Insights into the Mammalian Neuromuscular Junction Using the Torpedo Californica Electric Organ as a Model Tissue by Suzanne E. Mate A.S. in Health Science Laboratory Technology, August 2002, George Washington University B.S. in Biology, August 2007, George Washington University M.Phil. in Biochemistry & Molecular Genetics, May 2010, George Washington University A Dissertation submitted to The Faculty of Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 31, 2012 Dissertation directed by Eric P. Hoffman Professor of Pediatrics and of Biochemistry and Molecular Biology The Columbian College of Arts and Sciences of The George Washington University certifies that Suzanne E. Mate has passed the Final Examination for the degree of Doctor of Philosophy as of June 5, 2012 . This is the final and approved form of the dissertation. Insights into the Mammalian Neuromuscular Junction Using the Torpedo Californica Electric Organ as a Model Tissue Suzanne E. Mate Dissertation Research Committee: Eric P. Hoffman, Professor of Pediatrics and of Biochemistry and Molecular Biology Lubjica Caldovic, Associate Professor of Pediatrics, Committee Member Yetrib Hathout, Assistant Professor of Integrative Systems Biology, of Pediatrics, and of Biochemistry and Molecular Biology, Committee Member ii © Copyright 2012 by Suzanne E. Mate All rights reserved iii Motivated Dedicated To the soldiers of our armed forces. No pain. No gain. Train to Save iv Acknowledgments I, the author, wish to acknowledge: The vision of my mentor that led to the creation of the Center for Genetic Medicine Research at Children‘s National Medical Center. With a broad research focus and technologies that eneable multidimensional data queries, this Center is certainly a prelude to Integrative/Systems Biology. My breadth of knowledge acquired during my doctoral studies was unintentionally absorbed from exposure to the different expertise of many investigators and their philosophies on research strategies, the research from outside faculty invited to speak in one of several Seminar Series, and the skills of fellow students that enabled my personal performance. My growth and development into a scientist is undoubtedly a result of this rich, learning environment. I thank all personel whose efforts support the longevity of GenMed. As I search for a new lab, I recognize how spoiled I was to have the beadth of resources and experts readily available for direct research application and how limiting the absence of these resources is to meet basic research needs. This vision and design has left a lasting imprint on the ideal research setting that I hope to implement in my future career. The sweat of my mentor for his patience and persistence in transforming a soldier into a student. The amazing group of principle investigators, fellow graduate students, and research suport staff at GWU and CNMC that shaped my doctoral experience. v Abstract of Dissertation Insights into the Mammalian Neuromuscular Junction Using the Torpedo Californica Electric Organ as a Model Tissue The neuromuscular junction (NMJ) is a highly specialized sub-region of the myofiber cellular membrane, with unique structure and protein composition. Membrane surface area is increased at this specific location to accommodate motor neuron terminal endings and to support a high load of nicotinic acetylcholine receptors (AChR). Many proteins show exclusive localization to the endplate, in part due to nuclear domains (subsynaptic nuclei preferentially expressing synaptic and accessory genes). A limited repertoire of NMJ proteins has been characterized with regards to structure, expression, and function. We hypothesized that many additional NMJ protein components remain to be discovered, and the existing limited repertoire inhibits molecular understanding of the NMJ. The goal of this dissertation was to develop methods for identification of new NMJ components (increase the repertoire). The miniscule size and low abundance of the NMJ in muscle presents technical limitations in studying its proteome makeup and function. To overcome this, we used the Torpedo californica electric organ as a model– a highly specialized organ that is known to derive from expanded NMJs. We hypothesized that profiling the proteins of the electric organ will, by anatomical inference, reveal novel synaptic proteins that were overlooked in the mammalian NMJ due to their miniscule size and challenges for analyzing them. We conducted a non-targeted proteomics study to investigate the molecular constituents of the electric organ and assessed its concordance with the mammalian NMJ. We showed that the vi electric organ is a repository of candidate NMJ proteins, supporting our hypothesis. From the electric organ proteome, we selected one candidate protein for additional study, based upon high proteomic score and annotated function in receptor recycling pathways described by the current NMJ paradigm. Eps homology domain containing protein 1 (EHD1), and related family members, were selected for study in the electric organ and skeletal muscle. We found that EHD1 localized to the primary synaptic cleft of the NMJ, proving our initial hypothesis. We show that the function of EHD1 is not critical to maintaining the NMJ architecture as EHD1-/- endplates show normal morphology. However, EHD4 expression is increased in EHD1-/- skeletal muscle and likely compensates in function for the loss of EHD1 such that double knockout mouse models are necessary to assess the function of EHD1 at the NMJ. Many synaptic proteins are post-translationally modified by the addition of carbohydrates. We hypothesized that a proteomic approach targeting N-linked glycoproteins expressed in the electric organ could lead to novel biochemical insights regarding the structure/function of the NMJ. We identified the SITS-binding protein (SP105) as the most abundant N-linked glycoprotein expressed in the electric organ. Little is known regarding SP105 protein structure, localization, and function. Using a glycoproteomics approach, we identified five of the eight asparagine residues within consensus sequence N-X-(S/T) were glycosylated. Furthermore, glycans were composed of N-acetylhexosamine, mannose, galactose, and N-acetylneuraminic acids arranged in hybrid bi- and complex tri-antennary branches. The studies in this dissertation provide new knowledge concerning NMJ protein repertoire that will aid in presenting a more complete NMJ proteome to guide future studies on NMJ structure and function in health and disease. In addition, this work shows the conservation and vii diversity of N-linked glycan post-translation modifications of a highly abundant electrocyte protein, SP105. viii Table of Contents Dedication ............................................................................................................................................ iv Acknowledgments ................................................................................................................................ v Abstract of Dissertation ....................................................................................................................... vi List of Figures .................................................................................................................................... xiii List of Tables .................................................................................................................................. xvivi List of Abbreviations ........................................................................................................................ xvii Chapter 1: Introduction ........................................................................................................................ 1 1.1 The Neuromuscular Junction: Development, Structure, and Function ................................................................................................................................... .1 1.2 Electric Organs and Torpedo californica ........................................................................... 8 1.3 Modern High-throughput Technologies: Applications for Gene and Protein Discovery ............................................................................................................ 14 1.4 Muscle Transcriptome Profiling for Protein Inference ................................................... 15 1.5 Muscle Proteomic Profiling for Hypothesis Generation ................................................. 16 1.6 Displaying High-Throughput Proteomic Data: A Systems Biology Approach ................................................................................................................................ 17 1.7 In vivo Gene Transfer for Expression of Recombinant Proteins at the NMJ ................................................................................................................................... 18 1.8 Purpose and Significance ................................................................................................. 19 ix Chapter 2: Integrated genomics and proteomics of the Torpedo californica electric organ: Concordance with the mammalian neuromuscular junction. .............................. ..211 2.1 Abstract ..............................................................................................................................22 2.2 Introduction ........................................................................................................................23