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Glycosylation and Palmitoylation in Toxoplasma Gondii

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Glycosylation and Palmitoylation in Toxoplasma Gondii Role of Post Translational Modifications: Glycosylation and Palmitoylation in Toxoplasma gondii Flora C. Luk A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell and Developmental Biology Chapel Hill 2011 Approved by: Dr. James Bear, Dr. Con Beckers, Dr. Keith Burridge, Dr. Richard Cheney, Dr. Mohanish Deshmukh ©2011 Flora C. Luk ALL RIGHTS RESERVED ii ABSTRACT Flora C. Luk: Role of Post Translational Modifications: Glycosylation and Palmitoylation in Toxoplasma gondii (Under the direction of Con Beckers) Many eukaryotic proteins are post-translationally modified to regulate protein-protein interactions, protein stability, folding, localization, and proteolysis. Modified proteins play essential roles in cellular processes such as protein-protein interactions, signal transduction, and membrane association. Given the prevalence and importance of these modifications, post translational modifications such as protein palmitoylation and glycosylation have not been explored in Toxoplasma. At the time of our study, protein glycosylation was known to modify two proteins: GAP50 and gp23. And protein palmitoylation has only been experimentally confirmed for a few parasite specific proteins although many have been predicted to have cysteine residues that are modified. This body of work explored the contributions of two key post-translational modifications, glycosylation and protein palmitoylation, to Toxoplasma biology. Toxoplasma gondii is an obligate intracellular parasite that infects more than 60 million people in the U.S. Infection can cause congenital neurological defects, and severe disease in immuno-compromised individuals. Toxoplasma survival is dependent on parasite motility, which governs essential processes: invasion, egress, and dissemination throughout the host. We described the analysis of protein glycosylation in Toxoplasma. We began with iii the detection of N-glycosylation of myosin XIV motor complex anchor, GAP50, and proceeded to characterize the structure and immune-reactivity of Toxoplasma N-linked glycans. Subsequently, to identify palmitoyl proteins in Toxoplasma, we took advantage of recently developed biochemical and proteomic approaches allowing us to decrease the length of time of analysis and increase the number of proteins analyzed. The palmitoyl proteome of Toxoplasma included cytoplasmic and transmembrane proteins that also had additional acylation sites. The two analysis presented below would help us identify how post- translational modifications – glycosylation and palmitoylation – regulate parasite proteins to mediate important cellular processes essential to survival. iv To my family for their unconditional love and support v TABLE OF CONTENTS LIST OF TABLES…………………………………………………………………………....x LIST OF FIGURES…………………………………………………………………………..xi LIST OF ABREVIATIONS…………………………………………………………….......xiv CHAPTER 1: TOXOLPLASMA GONDII LITERATURE REVIEW ..................................... 1 Introduction ........................................................................................................................... 2 Apicomplexa ..................................................................................................................... 3 Life cycle of Toxoplasma gondii ...................................................................................... 4 Toxoplasmosis ................................................................................................................... 5 Toxoplasma gondii – the “model” apicomplexa parasite ................................................. 7 Post Translational Modifications .......................................................................................... 8 Glycosylation .................................................................................................................... 8 Protein Palmiotylation ...................................................................................................... 9 Toxoplasma Protein Palmitoyl Transferases (PPTs) ....................................................... 11 Figures and Legends ........................................................................................................... 14 References ........................................................................................................................... 18 CHAPTER 2: N-LINKED GLYCOSYLATION OF PROTEINS IN THE PROTOZOAN PARASITE TOXOPLASMA GONDII………………...…….20 Abstract ............................................................................................................................... 22 Introduction ......................................................................................................................... 23 Results ................................................................................................................................. 26 vi N and O-linked glycosylation of proteins is common in Toxoplasma gondii .......................................................................................................... 26 N and O-glycosylated proteins are found throughout parasite ............................................................................................................................ 27 N-glycosylation is essential for Toxoplasma viability .................................................... 28 Other N-glycosylated proteins of Toxoplasma ............................................................... 32 N-linked oligosaccharides of Toxoplasma differ from those in other eukaryotes ................................................................................................ 33 Discussion ........................................................................................................................... 35 Materials and Methods ........................................................................................................ 40 Figures and Legends ........................................................................................................... 45 References ........................................................................................................................... 63 CHAPTER 3: GLOBAL ANALYSIS OF TOXOPLASMA GONDII PALMITOYL PROTEOME…………………………………………………65 Abstract ............................................................................................................................... 66 Introduction ......................................................................................................................... 67 Results ................................................................................................................................. 70 Palmitoylation of Toxoplasma Myosin XIV Motor Complex ............................................................................................................... 70 Purification and identification of Toxoplasma palmitoyl proteins ........................................................................................................... 71 Validation of Candidate Palmitoyl Proteins ................................................................... 74 Toxoplasma small heat shock protein 20 Localizes to “stripe-like” structures ................................................................................ 75 Materials and Methods ........................................................................................................ 80 vii Figure and Legends ............................................................................................................. 88 References ......................................................................................................................... 123 CHAPTER 4: HOST CELL EGRESS AND INVASION INDUCED MARKED RELOCATIONS OF GLYCOLYTIC ENZYMES IN TOXOPLASMA GONDII TACHYZOITES……………….……………….125 Abstract ............................................................................................................................. 126 Introduction ....................................................................................................................... 128 Results ............................................................................................................................... 131 Glycolysis is a major source of energy for Toxoplasma motility .................................................................................................... 131 The Toxoplasma glucose transporter GT1 is found throughout the plasma membrane ................................................................................. 132 The subcellular location of aldolase-1 is different in intra and extracellular Toxoplasma ............................................................................... 133 Subcellular distribution of Toxoplasma aldolase-1 during the tachyzoite life cycle .................................................................... 135 Regulation and mechanism of aldolase-1 redistribution during host cell invasion and egress ............................................................................. 137 Localization of other glycolytic enzymes in intra and extracellular Toxoplasma ........................................................................... 141 Pellicle association of glycolytic enzymes in Toxoplasma ........................................... 143 Discussion ........................................................................................................................
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