Identification and characterization of novel proteins from a rare australian elapid snake drysdalia coronoides

IDENTIFICATION AND CHARACTERIZATION OF NOVEL
PROTEINS FROM A RARE AUSTRALIAN ELAPID SNAKE
DRYSDALIA CORONOIDES



SHIFALI CHATRATH

(M.Sc. (Biotechnology))




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


AUGUST 2010

i

Acknowledgements

At the very outset, I would like to thank God for providing me awesome load of
strength to endure the hardships of research life. I am indebted to National University
of Singapore for sponsoring my survival in Singapore by awarding research
scholarship. The vibrant research environment in NUS helped me shape as a skillful
researcher.

I express my sincere gratitude to my supervisor Prof. R. Manjunatha Kini for his
commendable support during my stay in ‘Protein Science Lab’. He has always been a
source of inspiration, encouragement and support. His critical comments on my
experimental designs have improved my way of thinking about science. Thanks Prof.!
for making me an independent researcher. I would like to especially thank him for his
promptness for hastening the process of my thesis submission.

I feel fortunate to be co-supervised by Prof. Prakash Kumar. His useful suggestions
during lab meetings, manuscript and thesis writing have greatly helped me improve
my writing skills. He has been a kind, humble and patient person who helped me
during ups and downs of research life.

I am also extremely thankful to Dr. J. Sivaraman, Dr. K. Swaminathan and Dr. Henry
Mok for always being available for advising me on structural studies of my project. I
also thank Dr. Lin Qingsong for helping me understand proteomics part of my
project.

I would also like to thank Dr. Hai Wei Song from Institute of Molecular and Cell
Biology (IMCB) for helping me with the set up of crystallization. My sincere thank
goes to our collaborator Prof. Daniel Bertrand from Department of Neuroscience,

University of Geneva, Geneva, Switzerland for carrying out a part of pharmacological
studies of drysdalin in his laboratory.

This acknowledgement would be incomplete without thanking Prof. Anjali Karande,
Indian Institute of Science, Bangalore India, Prof. Gurcharan Kaur and Prof.
Prabhjeet Singh from Guru Nanak Dev University, Amritsar, India, who always
guided me during my tough times. I am extremely grateful to them for giving me a
strong background in various fields of biotechnology.

I express my warm gratitude to all the past and present members of the ‘Protein
Science Lab’. Thanks to Dr. Rajagopalan Nandhakishore for guiding me through this
project, Dr. Cho Yeow for teaching me HPLC, Dr. Susanta Pahari, Dr. Robin Doley
and Dr. Md. Abu Reza for useful discussions, Dr. Raghurama Prabhakar Hegde for
helping me with the modeling of structures, Dr. Joanna Pawlak for useful suggestions
regarding my project and Dr. Alex Chapeaurogue for helping with the proteomics
part of my thesis. I would also like to thank Dr. Ryan, Dr. Guna Shekhar, Dr.
Pushpalatha, Shi Yang, Girish, Amrita, Bhaskar, Angelina, Sindhuja, Angie and Aldo
for their help during my stay in the lab. My special thanks go to Sheena for teaching

ii

me organ bath assays and Aarthi, Dr. Om Praba and Dr. Pushpa for being my lunch
and coffee buddies. I also acknowledge the help offered to me by ‘Plant
Morphogenesis Lab memebers’; Dr. Ramammoorthy, Vivek, Vijay, Mahesh and
Petra for useful comments on my work progress. I also thank Ms. Tay Bee Ling for
timely flow of reagents for my project. DBS non-academic staff also deserves a vote
of thanks for helping me with administrative stuff. I want to sincerely appreciate the
helps offered to me by Structure Biology Laboratory members including Dr. Karthik
for useful discussions, Dr. Zhang Jingfeng, Tzer Fong, Lisa, Veerendra, Pankaj,
Shaveta, Abhilash, Manjeet, Priyanka, Thangavelu. They were always there to

provide me things when I used to forget while coming one level up! Thanks to Pallavi
for helping me with primer designing when I used to get stuck. Thanks to Dr. Xing
Ding and Meng Kiat from Dr. Hai Wei’s lab, Mr. Mourier Gilles, CEA, Paris, France
for useful advice on refolding and Milena from Prof. Daniel’s lab.

I am extremely grateful to my aunt and her family in Singapore who provided me a
home away from home. I extend my thanks to my in-laws who have been very
supportive throughout. I have no words to express my gratitude for my brother who
supported my education after my father passed away. I would also like to thank my
God-fearing mother and sister for having faith in me that I can do it.

Above all, I am extremely thankful to my husband for being my pillar of strength. I
would not have come so far without his support and also my apologies for releasing
my frustration on him which he endured quite patiently.


Shifali Chatrath
August, 2010

iii


Table of Contents
Acknowledgements


i
Table of contents
iii
Summary

vii
List of figures
ix
List of tables
xii
Abbreviations
xiii
Chapter 1 Review of Literature


Introduction 1

Venomous snakes 1
Drysdalia coronoides; a rare Australian elapid
2

Snake venom 5
Venom composition, Enzymatic proteins, Non-enzymatic
proteins


Three-finger toxin family 12
Neurotoxins, Hannalgesin, Fasciculins, Muscarinic toxins,
Cardiotoxins (CTxs), Calciseptine and FS2 toxin,
Dendroaspin or mambin, Non-
venom proteins with
‘3FTx’ fold, 3FTx fold: molecular scaffold with multiple
missions

Nicotinic Acetylcholine Receptors (nAChRs) 29
Muscular type of nAChRs, Neuronal type of nAChRs,
Receptor-ligand interface revealed by AChBP (ACh-
binding protein)


Aim and scope of the thesis 38

Chapter 2 Venom Transcriptome and Proteome of Drysdalia
coronoides


Introduction 41

Materials and Methods 42

iv

Reagents and kits; Collection of venom and venom gland;
RNA isolation and cDNA synthesis; Cloning of ds cDNA;
Isolation of plasmids and verification of clones; cDNA
sequencing; Sequence analysis; 3’RACE of venom
proteins; In-solution tryptic digestion; HPLC separation
and mass spectrometric analysis of tryptic peptides;
Molecular modeling


Results and Discussions 50

Transcriptome

Construction of cDNA library; Composition of venom
gland transcriptome of D. coronoides; Three-finger toxins
family; serine protease inhibitors; Cysteine-rich secretory
proteins (CRISPs); Phospholipases A2 (PLA2s); Venom
nerve growth factor (VNGF); Phospholipase B (PLBs); a
new family of snake venom proteins; Snake venom
metalloproteases (SVMPs); Vespryns; Cellular
transcripts; Unknown and hypothetical proteins
50

Proteomics of crude venom of Drysdalia coronoides 69

Description of novel proteins 75

Chapter 3 Cloning, Expression and Purification of Recombinant
Drysdalin


Introduction 79
Overview of pET system; Choosing the host for
expression; Choosing the vector for expression; Factors
influencing expression and purification; Refolding of
proteins


Materials and Methods 85
Reagents and kits used; Bacterial strains and vectors
used; Columns used for purification; Cloning of synthetic
gene into pET-32a and pET-M; Transformation of cloning
and expression host strains; Expression of protein;

Purification of protein; Mass determination; Refolding of
protein


Results and discussion 97
Expression of the trx- fused drysdalin in pET-32a; Affinity
purification and cleavage of the fusion protein; RP-HPLC
of untagged drysdalin; Refolding and RP-HPLC of
untagged drysdalin; Expression of the His-drysdalin in
pET-M; Affinity Purification of His-drysdalin; RP-HPLC
purification of His-drysdalin, Refolding and RP-HPLC of


v

His-drysdalin; Expression, purification and refolding of
15
N-labeled His-drysdalin

Chapter 4 Structural and Functional Characterization of
Recombinant Drysdalin


Introduction 118

Functional characterization 118

Structural characterization 120

Materials and methods 123

Materials; Animals; In vivo toxicity studies; Ex vivo organ
bath studies; Electrophysiological studies; Measurement
of Circular Dichroism (CD) spectra; Crystallization; NMR
data acquisition


Results and discussion 129

Functional characterization 129
In vivo toxicity studies; Ex vivo toxicity studies; In vitro
refolded versus in vivo folded drysdalin; Reversibility
studies of drysdalin on neuromuscular junction;
Comparison of drysdalin to α-bungarotoxin (Bgtx); In
vitro electrophysiological studies of drysdalin


Structural characterization 142
Measurement of CD spectra; Crystallization; NMR studies

Chapter 5 Gene Regulation of Differentially Expressed Isoforms
of Drysdalin


Introduction 148

Materials and methods 153
Liver tissue; Kits and reagents used; Isolation of genomic
DNA; Construction of genome walker libraries; Genome
walking; Isolation and sequencing of clones

Results and discussion 155


Chapter 6 Gene structure of a novel protein 513V5

Introduction 173
Materials and Methods 175
Materials; Genomic DNA isolation and 513V gene


vi

sequencing

Results and discussion 176
Designing of primers for 513V; Analysis of 513V5 gene;
Accelerated Segment Switch in Exon to alter Targeting
(ASSET) in 513V genes; Novel protein encoded by 513V5
gene; Phylogenetic significance of 513V5 gene



Chapter 7 Conclusions and Future Prospects

Conclusions 189

Future Prospects 192

Bibliography 195

Appendix 224

Publications 238


vii

Summary
Identification and characterization of novel proteins from a rare Australian
elapid snake Drysdalia coronoides
Partial transcriptome from the venom gland of a rare Australian elapid snake
Drysdalia coronoides, whose venom composition was not known, was elucidated by
cDNA library approach and the results were corroborated by determining proteome
from the crude venom of the snake. Three novel proteins belonging to three finger
toxin (3FTx) super-family of the snake venom proteins were identified. They consist
of three clusters represented by the clones 13A, 342A and 513V5. One of them,
named drysdalin, possesses distinct structural features predicted by online server I-
Tasser, and was chosen for functional and structural characterization.
Drysdalin was expressed in E. coli followed by affinity chromatography,
reverse phase HPLC and refolding. It showed dose-dependent and time-dependent
neurotoxicity in mice with LD
50
of 0.775 mg/kg. It was found to be an irreversible
blocker of muscle-type and neuronal-type nicotinic acetylcholine receptors (nAChRs)
with EC
50
of 37 nM (~2.8 fold less than α-bungarotoxin) and 27 nM, respectively.
These data were in stark contrast to the substitutions of functionally conserved
residues which would lead to the decrease in binding affinity to nAChRs. Our data

suggest that despite these substitutions, there are some structural changes in drysdalin
that might have prevented the loss of binding affinity. The attempts to solve three-
dimensional structure of drysdalin by X-ray crystallography and NMR were initiated
because the C-terminus of drysdalin was predicted to acquire a unique fold. The
protein could not crystallize. However, 2D-NMR spectrum acquired with
15
N-labeled

viii

drysdalin could indicate the reasons for failure of crystallization attempts. The protein
sample used for 2D-NMR was suspected to exhibit heterogeneity and/or flexibility.
These results will help in future experimental designs for structural characterization
of drysdalin.
The gene structures of the differentially expressed, closely related isoforms of
drysdalin were studied by genome walking approach. The promoter regions of the
genes encoding these isoforms were highly similar with several random mutations
sharing no relation to their abundance in the transcriptome. This suggests that cis
elements in the proximity of the gene are not responsible for the differential
expression of these isoforms.
The second novel protein encoded by clone 342A exhibited a shorter loop II
lacking two of the critical residues implicated in binding to nAChRs. Therefore, this
3FTx is expected to have altered pharmaco-physiological properties. The third novel
protein encoded by clone 513V5 was identified from the genomic DNA. This search
was prompted due to the identification of a truncated clone 513A in the cDNA
library. Genomic DNA PCR revealed that 513V5 had a different open reading frame
than 513A transcript due to an insertion of 7 bp in exon II. Also, it had different exon-
intron boundary than other 3FTxs. These results imply the significance of deciphering
snake genome to search for new venom proteins.
Our study has significantly contributed to the field of snake venom research

with novel toxins identified from a combined transcriptomics, proteomics and
genomic approach. This work opens new avenues for various biochemical and
biophysical studies that may have biomedical applications in the long-term.

ix

List of Figures

Chapter 1
1.1
Drysdalia coronoides and its geographical distribution
4
1.2
Snake fang and the venom gland
6
1.3
Multiple sequence alignment of short-chain and long-chain toxins
13
1.4
Functional diversity of 3FTxs
14
1.5
Multiple sequence alignment of atypical long-chain neurotoxins and non-
conventional neurotoxins
18
1.6
‘3FTx fold’ in non-venom proteins
24
1.7
Gene organization of ‘3FTx fold’ containing protein from different

organisms
26
1.8
Cartoon representation of nAChRs
31
1.9
Schematic showing the events leading to neurotransmission followed by
muscular contraction
34
1.10
The three-dimensional structures of nAChR, AChBP alone and in complex
with α-cobratoxin
37

Chapter 2

2.1
Schematic showing construction of cDNA library
47
2.2
Agarose gel electrophoresis (1%) of RNA and ds cDNA from D. coronoides
52
2.3
Venom transcripts of D. coronoides
53
2.4
Three-finger toxins (3FTxs) from D.coronoides
56
2.5
Serine Protease Inhibitors (SPIs) from D.coronoides

59
2.6
Phospholipases A
2
(PLA
2
s)

from D. coronoides
62
2.7
Phospholipase B from D. coronoides
64
2.8
Snake venom metalloproteases (SVMPs) from

D. coronoides
66
2.9
2.10
SDS-PAGE analysis of D. coronoides venom
RP-HPLC profile of tryptic digest of the crude venom of D.coronoides
69
70
2.11
Schematic representing the sequence coverage of the proteins identified in
the venom of D. coronoides
72-73
2.12
Comparison of three dimensional structural models of the novel proteins to

the crystal structures of α-Cobratoxin (Cbtx) and erabutoxin (Ebx)
76
2.13
Schematic showing the summary of characterization of novel proteins from
D. coronoides
78

x



Chapter 3
3.1
Overview of pET systems
81
3.2
Comparison of optimized and native sequences encoding drysdalin
87
3.3
Schematic representation of expression constructs for drysdalin
88
3.4
Tris-Tricine PAGE (12%) analysis of trx-fused drysdalin
98
3.5
Peptide Mass Fingerprinting (PMF) of recombinant drysdalin after
enterokinase cleavage
100
3.6
Purification of untagged drysdalin

102
3.7
Purification of refolded untagged drysdalin
103
3.8
Tris-Tricine PAGE (15%) of His-drysdalin
106
3.9
Purification of His-drysdalin
109
3.10
Purification of reduced His-drysdalin
110
3.11
Purification of refolded His-drysdalin
111
3.12
RP-HPLC profiles depicting refolding kinetics of His-drysdalin
113
3.13
Purification of reduced
15
N-labeled His-drysdalin
115
3.14
Purification of refolded
15
N-labeled His-drysdalin
116
3.15

Schematic representation of the expression and purification
117

Chapter 4

4.1
Schematic showing different steps of ex vivo assay
126
4.2
In vivo toxicity studies of drysdalin
131
4.3
Neuromuscular block produced by untagged drysdalin
132
4.4
Neuromuscular block produced by His-drysdalin
134
4.5
Reversibility experiments on neuromuscular block produced by His-
drysdalin in CBCM
136
4.6
Dose-response curves of α-bungarotoxin and drysdalin on CBCM
137
4.7
Time-dependent inhibition of α-bungarotoxin and drysdalin on CBCM
138
4.8
Effect of drysdalin on human α7 nAChR expressed by Xenopus oocytes
141

4.9
Far UV-CD spectrum of drysdalin
143
4.10
1D (
1
H) NMR Spectrum of drysdalin.
144
4.11
Comparison of 1D (
1
H) NMR Spectra of drysdalin
145
4.12
2D (
1
H-
15
N) heteronuclear single quantum correlation spectrum of drysdalin
146

xi


Chapter 5


5.1
Strategy to obtain the putative promoter region of closely related LNTx
genes from D. coronoides

157
5.2
Alignment of promoter region and partial gene sequence of LNTx genes of D.
coronoides
159-168
5.3
Schematic showing the summary of genome walking and BLASTn results
of LNTx genes from D. coronoides
169
5.4
Cartoon representations of results obtained from genome walking of LNTx
genes from D. coronoides
172

Chapter 6

6.1
Comparison of partial cDNA sequence of 513A to other 3FTxs from D.
coronoides
174
6.2
Cloning of 513V5 gene
178
6.3
Differences across 513V genes
180
6.4
Gene sequence of a 513V5 gene
181
6.5

Summary of BLASTn results for 513V5 gene
183
6.6
Observation that led to the identification of a novel protein
185
6.7
Comparison of structure of protein encoded by 513V5 to erabutoxin b (Ebx)
187

Appendix

A.1
Amino acid sequence alignment of Interferon Gamma Inducible protein 30
(GILT)
224
A.2
Mass spectrum obtained for pro-peptide sequence of snake venom
metalloproteases (SVMPs) from D.coronoides
229
A.3
Vector map of pET-32a and pET-M
230
A.4
Partial cDNA sequences of long-chain neurotoxin (LNTx) isoforms from D.
coronoides
232
A.5
Alignment of partial gene sequence of LNTx genes of D. coronoides.
233-237

xii

List of Tables

Chapter 1
1.1

Classification and distribution of venomous snakes in the world
3
1.2

Enzymatic proteins from snake venoms
8 - 9
1.3

Non-enzymatic proteins from snake venoms
10 - 11
1.4

The ubiquitous world of ‘3FTx fold.’
27 - 28
1.5

Distribution and function of various nAChR subtypes
35
1.6

Intermolecular interactions between atoms of each partner in the complex
39

Chapter 2

2.1

Summary of the clones obtained from cDNA library of the venom gland of
D. coronoides
52

Chapter 4

4.1

Comparison of pharmacological parameters of drysdalin to Bgtx
138

Chapter 6

6.1

Comparison of exon-intron boundaries of 513V5 gene to other SNTxs
from D. coronoides
179

Appendix

A.1

Sequences of the peptides obtained from the trypsin digestion of the crude
venom of D. coronoides identified by tandem mass spectrometry

225-228
A.2

Tris-Tricine Gel
231





xiii

Abbreviations

Bioinformatics terms
BLAST Basic local alignment search tool
IPI International Protein Index
NCBI National center for biotechnology information
PDB Protein databank

Biological species
ACh Acetylcholine
AChBP Acetylcholine binding protein
AChE Acetylcholinesterase
ANP Atrial naturitic peptide
ATP Adenosine triphosphate
ATPase A class of enzyme that catalyses hydrolysis of ATP
Bgtx α-bungarotoxin
BMP Bone morphogenetic protein
BNP Brain naturitic peptide

BPP Bradykinin potentiating peptide
CBCM Chick biventer cerivicis muscle
Cbtx α-cobratoxin
CD Cluster of differentiation
cDNA complementary DNA
CLP C-type lectin related proteins
CNP C-type naturitic peptide
CNS Central nervous system
CRISP Cycteine-rich secretory protein
C-terminal Carboxy terminal
CTx Cardiotoxin
DA Dopamine
DNA Deoxyribonucleic acid

xiv

dNTP Deoxyribonucleotide triphosphate
Ebx Erabutoxin
ECM Extracellular matrix
EST Expressed sequence tag
gDNA Genomic DNA
GILT Gamma inducible lysosomal thiol reductase
GP Glycoprotein
GPI Glycophosphoinositol
G Proteins GTP binding proteins
GPCR G protein-coupled receptor
GTP Guanidine triphosphate
LAO L-amino acid oxidase
LGIC Ligand-gated ion channel
LNTx Long-chain neurotoxin

mAChR Muscrinic acetylcholine receptor
mRNA Messenger RNA
MT Muscarinic toxin
nAChR Nicotinic acetylcholine receptor
NADH Nicotinamide adenine dinucleotide (reduced form)
NGF Nerve growth factor
NPP Naturitic potentiating peptides
N-terminal Amino terminal
ORF Open reading frame
PDE Phosphodiesterases
PLA2 Phospholipase A2
PLB Phospholipase B
PNS Peripheral nervous system
RGD Arg-Gly-Asp- tripeptide
RNA Ribonucleic acid
rRNA Regulatory RNA
SA Swiss Albino

xv

SINE Short interspersed nuclear elements
SNTx Short-chain neurotoxin
SPI Serine protease inhibitors
SR Sarcoplasmic reticulum
SVMP Snake venom metalloproteases
TGF-β Tumor growth factor- β
trx Thioredoxin
TSS Transcription start site
uPAR Urokinase Plasminogen activator receptor
UTR Untranslated region

VF Venom factor
3FTx Three finger toxin

Techniques
CD Circular dichroism
ESI-MS Electrospray ionization-mass spectrometry
HPLC High performance liquid chromatography
HSQC Heteronuclear single quantum coherence
LC/MS Liquid chromatography/mass spectrometry
LD-PCR Long distance PCR
MALDI Matrix-assisted laser desorption/ionization
NMR Nuclear magnetic resonance
PCR Polymerase chain reaction
PAGE Polyacrylamide gel electrophoresis
RACE Rapid amplification of cDNA ends
RP-HPLC Reverse phase-HPLC
RT-PCR Reverse transcription PCR

Chemicals and reagents
ACN Acetonitrile
Amp Ampicillin

xvi

CCh Carbamylcholine (carbachol)
DTT Dithiothreitol
EDTA Ethylenediamine tetraacetic acid
GnHCl Guanidine hydrochloride
GSH Glutathione (reduced)
GSSG Glutathione (oxidized)

HCl Hydrochloric acid
IPTG Isopropyl β-D-thiogalactopyranoside
KCl Potassium chloride
LB Luria bertani
Ni-NTA Nickle-nitrilotriacetic acid
PBS Phosphate buffered saline
TCA Trichloroacetic acid
TFA Trifluoroacetic acid
TRIS Tris(hydroxymethyl)-aminomethane
X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside


Units and measurements
A Ampere
Å Angstrom
bp Base pair
°C Degree Celcius
cm Centi meter
Da Daltons
Ft Feet
g Grams
x g centrifugal force
h Hour
Hz Hertz
K Kelvin

xvii

k Kilo (1000)
kb Kilo base pair

kDa Kilo Daltons
kg Kilo gram
L Liter
m Meter
M Molar
mg Milli gram
MHz Mega Hertz
ml Milli liter
mm Milli meter
mM Milli molar
min Minute
ms Millisecond
m/z Mass to charge ratio
µg Micro gram
µl Micro liter
µM Micro molar
nm Nano meter
nM Nano molar
psi Pound per square inch
rpm Revolutions per minute
s Second
V Volt
Chemical Symbols
Na
+
Sodium
K
+
Potassium
Ca

2+
Calcium

Ni-NTA Nickle nitrolic acid
Zn2+ Zinc


xviii

Greek alphabets
α Alpha
β Beta
γ Gamma
δ Delta
ε Epsilon
κ Kappa
µ Micron

Others
1-D One-dimensional
2-D Two-dimensional
3-D Three-dimensional
EC50 Half maximal effective concentration (Dose which causes 50 % of
maximal effect after certain exposure time)
et al. et alii (and others)
i.p. Intra peritoneal
LD
50
Lethal Dose 50 (Dose which causes kill 50 % of a tested
population after certain exposure time)

MW Molecular weight
n Number of experiments
RT Room temperature
S.E. Standard error
URL Uniform Resource Locator
UV Ultraviolet











Chapter 1
Introduction and
Review of Literature
Chapter 1
1
Introduction
Snakes have fascinated scientists from different areas of research ranging from
biodiversity to genomics, proteomics, and drug development. It is the venom that
makes snakes the organism of choice for research by various laboratories world
wide. Snake venom has long been known to be employed in traditional Indian,
Chinese and Arabian medicine. Over the years, the perception regarding venom
has changed drastically from that of a deadly weapon to a pharmaceutically
important cocktail of bioactive proteins and polypeptides that act as lead

molecules for therapeutics development [1-3].
Snakes evolved from burrowing lizards during lower cetaceous period
(~100-150 million years ago) [4]. There are about 2,930 species of snakes
distributed on every continent except Antarctica, islands of Ireland, Iceland and
New Zealand [5, 6]. They have successfully colonized various habitats and feed
on small animals including lizards, snakes, rodents, small mammals, birds, eggs
and insects [7].
Venomous snakes
Around 1300 species of snakes are known to be venomous [2]. The venomous
snakes appeared during the Miocene period (less than 30 million years ago) which
saw drastic changes in prevailing environmental conditions including
development of more open habitats (savannahs), and radiation of rodents and
other potential prey species. This led primitive snakes with slow locomotion and
active immobilization strategy (i.e constriction) to adapt to rapid locomotion and
passive immobilization (i.e. pursuit and envenomation) [8]. During this period,
Chapter 1
2
the Duvernoy’s glands, the first venom producing apparatus were evolved.
However, at later stages, these glands became hypertrophied and evolved to form
specialized venom glands. Venomous snakes are classified into five families [9] :
Colubridae, Viperidae, Crotalidae, Elapidae and Hydrophidae (Table 1.1). Not all
of them are dangerous to humans. Colubridae, the largest snake family (~1000
species) produce small volumes of venom and have poorly developed venom
delivery apparatus [10]. Therefore, most of the snakes of this family are harmless
except a few like the African Boomslang (Dispholidus typus).
Drysdalia coronoides; a rare Australian elapid
Australian elapids are considered to be the most toxic snake species of the world,
with all the top 10 and 19 of the top 25 elapids with known LD
50
s residing

exclusively on this continent [11]. The venomous terrestrial snakes of the
Elapidae family have undergone an extensive radiation in Australia [12]. Elapid
snake genus Drysdalia from southern Australia is a group of rare snakes
comprising three species, namely, D. coronoides, D. mastersi and D. rhodogaster
[13]. These viviparous snakes live in a wide variety of climatic conditions. For
example, D. coronoides, (Figure 1.1A) the smallest of all, occupies the most
extensive geographical distribution (Figure 1.1B). It is the only species found in
Tasmania [14] where it is also referred to as ‘whip-snake’[15]. It is a small
slender snake which grows to about 40 cm in length [16]. Its body color varies
from light gray through olive-brown to almost black.
Chapter 1
3

Table 1.1 Classification and distribution of venomous snakes in the world.

Chapter 1
4


Figure 1.1 Drysdalia coronoides and its geographical distribution. (A) The
‘white-lipped snake’ (photo provided by Mr. Peter Mirtschin, Venom Supplies
Pty. Ltd., Australia.). (B) The regions (blue) in the Australian continent where the
snake can be found. The map downloaded from the URL:

and modified to show the distribution.
Chapter 1
5
The snake is easily recognized by the thin white stripe running along its upper lip,
continuing along the side of the head, fading out along the neck and hence the
name ‘white-lipped snake’ (Figure 1.1A) It feeds almost exclusively on scincid

lizards and frogs [14]. There are no reports of this snake biting humans.
Snake venom
Snake venom is produced in a highly developed secretory organ called venom
gland (a modified parotid salivary gland of other vertebrates) [4]. This gland is
situated on each side of the head below and behind the eye, wrapped with a
muscular sheath (Figure 1.2). Venom is stored in large alveoli before getting
channelled to the tubular fang through a duct from where it is ejected [17].
Venom composition
The peptides and the proteins constitute over 90% of the dry weight of the venom.
Minor non-protein compounds present in the venoms include metal ions, lipids,
nucleic acids, carbohydrates and amines [18, 19]. Magnesium, calcium and zinc
are the most common metal components, while copper has been detected in
certain venoms. These metal ions are mainly associated with the proteins and are
presumed to be enzyme cofactors [20]. Also, venoms from some snakes, e.g.
mamba (Dendroaspis ssp.), contain high levels of acetylcholine [21].
Venom proteins are used mainly to immobilize and kill the preys and
predators as well as to support the digestion of the food swallowed by the snake
[22]. Therefore, the venom composition varies in different snake families as well
as within a family depending on the feeding habits, geographical location and
environmental conditions [23-25].