzymatic strategy affords selective labeling of 40 glycosylated CREB Figure 2.7 Traditional methodology for O-GlcNAc detection fails to detect 41 α-crystallin by Western blotting Figure 2.8 The chemoenzymatic strategy permits rapid and sensitive 42 detection of αA-crystallin Figure 2.9 The Y289L enzyme may be able to utilize other unnatural 43 analogues for chemoselective ligation x Chapter 3 Figure 3.1 Strategy for identifying O-GlcNAc proteins from cells 63 Figure 3.2 Strategy for identifying O-GlcNAc glycosylation sites 66 Figure 3.3 Selective isolation of chemoenzymatically tagged O-GlcNAc 68 glycosylated proteins via streptavidin Figure 3.4 The transcriptional repressor MeCP2 is O-GlcNAc glycosylated 70 in neurons Figure 3.5 Enrichment of CREB O-GlcNAc peptides via the 72 chemoenzymatic strategy Figure 3.6 Identification of the O-GlcNAc modified peptide on CREB by 73 LC-MS/MS Figure 3.7 Application of the chemoenzymatic strategy toward the 74 identification of the αA-crystallin peptide Figure 3.8 Enrichment of OGT O-GlcNAc peptides via the 76 chemoenzymatic labeling strategy Figure 3.9 Identification of O-GlcNAc modified