O NEUROTOXINS NOVEL ANTAGONISTS OF NICOTINIC ACETYLCHOLINE RECEPTORS FROM SNAKE VENOM

Ω-NEUROTOXINS: NOVEL ANTAGONISTS OF NICOTINIC ACETYLCHOLINE RECEPTORS FROM SNAKE VENOM VARUNA H P (M.Sc (Biochemistry)) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE NATIONAL UNIVERSITY OF SINGAPORE JANUARY 2015 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Varuna H P 20thJanuary 2105 i Acknowledgements To begin with, I would like to thank the almighty for bestowing me with strength to tackle failures and persistence to learn from them I feel privileged to be supervised by Professor R Manjunatha Kini, who was not only a patient teacher but an inspiration I am thankful for his aspiring guidance, invaluably constructive criticism and friendly advice during the course of my research He has a great role in fine tuning my presentation skills, writing skills as well as my scientific thought process I would also like to thank him for providing me an opportunity to learn at various labs both in Singapore and during overseas attachments, which was a great deal of exposure for me to grow as a confident and independent researcher I would like to express my sincere gratitude towards Professor David J Adams, RMIT, Melbourne, Australia, for opening his lab doors for me to learn, conduct and discuss electrophysiology experiments I would like to thank Professor Adam’s group members, Dr Fe Abogadie, Dr Hartmut Cuny, Dr Peter Bartels, Joshua Ross Lawson, Dr Geza Berecki, Dr Han Shen Tea, Dr Shiva Nag and Dr Mahsa Sadeghi who have helped me in one way or the other during my stay at RMIT I owe a great debt of gratitude to Dr Shiva Nag, who has helped me patiently with my experiments and was always available for discussions I am thankful to Dr Han Shen Tea, who was a joyful neighbour and dependable senior for scientific discussions in the lab I would like to thank Professor J Sivaraman and his lab member, Dr Jobichen for being available to advice on the crystallisation studies of Oh9-1 I ii would like to thank Professor Peter Wong and his group members, Mrs Ting Wee Lee and Ms Su Jing for helping me with various aspects of pharmacology experiments I am grateful to Professor Cletus D’Souza, who believed in me and has guided me through my tough times I would also like to thank Professor B S Vishwanath, Professor Karun Kumar, Dr K Kemparaju, Dr Gopal Marathe and Dr K S Girish for being such wonderful teachers I would like to thank all the past and present lab members of Protein Science lab, Dr Ryan McCleary, Dr Aktar Chouduri, Dr Guna Shekhar, Dr Amrita Roy, Dr Vallerinteavide Mavelli Girish, Dr Tan Wei Ling Angelina, Dr Bhaskar Barnwal, Dr Sindhuja Shirdharan, Dr Foo Chun Shin, Dr Guillaume Blanchet, Dr Saravanan Vivekanandan, Aldo, Ritu Chandna, Bidhan Chandra Nayak, Summer, Iyer Janaki Krishnamoorthy, Ben Wong and Norrapat Shih for providing a professional lab environment for me to learn and develop as an independent professional in research My special thanks to Dr Guillaume for advising me on various aspects of my thesis and explaining me about phylogeny studies I would like to thank Stuart, Dr Foo Chun Shin and Ritu Chandna for initially teaching me CBCM experiments I extend my gratitude to Dr Ryan McCleary for being supportive and boosting confidence in me at difficult times I owe a great deal of gratitude to Ritu Chandna, who was not only an awesome friend but an invaluable senior for me She was my moral support, my escape destination for all the research related road-blocks and a jovial friend in and outside the lab throughout my Ph.D I thank you Ritu Chandna, for always being there, which meant a lot to me iii I extend my gratitude to all the members of Structural Biology Labs I-V who have helped me in my lab work both by lending chemicals whenever I used to run out of them and by co-operating with space in the common facilities, especially in the cold room I am grateful to NUS graduate research scholarship program, for the financial support during my Ph.D I sincerely thank Bee Ling and Xu Liyuan, who were always a mail away for all the purchases and management in the lab Thank you for your support and in time responses I would like to thank Mrs Reena, Mrs Priscilla, Mrs Chan and all the staff of Department of Biological Sciences, who have helped me with various aspects of my stay at NUS Thank you all for your patience and support Most importantly, none of this could have happened without my family My Dad (H K Puttaswamy Gowda), who is more a friend to me and has always, offered his encouragement and blessings My Mom (M.K Tulsi Devi), who taught me as much as she could in 12 years of our togetherness, which has always guided and protected me, in her absence My heartfelt thanks to my brother Panish H P, dearest friend Kouser and all Abhivyakthians, my well-wishers Renuka aunty, Sadhana and Sharath - every time I had doubts, you guys were always there encouraging me beyond my doubts I am forever grateful for that I am also thankful to my parents-in-law, Mr Ramaiah V and Mrs Yashoda Ramaiah, my sister-in-law, Indumukhi and the little one of the family, Arna, for their constant love and encouragement I sincerely thank my brother-in-law, Dr C.M Chandrashekar, Assistant Professor, Homi Bhaba National Institute, India, for helping me proof-read my thesis I am extremely iv thankful to my husband, Dr Pramodh Ramaiah, Physician, Transocean Oil and Gas Corporation; for his support, patience and active participation in scientific discussions His witty suggestions on various experiments have helped me in achieving time-effectiveness with less labour This thesis stands as a testament to my family’s unconditional love and encouragement Finally, I am using this opportunity to express my gratitude to everyone who supported me throughout the course of Ph.D Varuna H P January, 2015 v Table of contents DECLARATION i Acknowledgements ii Table of contents vi Summary xiii RESEARCH COLLABORATIONS xvi ACADEMIC CONFERENCES AND PRESENTATIONS………….xviii List of Tables xviii List of Figures xix Abbreviations xxii CHAPTER 1: INTRODUCTION 1.1 Venom research: A paradigm shift 1.2 Snakes: Venomous and non-venomous slithering serpents 1.3 Snake venom: Deadly cocktail of multifaceted components 1.3.1 Enzymatic snake venom proteins 1.3.2 Non-enzymatic proteins 1.4 Three-finger toxin family (3FTx) 10 vi 1.5 Three-finger neurotoxins 15 1.5.1 Curaremimetic toxins (α-neurotoxins) 16 1.5.2 κ-neurotoxins and other dimeric three-finger neurotoxins; 19 1.6 α-Neurotoxins: Delineation of molecular determinants 20 1.7 Nicotinic acetylcholine receptors (nAChRs) 29 1.7.1 Muscle type nAChRs 35 1.7.2 Neuronal nicotinic acetylcholine receptors 35 1.7.3 Significance of nAChRs 37 1.8 Ligands and ligand binding site of nAChRs 38 Agonists and agonist binding site: 38 Antagonists and their binding sites: 41 1.9 Antiquity of nAChRs: defined by predator-prey relationships 43 1.10 Rationale and Aims 46 1.10.1 Rationale: in the scope of nAChRs 46 1.10.2 Rationale: in the scope of snake venom research 47 1.10.3 Aims: specific aims 48 vii CHAPTER 2: CHARACTERIZATION OF STRUCTURE , FUNCTION OF Oh9-1 52 2.1 Introduction 53 2.2 Materials and Methods 55 Cloning of Oh9-1 gene into pET-22b vector 57 Transformation of clone and expression host strains 58 Plasmid DNA isolation 58 DNA sequencing and analysis 59 Protein expression 60 Preparation and solubilisation of inclusion bodies 61 Preparing samples for PAGE 62 Sodium dodecyl sulphate – polyacrylamide gel electrophoresis 63 Reduction of solubilised inclusion bodies 64 Protein purification on RP-HPLC 64 Molecular mass determination 64 Refolding of protein 65 Gel filtration chromatography 66 Circular dichroism (CD) spectroscopy 67 viii Ex vivo Organ bath 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thesis describes characterization of a novel class of neurotoxins from snake venoms, here I present a brief introduction to venomous snakes, snake venom composition,... Plenty Road, Bundoora, VIC 3083, Australia 2 Crystallographic studies of Oh9-1 Associate Professor Jayaraman Sivaraman and Dr Jobichen Chacko Structural Biology Laboratory 5, S3-04 Department of Biological Sciences National University of Singapore, Singapore 3 Ex-vivo Organ bath experiments of Oh9-1 Professor Wong Tsun Hon, Peter, Department of Pharmacology Yong Loo Lin School of Medicine National University... helpful in the isolation of nicotinic acetylcholine receptors (nAChRs) and understanding of their role in neurotransmission Since then, a number of new ligands for 4 nAChRs and other receptors have been isolated from snake venom and characterized The properties of these toxin ligands can be used to design various molecular tools to define the roles of various subtypes of receptors involved and as therapeutic... of venomous snakes Table 1.2 Classification of α -neurotoxins Table 1.3 Intermolecular interactions of α-cobratoxin Chapter 2 Table 2.1 Comparison of characteristics of Oh9-1 with α -neurotoxins Chapter 3 Table 3.1 Reduced and refolded mass of alanine mutants of Oh9-1 Table 3.2 Effect of alanine mutations in Oh9-1 on inhibition of nAChRs xviii List of Figures Chapter 1 Figure 1.1 Common enzymes found... to nAChRs, which act as competitive antagonists of nAChRs These neurotoxins may therefore be invaluable tools in studying nAChR physiology The structure-function relationships of the classical neurotoxins, named neurotoxins, which belong to three-finger toxin (3FTx) family of snake venom neurotoxins are competitive antagonists with nano-molar affinity and high selectivity towards nAChRs In addition,... be agonists or antagonists xix Chapter 2 Figure 2.0 Comparison between the amino acid sequences of Oh9-1 and other three-finger toxins from snake venom Figure 2.1 Schematic showing different steps of ex vivo assay Figure 2.2 Determination of the mode of neuromuscular blockade in the avian NMJ Figure 2.3 Schematic representations of TEVC experiments of Xenopus oocytes Figure 2.4 Comparison of unoptimized... unoptimized and optimized sequences encoding Oh9-1 Figure 2.5 Schematic representation of expression construct of Oh9-1 Figure 2.6 Recombinant expression and purification of Oh9-1 Figure 2.7 Pharmacological profile of Oh9-1 on CBCM Figure 2.8 Inhibition of nAChR subtypes expressed in Xenopus oocytes by Oh9-1 Figure 2.9 Oh9-1 is a competitive antagonist Figure 2.10 Interaction of Oh9-1 with acetylcholine binding... of Biological Sciences, National University of Singapore, Singapore 7 Snake venoms are rich in enzymes There are about 20 enzyme families detected in snake venoms and 12 are known to be found in most of the snake venoms (Table 1.2) (R.C Hider et al 1992) Some of the common snake venom enzymes are metalloproteinases, hyaluronidase, phospholipase A2 (PLA2), nucleotidases (5΄-nucleotidase, ATPase, phosphodiesterase... 145 Bibliography 160 xii Summary Various subtypes of nicotinic acetylcholine receptors (nAChRs) mediate ionotropic actions of acetylcholine in the central and peripheral nervous system Due to the lack of highly selective nicotinic ligands, the precise location, functional roles and various disorders associated with nAChRs remain unclear Neurotoxins isolated from snake venom have a natural... acetylcholine binding protein and its mutants Figure 2.11 Selectivity profile of Oh9-1 on other Cys-loop receptors Figure 2.12 Refolded Oh9-1 is a monomer at physiological pH Figure 2.13 Crystals of Oh9-1 obtained in Index HR2-144 condition xx Chapter 3 Figure 3.0 Showing the mutations to alanine on Oh9-1 protein sequence Figure 3.1 Production of alanine mutants of Oh9-1 Figure 3.2 Inhibition of nAChR subtypes ... characterization of a novel class of neurotoxins from snake venoms, here I present a brief introduction to venomous snakes, snake venom composition, molecular targets of snake venom components... structure of nicotinic acetylcholine receptors (nAChRs) Figure 1.10 Prominent subtypes of nicotinic acetylcholine receptors Figure 1.11 Structure of the acetylcholine (ACh)-binding site on theα7 nicotinic. .. of nicotinic acetylcholine receptors (nAChRs) and understanding of their role in neurotransmission Since then, a number of new ligands for nAChRs and other receptors have been isolated from snake