CONFORMATION BASED REAGENTS FOR THE DETECTION OF DISEASE-ASSOCIATED PRION PROTEIN By KRISTEN-LOUISE HATCHER Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Shu G. Chen, Ph.D. Department of Pathology CASE WESTERN RESERVE UNIVERSITY May, 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. To Grandad Dee, for teaching me to love science, and Grandad Hatcher, for always believing I could do it Table of Contents List of Tables 3 List of Figures 4 Acknowledgements 5 List of Abbreviations 6 Abstract 7 Chapter 1: Introduction 1.1 The Prion Protein 9 1.1.1 The Codon 129 Polymorphism 11 1.2 The Pathogenic Prion Protein 12 1.2.1 Prion Conversion 12 1.2.2 PK Sensitive PrPSc 14 1.2.3 Prion Strains 15 1.3 Creutzfeldt-Jakob Disease 16 1.3.1 Sporadic Creutzfeldt-Jakob Disease 16 1.3.2 Familial Creutzfeldt-Jakob Disease 19 1.3.3 Acquired Creutzfeldt-Jakob Disease 22 1.3.3.1 Iatrogenic Creutzfeldt-Jakob Disease and Kuru 22 1.3.3.2 Variant Creutzfeldt-Jakob Disease 23 1.4 Chronic Wasting Disease and Bovine Spongiform Encephalopathy 27 1.5 Current Prion Disease Diagnostics 30 1.5.1 Premortem Laboratory Diagnostics 31 1.6 Limitations of Current Methods 37 1.6.1 PrPSc in Blood 38 1.6.2 PrPSc in Urine 39 1.7 Emerging Diagnostic Technologies and Detection Reagents 40 Chapter 2: Cryptic Peptides Of The Kringle Domains Preferentially Bind To Disease- Associated Prion Protein Summary 44 Introduction 46 Methods 50 Results 54 Discussion 72 Chapter 3: Aptamers Selected against PrPSc show High Specificity to Prions from Different Species Summary 78 Introduction 80 Methods 84 Results 89 Discussion 107 1 Chapter 4: Summary and Conclusions 4.1 General Discussion 114 4.2 The Kringle-Based Peptides 116 4.3 The DNA Aptamers 117 4.4 Similarities and Differences 119 4.5 Future Directions 125 Appendix 1 Biography 130 References 131 2 LIST OF TABLES Table 1. Molecular subtypes of sCJD with corresponding phenotypic features Table 2. Six molecular subtypes of sCJD with corresponding sensitivity of the 14-3-3 test 3 LIST OF FIGURES Figure 2.1. The presence of the YRG motif in the kringle domains of plasminogen Figure 2.2 Capture of disease-associated PrP by P1 and P2 Figure 2.3. Capture of PrPSc from animal forms of prion disease by Peptides 1 and 2 Figure 2.4. P1 and P2 capture PrPSc from different subtypes of human prion diseases. Figure 2.5. Binding of P1 and P2 to the PK-resistant core fragment of PrPSc Figure 2.6. The binding of PrPSc to P1 is conformation dependent. Figure 2.7. Binding of P1 to PrPSc compared to binding of angiostatin (K(1+2+3)) Figure 2.8 Peptide 1 successfully competes with K(1+2+3) for prion binding Figure 2.9. Effect of substitution of arginine and tyrosine residues on the binding of P1 to PrPSc. Figure 2.10. Capture of spiked PrPSc from human plasma by P1 Figure 3.1 Structure and sequence of PrPSc-specific DNA aptamers Figure 3.2 Dose-dependent aptamer capture of hamster scrapie Figure 3.3 Aptamer capture of PrPSc from different subtypes of human prion disease Figure 3.4 Aptamers capture of PK digested human PrPSc shows strain differences Figure 3.5 Capture of prions from multiple animal species show PrPSc specificity Figure 3.6 Aptamer based gel shift analysis can detect prion infection in buffy coats Figure 3.7. The binding of PrPSc to 12A46 is has both sequence and conformation components Figure 3.9 Sequence alignment of prion protein from four species 4 ACKNOWLEDGEMENTS I would first like to thank my family for their support, including my parents, Peter and Frances Hatcher, and especially my husband, Rob Carter. It has not always been an easy journey, and I could not have done it without him. I would also like to thank my two baby boys, Kieran and Aidan, for allowing me to keep things in perspective, and for making me laugh. This work would not have been possible without the support of my advisor, Dr. Shu Chen, my committee, Dr. Robert Petersen, Dr. Qingzhong Kong, Dr. Michael Zagorski, Dr. Mark Smith, and Dr. Clive Hamlin. Their advice and guidance has been invaluable. Finally, I would like to thank Dr. Pierluigi Gambetti and the National Prion Disease Pathology Surveillance Center for providing facilities as well as all the tissue I needed. Kay Edmonds, Yvonne Cohen, Bikram Chakraborty and Mike Payne at the NPDPSC all provided technical support and helped make the NPDPSC a fun place to work. I would also like to thank our collaborators. Dr. Jian Zheng at Ortho Clinical Diagnostics provided the peptides and performed the BSE capture assay in Figure 2.3C. Dr. Srinand Sreevatsan and Dr. Ping Wang at the University of Minnesota performed the SELEX to isolate the aptamers, and are responsible for Figures 3.5 and 3.6. The aptamer project would not have been possible without their invaluable contribution. Thank you all. 5 LIST OF ABBREVIATIONS BSE Bovine spongiform encephalopathy CJD Creutzfeldt-Jakob Disease fCJD Familial Creutzfeldt-Jakob disease iCJD Iatrogenic Creutzfeldt-Jakob Disease sCJD Sporadic Creutzfeldt-Jakob Disease vCJD Variant Creutzfeldt-Jakob Disease CSF Cerebrospinal Fluid C-Type BSE “Classical” BSE CWD Chronic Wasting Disease DELFIA Dissociation-enhanced lanthanide fluorescence immunoassay EEG Electroencephalogram ELISA Enzyme-Linked Immunosorbent Assay EMSA Electrophoretic Mobility Shift Assay FFI Fatal Familial Insomnia GSS Gerstmann-Straussler-Scheinker disease H-Type BSE BSE in which unglycosylated band migrates higher than C-type K(1+2+3) The first three kringle domains of plasminogen (also called angiostatin) NMR Nuclear Magnetic Resonance PSWC Periodic Sharp Wave Complex PrP Prion protein PrPC Cellular prion protein PrPSc Disease-associated (scrapie) prion protein PK Proteinase K rhu PrPC Recombinant human cellular prion protein SELEX Systematic evolution of ligands by exponential enrichment 6 Conformation Based Reagents for the Detection of Disease-Associated Prion Protein ABSTRACT by KRISTEN-LOUISE HATCHER Prion diseases are a group of fatal, transmissible neurodegenerative diseases found in both animals and humans. Prion diseases are thought to arise when the normal cellular prion protein, PrPC, undergoes a conformational change from a primarily α-helical form into a form rich in β-sheets and resistant to protease, called PrPSc. Animal forms of prion disease include scrapie in sheep, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) in cattle. In humans, the diseases can take a sporadic, genetic or acquired form. One acquired form of human prion disease is variant Creutzfeldt-Jakob disease (vCJD), which emerged in the United Kingdom in 1995, and is thought to be caused by the consumption of BSE contaminated beef. The only definitive diagnostic techniques available at the present time use protease digested brain tissue. This thesis sought to develop a panel of diagnostic reagents for prion disease that enable the detection of PrPSc, but not PrPC, without protease digestion. We characterized two short peptide sequences, based on the Kringle domains of the serine protease plasminogen, for binding to PrPSc, following reports that plasminogen has PrPSc binding ability. The short peptides bound to all forms of PrPSc, both animal and human, and binding was retained in PrPSc-spiked human plasma. In addition, a panel of PrP binding DNA oligonucleotides, called aptamers, isolated through the SELEX technique, was used 7 in a capture assay. The aptamers bound to full length and PK treated PrPSc from hamster scrapie, as well as from sporadic CJD, vCJD, mouse scrapie, sheep scrapie, and white- tailed deer derived CWD. Binding was not observed to PrPSc from mule deer CWD, or from BSE, making these reagents among the first to show species or strain specificity. Strikingly, these aptamers were able to distinguish buffy coat samples derived from scrapie afflicted sheep from those of healthy animals in an electrophoretic mobility shift assay with 96% sensitivity and 100% specificity. These peptides and aptamers represent novel reagents with potential to open new avenues in prion disease research, surveillance, and therapeutics. 8 CHAPTER 1 INTRODUCTION 1.1 The Prion Protein The prion is the pathologic agent responsible for the transmission and propagation of prion diseases (1, 2). Prion diseases are a group of fatal, transmissible neurodegenerative disease, also known as transmissible spongiform encephalopathies (TSEs) that occur in both humans and animals(3). Prion diseases arise as result of one of three etiologies; sporadic, genetic or infectious. Animal prion diseases include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, as well as several rare forms of the disease arising in felines, mink and exotic ungulates. In humans, prion diseases most often occur in the form of sporadic Creutzfeldt-Jakob disease (sCJD), with an incidence of 1 case per million population annually (4). Creutzfeldt-Jakob disease may also arise as a result of a mutation in the gene encoding the prion protein (PRNP) in the form of familial Creutzfeldt-Jakob disease (fCJD)(5). Additional forms of genetic prion disease include fatal familial insomnia (FFI) and Gerstmann-Sträussler-Scheinker disease (GSS)(5). Finally, prion diseases in humans may occur as a result of infection, as in the case of iatrogenic Creutzfeldt-Jakob disease, which occurs during the course of certain medical procedures, and variant Creutzfeldt-Jakob disease (vCJD), which is believed to occur as a result of consumption of BSE infected tissue (6-10).