INSECT• AND VERTEBRATE•
SELECTIVE NEUROTOXINS FROM
AUSTRALIAN URO DACID AND
BUTHID SCORPION VENOMS:
LEAD COMPOUNDS FOR NOVEL
BIOPESTICIDES
Harry Wilson BSc, MSc
Neurotoxin Research Group
Department of Health Sciences
University of Technology, Sydney
2003
submitted in fulfilment of the degree requirements for Doctor of Philosophy (Science)
CERTIFICATE OF AUTHORSHIP
AND ORIGINALITY
I certify that the work in this thesis has not previously been submitted for a degree nor has it been submitted as part of the requirements for a degree except as fully acknowledged in the text.
I also certify that the thesis has been written and prepared by me. Any help that I have received in my research work and the preparation of the thesis itself has been acknowledged. In addition, I certify that all sources of information and literature used are indicated in the thesis.
~/ U_~ (signed) (date)
INSECT- AND VERTEBRATE-SELECTIVE NEUROTOXINS FROM AUSTRALIAN URODACID AND BUTHID SCORPION VENOMS: LEAD COMPOUNDS FOR NOVEL BIOPESTICIDES
Harry Wilson, Neurotoxin Research Group, Department of Health Sciences, University of Technology, Sydney
PhD Thesis, 2003
ABSTRACT
Scorpions from n1ne species were collected from locations across
Australia comprising four Lychas (Buthidae), four Urodacus (Urodacidae) and
one Cercophonius (Bothriuridae) . Three species had not been described
previously. Lychas and Urodacus species collected in sufficient numbers were
milked regularly for their venom, and characteristics of the venom determined.
Insect toxicity was qualitatively demonstrated for two Lychas species.
Insect and vertebrate toxicity was determined for Urodacus manicatus and U. hoplurus, and both species were shown to have very low mam1nalian toxicity
(>4nl.g/kg). Insect toxicity of these two venon1.s vvas estimated at 300-SOO~lg/ g
by injection into crickets (Acheta dOJnesticus). Veno1n profiles prepared by rp-
HPLC revealed that Lychas venom was simple in composition and resembles
that seen in overseas buthids. Venom composition of the Urodacus species was
extremely complex, complicating attempts to purify individual proteins.
Toxins were purified from the venom by rp-HPLC or size-exclusion
chromatography (SEC) followed by HPLC, and screened for activity in
vertebrate (chick biventer cervicis) and insect bioassays. Fractions from both
species showed selective excitatory toxicity towards insects and I or vertebrates.
Effects on vertebrate muscle included contracture and increased stimulated
twitch tension, and some fractions produced fasciculation. Further
investigation was undertaken to determine the effects on ion channels in
insects, as part of a search for potential biopesticide compounds.
In neurones isolated fron1 the American cockroach terminal abdominal
ganglion, the predominant effect was a block of whole-cell sodium current.
ACKNOWLEDGEMENTS
I would like to thank the many people who have assisted me in the preparation of this thesis. Firstly, I would like to acknowledge the role played by my supervisor A. Prof. Graham Nicholson, of the Department of Health Sciences, UTS. Without his guidance this would never have been finished. Secondly, I would like to acknowledge the assistance with theory, technique and equipment of my co-supervisors, A. Prof. Kevin Broady of the Department of Cell and Molecular Biology, UTS and Prof. Glenn King, UConn Health Centre, Farmington, CN, USA.
For direct assistance in several areas: Provision of Urodacus hoplurus scorpions: Peter Mirtschin, Venom
Supplies P.L., Tanunda, South Australia. Identification of Australian Scorpions: Erich Volschenk, Dept. of
Terrestrial Invertebrates, Western Australian Museum. QTOF Mass spectrotnetry: Dr. Peter Hains, Dept. Chemistry,
W ollongong, NSW. Initial assistance in ESI-MS: Mr. Matt Padula, Dept. Cell and
Molecular Biology, UTS. Sequencing of peptides: Dr Bernie Mcinerny, Australian Proteome
Analysis Facility, North Ryde, NSW.
For their support in less tangible ways, my fellow lab workers, of whom there have been too many to list fully, but particularly Simon Gunning, Andis Graudins, Y oumie Chong, Phil Lawrence and Qiao Yang. Thanks for all your help and kind words.
TABLE OF CONTENTS
CHAPTER ONE: INTRODUCTION 1.1 INSECTICIDES OF THE FUTURE 1.1.1 The Problem Facing Modern Agriculture 1.1.2 Current Methods of Agricultural Pest Control 1.1.3 Alternative Strategies for Agricultural Pest Insect Control 1.1.4 The Recombinant Baculovirus Approach 1.1.5 The Baculovirus System for Agricultural Insect Control 1.2 SCORPION BIOLOGY 1.2.1 Evolution of the Scorpions 1.2 .2 Scorpion Anatomy 1.2 .3 Scorpion Behaviour 1.2.4 Australian Scorpions 1.2.5 Australian Buthidae 1.2.6 The Urodacidae 1.3 TOXINS FROM ANIMAL VENOMS 1.3.1 Animal Venoms: Nature's Combinatorial Libraries 1.3.2 Toxin Applications as Therapeutic Agents 1.3.3 Toxin Applications as Pharmacological Tools 1.4 SCORPION VENOM 1.4.1 Components of Scorpion Venoms 1.4.2 Scorpion Venoms of Medical Importance 1.4.3 Scorpion Neurotoxins 1.5 NEUROTOXIN TARGETS: ION CI-fANNELS 1.5.1 Ion Channels: An Overview 1.5.2 Sodium Channel Structure and Subtypes 1.5 .3 Sodium Channel Function 1.5.4 Sodium Channel Toxins 1.5.5 The Scorpion a-Toxins 1.5.6 The Scorpion fl- Toxins 1.5.7 Potassium Channel Structure and Function 1.5.8 Potassium Channel Toxins 1.6 OBJECTIVES AND OUTLINE OF THIS THESIS
CI-IAPTER TWO: MATERIALS AND METHODS 2.1 SCORPION COLLECTION, HOUSING AND MILKING 2.1.1 Introduction 2.1.2 Collection of Scorpions 2.1.2.1 Buthidae 2.1.2.2 Urodacidae 2.1.2.3 Bothriuridae and Liochelidae 2.1.3 Scorpion Identification 2.1.4 Scorpion Housing and Maintenance 2 .1 .5 Venom Collection 2.2 CI-IROMA TOGRAPI-IY 2.2.1 Reverse-Phase HPLC 2.2.2 Shallow Gradient rp-HPLC 2.2.3 Size-Exclusion Chromatography 2.3. SCREENING FRACTIONS FOR ACTIVITY 2.3.1 Vertebrate Toxicity in vivo
1 1 2 4 5 6 8 8 12 13 19 19 21 23 23 24 26 27 27 29 30 38 38 39 45 45 52 53 54 58 60
61 62 62 62 62 65 67 68 69 71 73 73 74 75 76 76
2.3.2 Vertebrate Toxicity in vitro 77 2.3.3 Insect Toxicity in vivo 79 2.3.4 Calculation of KD 50, PD 50 and LD 50 Values 83 2.4 ELECTROPHYSIOLOGY OF VENOMS AND ISOLA TED TOXINS 84 2.4.1 Preparation of Isolated Cockroach TAG Neurones 85 2.4.2 Preparation of Isolated Rat DRG Neurones 89 2.4.3 Preparation of Electrodes 92 2.4.4 Patch-clamp Recording Set-Up 93 2.4.5 Solutions Used for Current Recording 95 2.4.6 Voltage-Clamp Stimulation Protocols 96 2.4.6.1 INa Protocol 97 2.4.6.2 IV Protocol 97 2.4.6.3 Steady-state inactivation protocol (hoo) 98 2.4.6.4 R/S Protocol- determination of predominant Na+ channel subtype in DRG neurones 99 2.4.7 Current Clamp protocol 100 2.4.8 Electrophysiology Data Analysis 100 2.5 BIOCHEMICAL TECHNIQUES 101 2.5.1 Determination of Molecular Mass by MS 101 2.5.2 Estimation of Protein Content 102 2.5.3 Determination of Protein Primary Sequence 103 2.5.3.1 Pyridylethylation 103 2.5.3.2 N-Terminal Amino Acid Sequencing 104 2.6 SOURCES OF CHEMICALS AND TOXINS 104 2.6.1 Scorpion Toxins 104 2.6.2 Spider Toxins 104 2.6.3 Poly-lysine hydrobromide 105 2.6.4 D-MEM (Dulbecco's Modified Eagles Medium) 105 2.6.5 CMF-PBS (Phosphate Buffered Saline, Ca2+ and Mg2+ free ) 105 2.6.6 Newborn Calf Serum (NCS) 106 2.6.7 lvfinor Chemicals and Solvents 106
CHAPTER THREE: RESULTS AUSTRALIAN SCORPION SPECIES AND CHARACTERISTICS OF THEIR VENOM 107 3.1 Collection of Australian Scorpions 107 3.2 Characteristics of the venom 113 3.3 Venom Yield Variation OfCaptive Urodacus Scorpions 117 3.4 HPLC Profiles of Australian Scorpion venom 120 3.5 Important considerations in venom collection 123 3.6 Conclusion 127
CHAPTER FOUR: RESULTS TOXICITY OF THE VENOM OF AUSTRALIAN SCORPIONS 130 4.1 Lychas Venom Toxicity Studies in Insects 130 4.2 Urodacus Venom Toxicity in Insects in vivo 131 4.3 Urodacus Whole Venom Toxicity in Vertebrates 137 4.3.1 Urodacus manicatus Whole Venom Toxicity in vivo 137 4.3 .2 Urodacus hoplurus Whole Venom toxicity in vitro 138 4.4 Conclusion 140
CHAPTER FIVE: RESULTS IDENTIFICATION OF SELECTIVELY TOXIC FRACTIONS IN URODACUS VENOM 140 5.1 Introduction 140 5.2 Identification of Selective Toxins in U. manicatus venom 141 5.2.1 Fractionation of venom by rp-HPLC. 141 5.2.2 Insect-active Toxins in U. manicatus venom 144 5.2.3 Vertebrate-active Toxins in U. manicatus venom 147 5.2.4 Selection of Specific Toxins from U. manicatus venom 148 5.3 SELECTIVE TOXINS IN URODACUS HOPLURUS VENOM 151 5.3.1 Separation of U. hoplurus Venom Fractions 151 5.3.1.1 Size-exclusion chromatography of U. hoplurus venom 151 5.3.1.2 Size-Exclusion Chromatography of U. hoplurus venom: effect of solvent 152 5.3.1.3 Characteristics of the Size Exclusion Chromatogram 155 5.3.1.4 Size Exclusion Chromatogram of U. manicatus Venom 155 5.3.2 Insect Toxicity of U. hoplurus venom Fractions 155 5.3.3 Vertebrate Toxicity of U. hoplurus venom Fractions 158 5.3 .4 Further fractionation of U. hoplurus venom 162 5.3.5 Screening for Selective Toxins in U. hoplurus venom 166 5.3.6 Selection of Specific Toxins from U. hoplurus venom 168 5.4 Further characterisation of selected Urodacus hoplurus fractions 170 5.5 Conclusions 173
CHAPTER SIX: RESULTS PURIFICATION OF TOXINS FROM URODACUS VENOM 173 6.1 Introduction 173 6.2 Purification of Insect and Vertebrate-Selective Toxins from Urodacus manicatus Venom 174 6.2.1 Vertebrate-selective toxins in Urodacus manicatus ven01n 174 6.2.2 Insect-selective fractions in U. manicatus venont: Purification by rp-HPLC 177 6.2.3 Insect-selective fractions in U. manicatus venom: Alternate Purification by SEC
180 6.3 Purification of insect and Vertebrate-Selective Toxins from Urodacus hoplurus Venom 183 6.3 .1 Vertebrate-selective Toxins in U. hoplurus venom 183 6.3 .2 Insect-selective Toxins in U. hoplurus venom 186 6.4 Determination of Toxin Primary Structure 194 6.4.1 Fraction UhF2.9 .1 194 6.4.2 Fraction UhF2 .10.1 199 6.5 Conclusions 200
CHAPTERSEVEN: RESULTS ELECTROPHYSIOLOGY OF URODACUS TOXINS ON INSECT NEURONES 200 7.1 electrophysiological investigation of the actions of whole venom and major fractions 200 7.1.1 Whole-cell currents in isolated TAG neurones 200 7.1.2 Initial determination of electrophysiological action 206 7.2 Electrophysiology of Purified Urodacus Toxins 209 7.2.1 Screening of Urodacus Toxins for activity 209 7.2 .2 Toxins with Low activity in TAG neurones 210 7.2.3 Toxins with High activity in TAG neurones 212
7.3 Further effects of Urodacus toxins on sodium current in TAG neurones. 215 7.3 .1 Effects on voltage-dependence of activation of sodium channels. 215 7.3 .2 Urodacus toxin effects on sodium channel steady-state inactivation. 218 7.4 Summary of Urodacitoxin Actions 218
CHAPTER EIGHT: RESULTS CHARACTERISATION OF BUTHIDAE SODIUM CHANNEL TOXINS 220 8.1: SODIUM CHANNEL TOXINS FROM A BUTHID SCORPION: BACKGROUND 220 8.2: Actions of LQHII and LQHIII on mammalian neurones 222 8.2.1: Lqhiii Effects on action potentials in rat DRG neurones 223 8.2 .2: Effects on tetrodotoxin-sensitive sodium channels in DRG neurones 226 8.2.3 Lqhii and Lqhiii Effects on the Voltage-Current Relationships ofTTX-S Sodium Channe~ 229 8.2.4 Lqhiii Effects on Steady-State Inactivation of TTX-S Sodium Channels 231 8.2 .5: Effects on tetrodotoxin-resistant sodium channels 232 8.3 Actions of Lqhiii on insect neurones 234 8.4 CONCLUSIONS 236
CHAPTER NINE: DISCUSSION 237 9.1 CHARACTERISTICS OF THE VENOM OF AUSTRALIAN SCORPIONS
237 9.1.1 New Scorpion Species 237 9.1.2 Venom Toxicity and Scorpion Biology 238 9.1.3 Venom Complexity and Profiles 241 9.1.4 Problerns Encountered During Fraction Purification 243 9.2 URODACUS VENOM AND TOXINS 245 9.2.1 Insect Actions of Urodacus Venom and Toxins 245 9.2 .2 Vertebrate Actions of Urodacus Venom and Toxins 248 9.2.2 Electrophysiology of Urodacus Venom and Toxins in TAG Neurones 251 9.2.3 Primary Structure of the Urodacitoxin UD.IT-Uh1a 252 9.2.4 Structural homology of UDIT-Uhla with other scorpion toxins 254 9.2.5 Implications for Sodium Channel Pharmacology 256 9.3 ACTIONS OF LEIURUS ALPHA-TOXINS 257 9.3.1 Differential Actions of Lqhii and Lqhiii on Sodium Channel subtypes in the Rat
9.3.2 Lqhiil has Scorpion alpha-Toxin Activity in the Cockroach TAG 9.4 Conclusions and Future Directions
APPENDIX: CITED REFEREN CES
257 258 259
262
LIST OF TABLES A N D FIGURES
Chapter One: Introduction Figure 1.1: Evolutionary relationships and classification of arachnids. 9 Figure 1.2: Scorpion anatomy. 10 Figure 1.3: Bothriuridae and their Australian distribution. 14 Figure 1.4: Liochelidae and their Australian distribution. 15 Figure 1.5: Buthidae and their Australian distribution. 16 Figure 1.6: Urodacidae and their Australian distribution. 17 Figure 1.7: Sodium channel (VGSC) structure and function. 40 Figure 1.8: Identified VGSC Neurotoxin receptor sites. 44 Figure 1.9: Toxins that target the VGSC. 46 Figure 1.10: Toxins that target the VGKC. 47 Figure 1.11: Potassium channel (VGKC) structure and function. 53
Table 1.1: Toxicity of medically-important scorpion venoms. 29 Table 1.2 Identified Scorpion Toxins. 32 Table 1.3: Identified Mammalian Sodium Channel Subtypes. 39 Table 1.4 Identified VGSC Neurotoxin receptor sites. 44
Chapter Two: Materials and Methods 60
Figure 2.1 Scorpion collection areas. Figure 2.2 Collection of venom. Figure 2.3 Insect toxicity in vivo. Figure 2.4 Preparation of Cockroach TAG neurones.
Table 2.1: Categories and signs of toxicity in mice Table 2.2: Categories and signs of toxicity in crickets Table 2.3: Categories and signs of toxicity in blowfly larvae Table 2.4: Trypsin incubation times for rat DRG
Equation 2.1 Equation 2.2 Equation 2.3
62 70 79 86
75 78 81 90
83 97 98
Chapter Three: Australian Scorpions and Characteristics of their Venom 107
Figure 3.1: Lychas Species Collected 110 Figure 3.2:Urodacus Species Collected 111 Figure 3.3: Features of Urodacus Species, Cercophonius squama 113 Figure 3.4: Variation in Venom Yield of Captive Urodacus Scorpions 117 Figure 3.5: Comparative Lychas Whole Venom profiles 120 Figure 3.6: Comparative Urodacus Whole Venom Profiles 121 Figure 3.7: Sex Differences in U. hoplurus Venom HPLC Profiles 124 Figure 3.8: Individual U. hoplurus compared to Pooled Venom 125 Figure 3.9: Batch Variation in Lirodacus manicatus Whole Venom 127 Figure 3.10: Venom Variation Between Groups of U. hoplurus 128
Table 3.1: Australian Scorpion species collected and identified 108 Table 3.2: Characteristics of Venom from Australian Scorpions 115
Chapter Four: Toxicity of the Venom of Australian Scorpions 130
Figure 4.1: Urodacus manicatus whole venom insect toxicity. 132 Figure 4.2: Urodacus hoplurus whole venom insect toxicity. 133 Figure 4.3: Timecourse of toxicity of U. hoplurus venom on crickets. 135 Figure 4.4: Effects of U. hoplurus venom on chick muscle in vitro. 138
Table 4.1: Insect Toxicity of Australian Scorpion Venoms 131
Chapter Five: Selectively Toxic Fractions in Urodacus venom 140 Figure 5.1 Representative rp-HPLC of ll. manicatus venom 142 Figure 5.2: Toxicity profile of U. manicatus venom fractions 149 Figure 5.3: Effect of Solvent pH on Protein Elution 152 Figure 5.4 Size Exclusion Chromatograms of Urodacus Venoms 153 Figure 5.5: Vertebrate activity of U. hoplurus venom fractions 159 Figure 5.6: Detail of U. hoplurus effects on vertebrate muscle 160 Figure 5.7: rp-HPLC of U. hoplurus venom fractions 1 and 3 163 Figure 5.8: rp-HPLC of U. hoplurus venom fraction 2 164 Figure 5.9 Effects of fractions of U. hoplurus venom on chick muscle 168 Figure 5.10: Details of effects of active U. hoplurus fractions . 170
Table 5.1: Insect and vertebrate activity U. manicatus fractions 144 Table 5.2 Effects of U. hoplurus size-exclusion fractions on insects. 156 Table 5.3: Vertebrate activity of U. hoplurus venom fractions . 158
Chapter Six: Purification of Toxins from Urodacus Venom 173
Figure 6.1: Vertebrate-active UmF29.4 from U. manicatus venom. 175 Figure 6.2: Multiple components of insect-active fractions. 178 Figure 6.3: Size-exclusion chromatography of U. manicatus fractions. 181 Figure 6.4: Components of UhF2.0. 184 Figure 6.5: Components and Purification of UhF2.3 and UhF2.4 187 Figure 6.6: Purification of UhF2.8 and 2.9 from U. hoplurus. 189 Figure 6.7: Purification of Multiple Components of UhF2.10 and 2.12 192 Figure 6.8: Comparison of UhF2.9.1 primary sequence 196 Figure 6.9: Secondary structure of UDIT-Uh1a and buthid toxins. 197
Table 6.1: Characterisation of purified Urodacus toxins. 200
Chapter Seven: Electrophysiology of Urodacus Toxins on Insect Neurones
Figure 7.1: Characterisation of TAG neurones. 202 Figure 7.2: Effects of Urodacus hoplurus whole venom and fractions. 206 Figure 7.3: U. hoplurus toxins with low activity in TAG neurones. 210
Figure 7.4: Urodacus toxins with high activity on TAG neurones. 212 Figure 7.5: Concentration dependence of Urodacus toxin effects. 213 Figure 7.6: Urodacus toxin effects on voltage-dependence of activation of
TAG neurone sodium channels. 216 Figure 7.7: Effects of Urodacus toxins on steady-state inactivation of TAG
neurone sodium channels. 218
Table 7.1: Activity of Purified Urodacus Fractions in TAG neurones 215 Table 7.2: Effects of purified Urodacus fraction on current-voltage
relationships of whole-cell sodium current in TAG neurones. 219
Chapter Eight: Characterisation of Buthidae Sodium Channel Toxins 220
Figure 8.1: Effect of Lqhiii on action potentials in rat DRG neurones. 224 Figure 8.2: Dose-dependent effects of Lqhll and Lqhiii on TTX-S sodium
currents in DRG neurones. 227 Figure 8.3: Typical effects of Lqhiii on current-voltage relationships of
TTX-S currents in DRG neurones. 229 Figure 8.4: Lqhii and Lqhiii have no effect on TTX-R sodium currents in
the rat DRG. 232 Figure 8.5: Effect of Lqhiii on isolated cockroach TAG neurones. 234
Table 8.1: Summary of Lqhii and Lqhiii effects on DRG TTX-S sodium currents. 230