Elucidating the Molecular Mechanisms Involved in Assembly/Folding and Targeting V-ATPase a-subunit Isoforms to their Functional Destinations By Sally Esmail A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Faculty of Dentistry University of Toronto © Copyright by Sally Esmail 2017 ii Elucidating the Molecular Mechanisms Involved in Assembly/Folding and Targeting V-ATPase a-subunit Isoforms to their Functional Destinations Sally Esmail Doctor of Philosophy Faculty of Dentistry University of Toronto 2017 Abstract Vacuolar H+-ATPases (V-ATPases) are proton pumps distributed across membranes of specialized cells and luminal compartments. V-ATPases in the plasma membrane of osteoclasts are responsible for acidifying the surface of bone, essential for bone resorption. V-ATPases in the plasma membrane of metastatic cells acidify the extracellular space to facilitate invasion. The V-ATPase a subunit has four isoforms (a1-a4) that localize to distinct compartments. In invasive cancer, plasma membrane expression of a3 and a4 are required for metastasis while a3 is specific for the plasma membrane of osteoclasts. Thus, both isoforms are potential therapeutic targets. Sequences of a isoforms reveal putative N-glycosylation sites within extracellular loop II (ELII). Upon PNGase F and Endo H treatment, all a isoforms showed faster mobility on SDS- PAGE indicating the presence of N-linked oligosaccharide. Using site-directed mutagenesis, I showed that deglycosylated a1–a4 had shorter half-lifes, more rapid proteasomal degradation, endoplasmic reticulum (ER) retention, defective Golgi trafficking, and an inability to associate with ER assembly factor, VMA21. In addition, deglycosylated a4 showed defective cell-surface iii expression and assembly. Cutis laxa type II, osteopetrosis and distal renal tubular acidosis (dRTA) result from mutations within the a2, a3 and a4 subunits, respectively. To further map critical domains essential for V-ATPase structure and function, I studied human disease-causing missense mutations that affect conserved residues of a isoforms, specifically: a2P405L, a4R449H and a4G820R. a4R449H and a2P405L were unstable and degraded by the proteasomal pathway. The data also indicated that a2-P405 is required for Golgi trafficking while a4-R449 is essential for ER exit and cell-surface expression. a4R449H shows increased association with the assembly factor, VMA21. Molecular modeling of a4 predicts a4G820R would interfere with proton translocation through the cytoplasmic half channel formed by the a subunit. This work enhances our knowledge of a isoforms structure and informs possible therapeutic interventions against cancer metastasis and lytic bone diseases. iv Acknowledgments First, I would like to express my sincere gratitude to my supervisor, Prof. Morris F Manolson for giving me the chance to join his lab and the tremendous support, motivation and guidance during the course of my PhD program. His guidance and scientific expertise greatly helped me in all aspects of my PhD research and writing of this thesis. I have been extremely lucky to have a supervisor who cared so much about my research. I would also like to extend my gratitude to my co-supervisor Prof. Reinhart A.F. Reithmeier; his expertise and deep knowledge of the research subject inspired me. He helped me to conceptualize and analyze all the data presented in this thesis. His careful and critical editing positively impacted the production of this thesis. I feel exceedingly lucky and could not have imagined better advisors. Completing this work would have been more difficult were it not for the support of Dr. Norbert Kartner. His critical thinking and guidance helped me in designing all the experiments and in interpretation of data. I am deeply thankful to his scientific expertise and professional academic writing skills, without which I would not have publishable research. I would like to thank my thesis committee member Prof. Boris Hinz for his constructive criticism, insightful comments and encouragement, but also for the hard questions which encouraged me to expand my horizons from various research perspectives. I must also thank all lab members of the Manolson’s lab and the Reithmeier’s lab, with special thanks to Dr. Yeqi Yao and Jing Li for all their help and technical support. Finally, I would like to thank my family and friends who always believed in me and encouraged me to come over to Canada to pursue my research path and remained patient and positive. v Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents ........................................................................................................................... iv Original Contribution by author ..................................................................................................... ix List of Figures and Tables.............................................................................................................. xi Abbreviations ............................................................................................................................... xiii 1. Introduction–Thesis Rationale ....................................................................................................1 2. Literature Review.........................................................................................................................3 2.1. Recent Advances in the Understanding of Membrane Protein N-glycosylation Structure, Function, and Regulation in Health and Disease ................................................3 2.1.1. Biosynthesis and elongation of mammalian N-linked glycan ...................................3 2.1.2. Role of N-glycans in protein folding, stability, and quality control in the ER ..........6 2.1.3. Regulation of mammalian N-glycosylation ...............................................................9 2.1.4. N-glycosylation in cell biology, signal transduction, and immunity .......................10 2.1.5. N-glycosylation in diseases......................................................................................11 2.1.6. N-glycosylation as a potential therapeutic target .....................................................12 2.1.7. Future prospectives: Advancing the knowledge of N-glycan 3D structure .............13 2.2. V-ATPase structure, function, regulation and drug targeting ............................................14 2.2.1. V-ATPase Function .................................................................................................14 2.2.2 V-ATPases Structure ................................................................................................16 2.2.3 V-ATPases subunits isoforms ...................................................................................17 2.2.4. Regulation of V-ATPase assembly and trafficking .................................................20 2.2.5 Involvement of V-ATPases in human diseases ........................................................20 2.2.6 V-ATPase a subunit topology and atomic models ...................................................21 2.2.7 V-ATPase inhibitors and their limitation ..................................................................27 vi 2.2.8 V-ATPase a subunit as a potential therapeutic target ...............................................28 3. Research Objectives and Hypotheses ........................................................................................29 Hypotheses. ....................................................................................................................................29 Central Objective ...........................................................................................................................30 Aim 1 ....................................................................................................................................30 Aim 2 ....................................................................................................................................30 Significance....................................................................................................................................30 + 4. N-Linked Glycosylation Is Required for Vacuolar H -ATPase (V-ATPase) a4 Subunit Stability, Assembly , and Cell Surface Expression .....................................................................31 4.1 Abstract ...............................................................................................................................32 4.2. Introduction ........................................................................................................................32 4.3. Materials and Methods .......................................................................................................34 4.4. Results ................................................................................................................................39 4.4.1. The Human V-ATPase a4 subunit is N-glycosylated...................................................39 4.4.2. N-glycosylation is required for a4 stability .................................................................40 4.4.3. Un-glycosylated a4 is degraded in proteasomal and lysosomal pathways ....................41 4.4.4. Un-glycosylated a4 is mostly retained in the ER .........................................................42 4.4.5. Un-glycosylated