A novel protein isolated from the venom of ophiophagus hannah (king cobra) showing beta blocker activity

β-CARDIOTOXIN: A NOVEL PROTEIN ISOLATED FROM THE VENOM OF OPHIOPHAGUS HANNAH KING COBRA SHOWING BETA-BLOCKER ACTIVITY NANDHAKISHORE RAJAGOPALAN M.. LIST OF FIGURES PAGE Chapter One Fig

β-CARDIOTOXIN: A NOVEL PROTEIN ISOLATED FROM THE VENOM OF OPHIOPHAGUS HANNAH (KING COBRA)

SHOWING BETA-BLOCKER ACTIVITY

NANDHAKISHORE RAJAGOPALAN

(M Sc (Life Sciences))

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY AT THE NATIONAL UNIVERSITY OF SINGAPORE

DEPARTMENT OF BIOLOGICAL SCIENCES, FACULTY

OF SCIENCE, NATIONAL UNIVERSITY OF SINGAPORE

JANUARY, 2008

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tÇw àxtv{xÜá

ii

iii

he has given me in thinking and designing my work This has molded me as an independent researcher over the past few years

Next, I would like to thank my co-supervisor Associate Professor Prakash Kumar

He has been a pillar of support throughout my time in Singapore His useful suggestions during the manuscript preparation are not only reflected in the published article but will also influence the way I write in future

I would like to thank the graduate program run by the National University of Singapore for their financial support I would also like to thank the Biomedical Research Council (BMRC) for their grant which funded the research

All this work would not have been possible without the support of our able collaborators I would like to thank Associate Professor Peter Wong for helping

me out whenever I had some experiments to be done at the Department of Pharmacology I would also like to thank Dr Zhu Yi Zhun for opening the doors

of his lab for my work I would like to thank the staff and students from the Department of Pharmacology for their technical support Thanks to Assistant Professor Bian Jinsong, Dr Wong Zong Jing, Mrs Ting Wee Lee, Ms Pei Ling,

Ms Kay Lee and Ms Ning Li I would also like to thank Assistant Professor Jayaraman Shivaraman and Ms Sunita for their kind support for the structural studies

I would like to thank all the teachers who made a difference in my life I would like to thank Mrs Radha, Mrs Meera, Mrs Vijayalakshmi, Mrs Saraswathi

iv

Chandrasekaran, Dr Akbar Sha, Dr M Krishnan, Dr A S Rao and Dr Chellam Balasundaram

I would like to express my gratitude to my seniors Dr Selvanayagam Nirthanan (Niru) and Dr Rajamani Lakshminarayanan (Lakshmi) for their valuable suggestions and constant motivation

I would like to thank all my lab mates for making my stay fun and entertaining Thanks to Dr Pung Yuh Fen, for guidance during my stint as a trainee, Dr Yajnavalka Banerjee, for providing great support and guidance, Dr Md Abu Reza,

Dr Dileep Gangadharan, Dr Syed Rehana, Dr Joanna Pawlak, Dr Kang Tse Siang, Rocky, Shifali and Dr Raghurama Hegde for all the help they have done I would like to thank Cho Yeow and Shi Yang for organizing great lab parties and

Dr Robin Doley for managing the complicated finances for these parties I would like to thank Amrita and Girish for keeping me highly entertained during the writing of the thesis I would also like to thank the “Kids Army” (a.k.a the undergrads) members Sin Min, Ee Xuan, Ming Zhi, Bee Har and Maulana for spreading their infectious enthusiasm Last but not the least I would like to thank

Ms Tay Bee Ling for maintaining the lab finances and making sure we get things

on time

I am grateful to my family for their support Thanks to my father Mr Rajagopalan and my mother Mrs Lalitha Rajagopalan, my brothers Mr Prasanna and Mr Vasanth and my cousins Ms Aarthi Ravichandran and Ms Madhulika Ravichandran

Nandhakishore Rajagopalan

January, 2008

1.3.10 3FTXs from Viperidae snakes 36

1.3.13 The three-finger fold: ‘leprechauns 1’ of molecular

CHAPTER TWO: IDENTIFICATION AND ISOLATION OF

2.2.4 Construction of cDNA library using 5’-RACE ready

2.2.9 Verification of clones using restriction digestion 74

2.2.14 Molecular mass determination 75

2.3.3 Isolation and characterization of novel proteins 83

viii

3.3.2 Anticoagulant activity 107

CHAPTER FOUR: CHARACTERIZATION OF A MOLTEN

4.2.2 Thermal denaturation studies using CD spectroscopy 124

4.2.3 Chemical denaturation studies using CD spectroscopy 125

4.2.5 Combined effects of pH and temperature on

4.3.4 Combined effects of pH and temperature on

β-CARDIOTOXIN 143

6.2.2 Site-directed mutagenesis studies to determine the

6.2.3 Characterization of the α-helical ‘molten globule’

6.2.6 Characterization of other novel proteins identified

x

Bibliography 168

SUMMARY

Snake venoms have provided a number of novel ligands with therapeutic potential These proteins have been classified into a small number of super-families that may be enzymatic or non-enzymatic In the past, the most abundant or the most active proteins from the snake venoms have been well characterized In recent times many new and less abundant molecules from snake venoms have been isolated and characterized by employing more sophisticated techniques from proteomics and genomics

We have constructed a cDNA library from the venom gland mRNA of

Ophiophagus hannah (king cobra) and identified five novel proteins From the

Cys numbers and pattern we concluded that all these new proteins belong to the three-finger toxin family of snake venom proteins We have isolated one of these proteins with a mass of 7012.43 ± 0.91 Da from the venom using a two-step chromatography approach with size-exclusion chromatography as the first step and reverse phase high performance liquid chromatography as the second one

This protein was non-lethal in mice up to an intra-peritoneal dose of 10 mg/kg It

is also non-hemolytic and does not show anti-coagulant activity It caused a decrease of heart rate in anesthetized rats as well as in rat isolated perfused heart,

in contrast to conventional cardiotoxins that increase the heart rate Radioligand displacement studies showed that this protein targets β-adrenergic receptors with a

Ki of 5.3 and 2.3 µM toward human β1 and β2 subtypes, respectively, and hence

xii

we named the protein as β-cardiotoxin This is the first report of an exogenous protein beta-blocker

We have identified a ‘molten globule’ intermediate in the thermal unfolding pathway and characterized it using CD spectroscopy β-Cardiotoxin undergoes a structural transition from β-sheet to α-helix at higher temperatures This α-helical intermediate does not occur in the chemical denaturation of the protein We have also identified and optimized a condition for obtaining diffraction quality crystals

of β-cardiotoxin

Finally, we have initiated structure-function relationship studies for identifying the functional site of the protein This information along with the 3-D structure of the protein will enable us to design novel bioactive peptides with therapeutic potential for treatment of cardiovascular diseases

RESEARCH COLLABORATIONS

The following laboratories provided invaluable technical assistance in performing some of the experiments discussed in this thesis Their contribution is gratefully acknowledged

• Associate Professor Peter Tsun Hon Wong and Mrs Ting Wee Lee Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore

• Associate Professor Zhu Yi Zhun, Dr Wang Zong Jing, Ms Ho Pei Ying and Ms Ning Li Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore

• Assistant Professor Jayaraman Shivaraman, Ms Sunita, Dr Sundaramurthy Kumar and Mr Jobichen Chako Structural Biology Laboratories, Department of Biological Sciences, National University of Singapore

xiv

ACKNOWLEDGEMENT OF COPYRIGHT

o Dr Robert A Barish MD, Vice dean for Clinical Affairs, University of Maryland School of Medicine, USA for permission to reproduce Figure 1.1 and Table 1.1 in Chapter 1

o Professor R Manjunatha Kini, Department of Biological Sciences, National University of Singapore for permission to reproduce Figure 1.4 in Chapter 1 and Figure A.5 in Appendix

o Professor Miyano Masashi, Chief Scientist and Head, Structural Biophysics Laboratory RIKEN SPring-8 Center, Japan for permission to reproduce Figure 1.14 in Chapter 1 and for kindly providing Figure 1.20 in Chapter 1

o Professor Brian Kobilka, Department of molecular and cellular physiology, Stanford University School of Medicine, USA for permission to reproduce Figure 1.15, 1.16, 1.21, 1.22 and 1.23 in Chapter 1 and Figure 5.3 in Chapter 5

o Professor Raymond C Stevens, Departments of Molecular Biology, Chemistry, The Scripps Research Institute, USA for permission to reproduce Figure 1.21, 1.22 and 1.23 in Chapter 1

o Dr Howard Rockman, MD, Professor of Medicine, Cell Biology and Molecular Genetics, Chief of Cardiology, Duke University Medical Center, USA for permission to reproduce Figure 1.17, 1.18 and 1.19 in Chapter 1

o Professor Stephen Sprang, Director, Center for Biomolecular Structure and Dynamics and Professor, Division of Biological Sciences, University of Montana, USA for permission to reproduce Figure 1.24 in Chapter 1

PHOTO COURTESY

¾ Mr Shiyang Kwong, National University of Singapore, Singapore for

kindly providing photo of Bitis gabonica (Gaboon viper) for Figure 1.2 in

Chapter 1

¾ Mr Duncan MacRae Phyto Medichem Singapore Pte Ltd for kindly

providing photo of Ophiophagus hannah (King cobra) for Figure 1.2 in

Chapter 1

¾ Ms Susan Scott for kindly providing photo of Pelamis platurus

(Yellow-bellied sea snake) for Figure 1.2 in Chapter 1

¾ Mr Peter Mirtschin, Venom Supplies Pte Ltd., Australia for kindly

providing photo of Ophiophagus hannah (King cobra) for Figure 1.3 in

Chapter 1

xvi

LIST OF FIGURES

PAGE

Chapter One

Figure 1.1 Venom gland and venom delivery apparatus 3

Figure 1.2 Venomous snakes from different families 6

Figure 1.3 Ophiophagus hannah (King cobra) 8

Figure 1.4 Structural similarities between ‘sibling’ three-finger toxins 15

Figure 1.10 Calciseptine and FS2 toxins 30

Figure 1.12 3FTXs from Colubridae venoms 34

Figure 1.13 Non-venom proteins with the three-finger fold 38

Figure 1.14 Two-dimensional model of bovine rhodopsin 46

Figure 1.15 Schematic structure of β2-AR 47 Figure 1.16 The secondary structure and location of agonist binding

Figure 1.17 Overview of GPCR signaling 51

Figure 1.18 Gs coupled receptor signaling 54

Figure 1.19 Gi coupled receptor signaling 55

Figure 1.20 Ribbon drawings of inactive bovine rhodopsin 57

Figure 1.21 Comparison of crystal structures of β2-AR-T4L fusion

protein and the complex between β2-AR365 and a Fab

conserved E(D)RY motif 62 Figure 1.24 Cartoon representation of the β2-AR structure 63

Chapter Two

Figure 2.1 Construction of cDNA library 79

Figure 2.2 Abundance of various genes from cDNA library 81

Figure 2.3 Multiple sequence alignment of novel proteins 82

Figure 2.4 Gel filtration of O hannah venom 84 Figure 2.5 Purification of β-cardiotoxin 84

Figure 2.7 Purification of WTX DE1 homolog 1 and MTLP-3

Figure 2.8 Comparison of secondary structure of β-cardiotoxin

Figure 2.9 Far-UV CD spectrum of WTX DE1 homolog 1 89

Figure 2.10 Far-UV CD spectrum of MTLP-3 homolog 89

Figure 2.11 Changes in residues between β- cardiotoxin and its

Chapter Three

Figure 3.1 Substitutions in the loop regions of β-cardiotoxin 99

Figure 3.2 Effect of β-cardiotoxin on blood coagulation 109

Figure 3.3 Hemolytic activities of β-cardiotoxin and CM18 109

Figure 3.4 Effects of β-cardiotoxin on cardiac function 110

Figure 3.5 Induction of bradycardia by β-cardiotoxin 111

Figure 3.6 Effects of β-cardiotoxin on Langendorff perfused hearts 113

Figure 3.7 Changes in heart rate after treatment with β-cardiotoxin 114 Figure 3.8 Interaction of β-cardiotoxin with β-ARs 116

Chapter Four

Figure 4.1 Effect of temperature on the secondary structure of

xviii

Figure 4.2 Effect of temperature on the secondary structure of

Figure 4.7 Combined effects of temperature and pH on the

secondary structure of β-cardiotoxin 135 Figure 4.8 Combined effects of temperature and pH on the

tertiary structure of β-cardiotoxin 136 Figure 4.9 Prediction of secondary structure of β-cardiotoxin 140

Chapter Five

Figure 5.5 Purification of synthetic peptides 154

Figure 5.6 ESI-MS of purified synthetic peptides 155

Figure 5.7 Interaction of synthetic peptides with β-ARs 156

LIST OF TABLES

PAGE

Chapter One

Table 1.1 Venomous snakes of the world 5

Table 1.2 Enzymatic proteins from snake venoms 11

Table 1.3 Non-enzymatic proteins from snake venoms 12

Table 1.4 Diversity of the three-finger fold 40-42

xx

ABBREVIATIONS

Single letter and three letter abbreviations of amino acid residues were followed

as per the recommendations of the IUPAC-IUBMB Joint Commission on Biochemical Nomenclature

Chemicals and reagents

MES [2-(N-morpholino) ethanesulfonic acid]

MOPS [3-(N-morpholino) propanesulfonic acid]

PEG-PS Polyethylene glycol-polystyrene

BLAST Basic local alignment search tool

CICR Ca2+ induced Ca2+ release

CRISP Cycteine-rich secretory protein

EBI European bioinformatics institute

ESI-MS Electrospray ionization-mass spectrometry

G Proteins GTP binding proteins

GPCR G protein-coupled receptor

i.t.v intra tail vein

IC50 Dose which causes 50 % inhibitory effect

LC/MS Liquid chromatography/mass spectrometry

LD50 Lethal dose causing death of 50 % of animals tested

mAChR Muscarinic acetylcholine receptor

MTLP Muscarinic toxin like protein

nAChR Nicotinic acetylcholine receptor

NCBI National center for biotechnology information

NMR Nuclear magnetic resonance

NOESY Nuclear Overhauser effect spectroscopy

PCR Polymerase chain reaction

PKA cAMP-dependant protein kinase

RACE Rapid amplification of cDNA ends

RP-HPLC Reverse phase-high performance chromatography

Chapter One

Introduction

Chapter One Introduction

CHAPTER ONE Introduction

Fossil evidence suggests that snakes evolved from lizards probably during the Cretaceous age (100 to 120 million years ago) (Harris, 1991) The earliest snake species might have employed the slow locomotion and active immobilization strategy (i.e., constriction) However, during the Miocene period (less than 30 million years ago) there was a rapid shift in prevailing environmental conditions, like development of more open habitats (savannahs) and radiation of rodents and other potential prey species In response to this, snakes evolved the strategy of rapid locomotion and passive immobilization (i.e., pursuit and envenomation) (Savitzky, 1980) The Duvernoy’s glands were the first venom producing apparatus that evolved during this period Subsequently, these glands were hypertrophied and became very specialized venom glands Advanced venom delivery apparatus like front fangs evolved in elapidae (cobras and kraits), hydrophiidae (sea snakes), viperidae (vipers) and crotalidae (rattle snakes); among which, the viperidae and crotalidae have the most sophisticated venom delivery apparatus with retractable fangs (Figure 1.1)

Presently, there are about 3200 species of snakes and approximately 1300 of them

are venomous (Hider et al., 1991) Of the 3200 snake species, only 15 percent are considered to be dangerous to humans (Gold et al., 2002) This is due to the fact

that the majority of venomous snakes (~ 1000 species) belong to the Colubridae

2

Figure 1.1 Venom gland and venom delivery apparatus The most

sophisticated venom delivery apparatus found in viperidae snakes Reproduced with permission from Dr Robert A Barish MD, Vice dean for Clinical Affairs,

University of Maryland School of Medicine, USA (Gold et al., 2002).

Chapter One Introduction

family (Table 1.1), which produce small volumes of venom and have poorly developed venom delivery apparatus

1.1.1 Classification of venomous snakes

The systematic classification of snakes presents many challenges and broadly 11

to 13 distinct families are known Five of these families are known to have venomous species of snakes (Table 1.1) (Harris, 1991) Colubridae is the largest family of snakes (Figure 1.2 A) They dominate most parts of the world except Australasia Most snakes in this family are harmless except a few like the African

Boomslang (Dispholidus typus) Viperidae (true vipers) are the most widespread

snakes found throughout Europe, Africa, Asia and Americas, but absent in Australia (Figure 1.2 B) They are heavier and bulkier compared to other families and have a distinct triangular head Their venom delivery apparatus is very advanced and they possess retractable fangs Crotalidae is sometimes considered

as a sub family of viperidae Crotalids are similar to true vipers, except for the presence of heat sensing pits between the nostril and the eye (Figure 1.2 C) This gives them the ability of thermal imaging of warm-blooded prey at night Elapidae

is a diverse family of venomous snakes found in Americas, Asia, Africa and Australia (Figure 1.2 D) This family includes cobras, kraits, mambas, coral snakes and a large diversity of Australian elapids They possess short, fixed fangs

in the front of the mouth Hydrophidae, consisting of sea snakes, are considered as

a sub family of elapidae by some experts (Figure 1.2 E) (Harris, 1991) They

4

Fangs similar to those of elapidae; highly neurotoxic venom; rarely bite humans

Indopacific region: Pelamis platurus (pelagic sea snake), Laticauda colubrina (colubrine sea krait or yellow-lipped sea krait), Laticauda semifasciata (Erabu black-banded sea krait)

Hydrophinae(true sea snakes)Hydrophidae

Short, fixed fangs; venom injected by succession of chewing movements

Tropical and warm temperate zones: Naja species (cobras), Dendroaspis species (mambas), Bungarus species (kraits), Micrurus, Calliophis, and Maticora species (coral snakes), and

most venomous snakes of AustraliaElapidae

Heat-sensing foramen “pit” between each eye and nostril; elliptical pupils; retractable, canalized front-fangs

North America: Crotalus and Sistrurus species (rattlesnake), Agkistrodon species (cottonmouth, copperhead)

Central and South America: Crotalus species (rattlesnake), Agkistrodon species (copperhead), Bothrops species (fer-de- lance), Lachesis muta (bushmaster)

Crotalinae(pit vipers)Viperidae

No heat-sensing pit

Africa, Europe, Middle East: Bitis arietans (puff adder), B

gabonica (Gaboon viper), B nasicornus (rhinoceros-horned viper), Echis species (saw-scaled viper), Cerastes species (horned or desert vipers), Vipera species (vipers)

Indian subcontinent and Southeast Asia: Daboia russelli

(Russell’s viper)

Viperinae(true vipers)Viperidae

Largest family of snakes Venomous but rarely harmful to humans Primitive rear-fangs

Southeast Asia, India: Boiga species Sub-Saharan Africa:

Dispholidus typus (boomslang)

Colubridae

COMMENTS DISTRIBUTION AND EXAMPLES

SUBFAMILY FAMILY

Table 1.1 Venomous snakes of the world Table is adapted from Gold et al., 2002.

Figure 1.2 Venomous snakes from different families (A) Boiga dendrophila

(Mangrove cat snake) (Family: Colubridae), (B) Bitis gabonica (Gaboon viper) (Family:

Viperidae) (Photo provided by Mr Shiyang Kwong, National University of Singapore,

Singapore), (C) Tropidolaemus wagleri (Wagler’s viper) (Family: Crotalidae), (D)

Ophiophagus hannah (King cobra) (Family: Elapidae) (Photo provided by Mr Duncan

MacRae Phyto Medichem Singapore Pte Ltd) and (E) Pelamis platurus (Yellow-bellied

sea snake) (Family: Hydrophidae) (Photo provided by Ms Susan Scott)

Chapter One Introduction

possess some distinct features compared to elapidae that enable them to live in a marine habitat Their venoms are extremely potent and highly neurotoxic

This thesis deals with proteins isolated from the venom of Ophiophagus hannah

(King cobra) This snake belongs to the family elapidae and it is the longest venomous snake in the world, growing to a maximum length of 18 feet (Figure

1.3) They also have a relatively long lifespan of up to 25 years (Veto et al., 2007)

King cobra is distributed in parts of Southeast Asia, South China and India (Tan

and Saifuddin, 1989) The snake derives its name Ophiophagus (‘Ophis’ and

‘phagein’ meaning ‘snake’ and ‘eat’, respectively in Latin) because of the fact that

it preferentially feeds on other venomous and non-venomous snakes The snake is found in dense rain forests, bamboo thickets and mangrove swamps

The LD50 of the crude venom of O hannah in mice is 1.5 to 2.18 mg/kg via subcutaneous injection (Broad et al., 1979) and 1.28 mg/kg via intravenous injection (Ganthavorn, 1969) O hannah venom shows low lethality compared to

some of the other elapids (0.025 to 1.2 mg/kg) and sea snakes (0.09 to 0.71 mg/kg)

(Ganthavorn, 1969 and Broad et al., 1979) This relatively low lethality of the O hannah venom is compensated by the large volume, nearly 420 mg dry weight, of

venom yield per milking of the snake (Ganthavorn, 1969)

Figure 1.3 Ophiophagus hannah (King cobra) Photo provided by

Mr Peter Mirtschin, Venom Supplies Pte Ltd., Australia.

8

Chapter One Introduction

O hannah envenomation in humans is a very rare occurrence as these snakes are

generally shy and stay away from human dwellings However, due to the ever increasing trend of keeping exotic species of animals in zoos and private collections in the developed nations, there have been several well reported

envenomation cases (Ganthavorn, 1971, Wetzel and Christy, 1989, Pe et al., 1995, Gold and Pyle, 1998 and Veto et al., 2007) One third of the 35 cases of reported

O hannah envenomations have been fatal (Gold and Pyle, 1998) The general

symptoms observed upon envenomation are drowsiness, nausea, headache, abdominal pain, hypotension and occasionally shock Subsequently, massive swelling of bite areas and extensive tissue necrosis is observed (Gold and Pyle, 1998)

Chapter One Introduction

metal ions, peptides, lipids, nucleosides, carbohydrates and amines (Hider et al.,

1991) Thus envenomation will cause a simultaneous assault of various systems

leading to multiple organ failure and often death (Torres et al., 2003)

1.2.1 Classification of venom proteins

Snake venoms may contain more than 100 different proteins and these proteins belong to a limited number of protein families These families are broadly classified into two major classes: enzymatic and non-enzymatic (Table 1.2 and 1.3) (Kini, 2002) Members within a family of snake venom toxins share a high sequence homology and common structural scaffold, however, their molecular targets in the prey and hence the induced pharmacological effects may vary remarkably (Kini, 2002)

1.2.1.1 Enzymatic proteins

Snake venoms are rich sources of enzymes Many of these are hydrolases and they play a role in the digestion of the prey even before the snake swallows it More

10

Snake venom phosphodiesterase shares a number of mechanistic features in common with the nucleotidyl transferases All of these enzymes contain zinc, are activated by magnesium, and catalyze α-β phosphoryl bond cleavage.

Ubiquitously present in all venoms Phosphodiesterases

5'nucleotidase is known to affect hemostasis by inhibiting platelet aggregation Ubiquitously present in all venoms

Found widely in elapidae and viperidae venoms

Metalloproteinases

Snake venom serine proteinases, in addition to their contribution to the digestion

of prey, affect various physiological functions They affect platelet aggregation, blood coagulation, fibrinolysis, the complement system, blood pressure and the nervous system.

Found widely in elapidae and viperidae venoms

Serine Proteinases

Flavo enzymes catalyzing the conversion of L-amino acid substrates to α-keto acids LAOs from snake venoms also induce pharmacological effects like induction or inhibition of platelet aggregation, association with mammalian epithelial cells and induction of apoptosis and anti bacterial activity.

Found widely in elapidae, viperidae and crotalidae venoms

L-amino acid oxidase (LAO)

Esterolytic enzymes hydrolyzing 3-sn-phosphoglycerides Unlike mammalian

PLA2, snake PLA2enzymes induce various pharmacological effects like pre- and postsynaptic neurotoxicity, myotoxicity, cardiotoxicity, hemolytic activity, anticoagulant activity, antiplatelet activity, hypotension, hemorrhage and edema

Abundant in elapidae, hydrophidae, viperidae and crotalidae venoms Phospholipase A2 (PLA2)

Description Distribution

Family

Table 1.2 Enzymatic proteins from snake venoms

Bind to integrins through the RGD motif and prevent platelet aggregation.

Abundant in viperidae venoms Disintegrins

Ohanin shows potent hypolocomotion and hyperalgesia in animals.

First identified from Ophiophagus hannah

venom Also found in other elapidae and viperidae venoms

Potent hypotensive and vasorelaxant properties.

Reported from the venoms of Dendroaspis angusticeps, Micrurus corallinus, Pseudechis australis and Trimeresurus gramineus

Natriuretic peptides

They are 9 to 13 residues long which are rich in Pro residues They induce hypotension by acting on smooth muscles

Reported from viperidae and crotalidae venoms only

potentiating peptides

Bradykinin-These isopeptides are structurally and functionally related to mammalian endothelins They are potent vacoconstrictors acting on cardiac muscles and brain.

Isolated from the venom of Atractaspis engaddensis

Sarafotoxins

Cyeteine-rich secretory proteins (CRISPs) or helothermine-related venom proteins are proteins with 16 conserved Cys residues They are known to modulate the activity of various ion channels.

Identified in all families of snake venoms Helveprins/CRISP

Divided into four groups, they range in size from ~ 25,000 Da to ~ 35,000 Da and may exist as homodimer and contain carbohydrate moiety The exact role of these molecules in venoms is unknown However, some suggest that they may act as carriers of other molecules like neurotoxins to the CNS.

First identified from the venom of

Agkistrodon piscivorus (cottonmouth, water

moccasin) Later isolated from venoms of viperidae, crotalidae and elapidae.

Nerve growth factors (NGF)

These anticoagulant proteins inhibit prothrombin activation by non-enzymatic mechanisms.

Mostly found in viperidae and crotalidae venoms

C-type related proteins

Lectin-They range in size from ~ 5000 Da to ~ 6000 Da and has three disulphide bridges, and belong to the Kunitz pancreatic trypsin-inhibitor family As all the proteinases in blood coagulation and fibrinolysis are serine proteinases, these group polypeptides are thought to be potential anticoagulants.

Distributed widely in many snake venoms Serine proteinase

inhibitors

Members show similar molecular scaffold but varying functions like neurotoxicity, cardiotoxicity, anticoagulant activity and antiplatelet aggregation activity Similar structures are found in a wide range of species from plants to mammals.

Abundant in elapidae and hydrophidae venoms

Three-finger toxins (3FTXs)

Description Distribution

Family

Table 1.3 Non-enzymatic proteins from snake venoms

Chapter One Introduction

than 20 different enzymes have been identified in snake venoms and their distribution varies according to the family of snakes Generally, viperid and crotalid venoms contain large proportions of enzymes (80 – 95 %), while elapids have lower amounts (25 – 70 %) Hydrophid venoms have the lowest amounts of

enzymatic proteins (~ 20 %) (Hider et al., 1991) The major groups of enzymatic

venom proteins are phospholipase A2 (PLA2), L-amino acid oxidase (LAO), serine proteinase, metalloproteinase, phosphodiesterase, acetylcholinesterase,

nucleotidase and glycosidase (Table 1.2) (Torres et al., 2003) Apart from their

catalytic action, these enzymes exert various pharmacological activities including neurotoxic, myotoxic, cardiotoxic, hemorrhagic, hemolytic, procoagulant and

anticoagulant effects (Torres et al., 2003)

1.2.1.2 Non-enzymatic proteins

Snake venoms (especially hydrophid and elapid) are abundant in non-enzymatic proteins which act as toxins More than 1000 non-enzymatic protein toxins have been isolated and characterized and these have been grouped into a few families like three-finger toxins (3FTXs), serine proteinase inhibitors, lectins, nerve growth factors (NGFs), helveprins (CRISPs), sarafotoxins, Bradykinin-potentiating peptides, natriuretic peptides, waprins, vespryns and disintegrins

(Table 1.3) (Torres et al., 2003)

Chapter One Introduction

Among the non-enzymatic proteins, three-finger toxin (3FTX) family is the most abundant and well-characterized family β-cardiotoxin, the protein under investigation in this thesis belongs to this family All members of this family share a similar fold consisting of three finger-like loops made of β-sheet structure, projecting from a globular core and stabilized by four conserved intramolecular disulfide linkages giving rise to the compact “β-cross” motif (Endo and Tamiya,

1987, Nirthanan et al., 2003a, Tsetlin, 1999 and Harrison and Sternberg, 1996)

These proteins are 60 – 75 amino acids long and belong to either the short-chain (four disulfides, 60 – 64 residues) or the long-chain (five disulfides, 66 – 75 residues) type (Tsetlin, 1999) Despite the similar structural fold, different 3FTXs have diverse molecular targets in the prey (Figure 1.4) For example, short- and long-chain α-neurotoxins target α1 nicotinic acetylcholine receptors (nAChRs), long-chain α-neurotoxins also target α7 nAChR, κ-bungarotoxins target α3 and α4 nAChRs (Tsetlin, 1999), muscarinic toxins target muscarinic AChRs (Potter,

2001), fasciculin targets acetylcholinesterase (Karlsson et al., 1984), calciseptine

and FS2 toxin target L-type calcium channel (Yasuda et al., 1994), dendroaspin

targets integrin αIIbβ3 (McDowell et al., 1992), cardiotoxins target phospholipids and glycospingolipids (Kumar et al., 1997), hemextin AB complex targets coagulation factor VIIa (Banerjee et al., 2005a), and cardiotoxin A5 targets

integrin αvβ3 (Wu et al., 2006) This observation has given rise to the notion that

they act like ‘sibling toxins’ sharing the same ‘molecular moulds’ while performing ‘multiple missions’ (Figure 1.4) (Kini, 2002) However, there

14

Figure 1.4 Structural similarities between ‘sibling’ three-finger

toxins (A) Erabutoxin a (1QKD), (B) α-bungarotoxin (2ABX), (C)

cardiotoxin V4 (1CDT), (D) κ-bungarotoxin (1KBA), inset, dimer, (E)

candoxin (1JGK), (F) fasciculin 2 (1FAS), (G) muscarinic toxin MT-2

(1FF4), (H) FS2 toxin (1TFS), (I) dendroaspin (1DRS) Note all these

‘sibling’ toxins share a similar structural fold; the core at the top of the

molecules contains all four conserved disulphide bridges and three β-sheeted

‘fingers’ start from the core Some toxins, such as α-bungarotoxin (B) and

κ-bungarotoxin (D) have the fifth disulphide bridge in loop II In contrast

candoxin (E) has the fifth disulphide bridge in loop I However, these toxins

differ from each other in their biological activities Figure was reprinted with

permission from Prof R Manjunatha Kini, Department of Biological

Sciences, National University of Singapore (Kini, 2002).

Chapter One Introduction

are a number of “orphan groups” of 3FTXs whose molecular target in the prey

and hence their functional roles in the snake venoms are not yet known (Fry et al.,

2003a) Thus every new 3FTX isolated should be considered a new challenge as it might show unique pharmacological properties and have a new molecular target The molecular targets and structure-function relationships of some of the well studied 3FTXs are described in the following sections

1.3.1 Neurotoxins

Snake venom α-neurotoxins or curaremimetic toxins bind to the muscle (α1) nicotinic acetylcholine receptor (nAChR) and inhibit acetylcholine binding to the receptor This action alters the nerve-muscle transmission, provoking paralysis of skeletal muscles, including diaphragm and induce death as a result of respiratory

failure (Tremeau et al., 1995, Antil et al., 1999 and Kini, 2002) In this functional

aspect they resemble the plant alkaloid curare and hence the name

‘curaremimetic’ toxins α-neurotoxins are classified into two groups based on their sequence length and number of disulfide linkages They may be either short-chain neurotoxins with a chain length of 60-62 residues and consisting of four disulfide bonds (Figure 1.5 A) or long-chain neurotoxins with a chain length of 66-74 residues and composed of five disulfide linkages (Figure 1.5 B) The fifth disulfide bond in long-chain neurotoxins is found near the tip of loop 2 (Figure 1.4 B) Although they are of different chain lengths, long-chain and short-chain neurotoxins bind to nAChR in a mutually exclusive manner (Endo and Tamiya,

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