Ohanin a novel protein from king cobra (ophiophagus hannah) venom

Chapter I Literature Review


1

CHAPTER I
LITERATURE REVIEW

1.1 SNAKES
Snakes (class Reptilia and order Squamata) first appeared on earth during the Lower
Cretaceous period probably 100 to 150 million years ago based on the oldest ‘snake-like’
fossils found in sandstone beds of Algeria (Harris 1991; Rage 1984). Biologists generally
agree that snakes arose from lizard-like ancestors. Their long body shape and lack of
limbs probably evolved to enable their smooth movement in dense vegetation and forest.
There are about 2930 species of snakes at present (Stafford 2000). They range from
giants, like the anacondas, pythons and boas that can grow up to 7 m (23 feet), to the
smaller-sized snakes, like the burrowing blind snakes that may be as small as 10 cm (4
inches) long. Although they all vary in length, the features that collectively distinguish
snakes as a unique family are clearly recognizable. Generally snakes have highly flexible
bodies with no eyelids, shoulder and sternum. Interestingly in pythons, boas and some
other primitive snakes, some traces of the pelvis and horn-like claws at the base of the tail
which resemble the hind limbs, can still be seen.

1.2 VENOMOUS SNAKES
Approximately 1300 snake species are venomous (Hider et al. 1991; Stafford 2000). The
evolution of the venomous form, however, was much more recent, possibly as late as the
Miocene period (less than 30 million years ago) (Harris 1991). Venomous snakes are
Chapter I Literature Review


2

usually defined as those that have venom glands and specialized venom conducting fangs
which enable them to inflict fatal bites upon their victims (Klemmer 1968).

1.2.1 Classification and distribution of venomous snakes
The systematic classification of both venomous and non-venomous snakes still presents
many problems. Most taxonomists and authorities would only recognize 11 to 13 distinct
families. However, venomous snakes are generally identified in only five families. They
are the Elapidae, Hydrophiidae, Viperidae, Crotalidae and Colubridae (Harris 1991). It is
interesting to note that snakes are widely distributed on all continents in the world except
Antarctica, New Zealand, Madagascar, Ireland, Greenland, the Azores and Canaries
(Phelps 1981). They have successfully evolved into efficient predators and colonized
various habitats from mangrove swamps, estuaries, freshwater lakes, streams, dunes,
grasslands to forests (Garl and Roger 1989). The classification and distribution of
venomous snakes in the world are shown in Table 1.1.

1.2.2 King cobra (Ophiophagus hannah)
King cobra, also known as Ophiophagus hannah (Figure 1.1), belongs to the Elapidae
family. It is the longest venomous snake in the world. King cobra has an average size
ranging from 10 to 12 feet, but sometimes can grow up to 18 feet (5.5 meter) long (Zhao
1990). It is widely distributed in the northern parts of India, southern China (Hainan,
Fujian, Guangdong, Hainan, Guangxi, Guangzhou and Hong Kong) and southeast Asia
(Malaysia, Indonesia, Burma, Thailand, Philippines and Singapore) (Ganthavorn 1971;
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3
Zhao 1990). King cobra is generally found in dense or open rainforests, as well as
mangrove swamps, bamboo thickets, savannas and even around human settlements.

Its genus name, Ophiophagus, means snake eater, with ‘ophis’ and ‘phagein’

representing ‘snake’ and ‘to eat’, respectively in Latin. Hence king cobra preferentially
feeds on snakes and small reptiles. These preys sometimes can be as huge as 10 feet in
length. In addition to snakes, it also feeds on mice, rats, birds, frogs and fishes. The king
cobra kills the prey by injecting a lethal amount of venom with its fangs. It then swallows
its preys as a whole. It hunts both during the day and night time.

King cobra yields an average of 420 mg of crude venom in dry weight per
milking (Ganthavorn 1971). The LD
50
in mouse is ~1.2 to 3.5 mg/kg via intravenous
injection (Mebs 1989). The relatively low toxicity of king cobra’s venom is compensated
by the large amount of venom produced and injected into the preys each time. King
cobra’s venom shows predominantly haemotoxic and neurotoxic effects. The clinical
manifestations upon envenomation are: drowsiness, stupor, ptosis, dysarthria, dysphagia
and general muscular weakness (Ganthavorn 1971). In severe envenomation, impairment
of cardiovascular function can occur (Reid 1968; Wetzel and Christy 1989).
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Characteristics
Small head with short, fixed fangs mounted at the front of the
jaw (proteroglyphous)
Similar in general form to the Elapidae except: the nostrils are
mounted dorsally on the head and are equipped with closing
mechanism, the tail is laterally
flattened, the tongue is reduced, and salt glands have evolved

Heavier and bulkier built than the Elapidae; sluggish; possess a
large, flattened triangular head and a characteristic dentition;
venom fangs are large, grooved and mounted on the maxillary
bone
Similar in general form to the Viperidae except: possess heat-
sensitive pits situated on each side of the head between the
nostrils and the eye
Venom fangs are typically grooved and mounted at the rear of
the upper jaw (opisthoglyphous)

Distribution
America, Africa, Asia, Australia
Coastal waters of Asia and Australia,
Pacific to the western seaboards of
central and south America
Europe, Africa, Asia, America

America, parts of southeast Asia
All parts of the world except for
Australia, Antarctica, New Zealand,
New Zealand, Madagascar, Ireland,
Greenland, Azores and Canaries
Examples
Cobras, kraits, mambas,
coral snakes
Sea snakes
Vipers
Rattlesnakes
Tree snakes, mangroves
snakes
Family
Elapidae
Hydrophiidae
Viperidae
Crotalidae
Colubridae
Table 1.1 Classification and distribution of venomous snakes in the world.
Table is adapted from Harris (1991).
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5


























Figure 1.1 King cobra (Ophiophagus hannah). Photo is reprinted with the permission of
Mr. Peter Mirtschin from Venom Supplies Pty. Ltd., Australia.

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6
1.3 SNAKE VENOMS
Snake venoms are secretory products of venom glands (Oron and Bdolah 1973). Typical
venom glands consist of three major cell types, namely basal cells, conical mitochondria-
rich cells and secretory cells. Venom is only produced by secretory cells in the glands
(Oron and Bdolah 1978). It is further carried from the glands to the fangs by the ducts
that flow through the accessory glands. The function of the accessory glands is to prevent
wasteful flow of the secretions. Venom production appears to be regulated by the glands
themselves and is independently of neural control.

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 (Aird 2002). The
composition of venom components varies with the time of secretion into the glands. For
example, venom that is freshly secreted into the glands has a different composition than
venom that has been allowed to mature (De Lucca et al. 1974; Kochva and Gans 1965). It
should be noted also that the variation in the population or individual, age, diet,
geographical distribution and climate, can easily influence the venom’s composition
quantitatively and qualitatively even within the same species (Sasa 1999).

The composition of the venoms also differs between different families of
venomous snakes. For example, elapid and hydrophid venoms are rich in neurotoxic
proteins and peptides. They have been known to induce effects at the nervous systems
(Chang 1979). On the contrary, crotalid and viperid venoms are rich in proteinases. These
proteinases, such as the serine proteinases and metalloproteinases, tend to cause
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7
hemolytic effects and are largely responsible for the necrosis following the snake bite.
However, in general, the closer the phylogenetic relationships of the snakes, the more
similar are the venom properties and compositions (Tu 1996).

Snake venom proteins have evolved to target different tissues, organs and
physiological systems. Hence, a diversity of symptoms arises after a snake bite which
will ultimately lead to failure of multiple tissues, organs and systems and often death
(Torres et al. 2003). Some of the major clinical symptoms are intense localized pain, loss
of consciousness, drowsiness, headache, vomiting, inflammation, bleeding, shock,
hemorrhage, necrosis and muscular paralysis (Campbell 1979; Efrati 1979; Reid 1979;
Russell 1979).

1.3.1 Compositions and properties of snake venoms
Although snake venoms have always been of great interest for studies, it is only in the
recent years serious attempts have been made to fractionate individual venoms. These
studies have shown that snake venoms consist of proteins as well as non-protein
components. The minor, non-proteinaceous components of snake venoms are metals,
lipids, nucleotides, carbohydrates and amines. The proteinaceous components, which
consist of ~ 90 to 95 % of the total dry weight of the venom, can be further grouped as
enzymatic and non-enzymatic peptides and proteins (Hider et al. 1991).

The major enzyme groups found in snake venoms include phospholipases A
2
(PLA
2
), serine proteinases, metalloproteinases, phosphodiesterases, acetylcholinesterase,
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8
L-amino acid oxidases, glycosidase, hyaluronidase and nucleotidases (Torres et al. 2003).
Generally, enzymes in the venom have molecular mass ranging from 13,000 Da to
150,000 Da. Most of these are hydrolases and possess a digestive role. There are also
over 1000 non-enzymatic venom proteins that have been characterized. They are grouped
into three-finger toxins, serine proteinase inhibitors, C-type lectin-related proteins,
disintegrins, helveprins/ CRISPs, waprins, sarafatoxins, nerve growth factors, natriuretic
peptides and bradykinin-potentiating peptides (Kini 2002; Mochca-Morales et al. 1990;
Torres et al. 2003; Yamazaki et al. 2003). The first category of non-enzymatic venom
peptides and proteins has a molecular mass around 1,000 Da to 25,000 Da and are rich in
disulfide bonds. Therefore, they are robust and are relatively stable once isolated.
Another category is the low molecular mass compounds having the molecular mass of
less than 1,500 Da. They are less active biologically and are likely to be enzyme
cofactors (Bieber 1979). Some of these families are selected and discussed in the
subsequent literature review.

1.3.1.1 Phospholipases A
2
(PLA
2
)
Phospholipases are esterolytic enzymes that hydrolyze 3-sn-phosphoglycerides.
According to the sites of hydrolysis, they are classified as phospholipase A
1
, A
2
, B, C and
D (Kini 1997). Snake venoms are one of the richest sources of secretory phospholipases.

Most of the snake venom phospholipases are PLA
2
as they

hydrolyze the sn-2 ester bond
of 3-sn-phosphoglycerides, releasing lysophospholipids and fatty acids (Kini 1997).
Generally, snake PLA
2
enzyme is a single chain polypeptide of approximately 118 to 130
amino acid residues with high cysteine content (seven disulfide bonds) (Scott 1997).
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9

Snake venom PLA
2
enzymes can be divided into classes I and II. Class I enzymes
are abundant in Elapidae and Hydrophidae snake venoms, whereas class II proteins are
mainly isolated from Crotalidae and Viperidae venoms. Class I can be further classified
into classes IA and IB enzymes, based on the presence or absence of the pancreatic loop.
In the region 52 to 65 (bovine pancreatic PLA
2
sequence numbering) (Dufton and Hider
1983; Renetseder et al. 1985), class I proteins display an insertion of two to three amino
acid residues (the ‘elapid’ loop), which is extended by a further five amino acid residues
in the case of mammalian pancreatic PLA
2
s (the ‘pancreatic’ loop). This loop is absent in
class II PLA

2
.

The position of one of the seven disulfide bonds is also different between
class I and II PLA
2
s. Class I PLA
2
s have the Cys11-Cys77 disulfide bridge which is
absent in class II. But class II PLA
2
s possess an alternative disulfide bridge between
Cys51-Cys133 at the C-terminal extension (Dufton and Hider 1983).

So far, the protein and cDNA sequences of over 280 snake PLA
2
enzymes have
been determined (Danse et al. 1997; Tan et al. 2003). These sequences indicate that
snake PLA
2
contain multiple isoenzymes. Gene sequences determined further
demonstrate that these isoenzymes are from different but closely related PLA
2
genes
likely to have evolved from the physiological PLA
2
(Kordis and Gubensek 1996;
Nakashima et al. 1993). Generally, the primary sequence similarity among snake venom
PLA
2

isoenzymes can reach ~40 to 99 %. Furthermore, they also share high similarities in
their secondary structures and overall foldings (Figure 1.2) (Scott 1997).

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10
Interestingly, unlike mammalian PLA
2
enzymes which are only involved in
catalysis, snake venom PLA
2
isoenzymes are able to induce wide arrays of
pharmacological actions including presynaptic and postsynaptic neurotoxicity (Strong et
al. 1976), myotoxicity (Gopalakrishnakone et al. 1984; Ponraj and Gopalakrishnakone
1995), cardiotoxicity (Lee et al. 1977), hemolytic (Condrea et al. 1981), anticoagulant
effect (Verheij et al. 1980), antiplatelet (Chen and Chen 1989), hypotension (Huang
1984), internal hemorrhage (Vishwanath et al. 1987), organ or tissue damage and edema
(Vishwanath et al. 1987, 1988). The high affinity interaction between PLA
2
isoenzymes
with their acceptor(s)/receptor(s) is likely due to the complementarity of the contact
surfaces in terms of the ionic charges, hydrophobicity and van der Waals force (Kini
2003). Hence, snake PLA
2
isoenzymes are able to induce a wide spectrum of
pharmacological effects, by the mechanisms either dependent on or independent of their
catalytic activity, upon binding to the targets (Kini 2003). Among these pharmacological
actions, only neurotoxic, myotoxic and anticoagulant effects have been well-studied, thus
providing a great challenge to protein chemists to solve the complex puzzle in the

structure-function relationships and mechanisms of action (Kini 2003).

1.3.1.2 Snake venom L-amino acid oxidases

L-amino acid oxidase (EC1.4.3.2) (LAAO) is a flavoenzyme that catalyses the L-amino
acid substrate to an α-keto acid along with the production of ammonia and hydrogen
peroxide. LAAOs are found in many different organisms, such as snakes, bacteria, fungi
and plants. Snake venom L-amino acid oxidases (SV-LAAOs) represent the best studied
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11
ABAB

Figure 1.2 Snake Phospholipases A
2
isoenzymes. Three-dimensional structures of snake
PLA
2
s, class I (A) and class II (B). Arrow indicates the extra pancreatic or elapid loop
region which is only present in class I enzymes. Figure is generously given and reprinted
with the permission from Prof. R. Manjunatha Kini.







Chapter I Literature Review


12
members of this protein family. They are non-covalently bound homodimeric
glycoproteins of around 110 to 150 kDa with FAD-binding motif (Du and Clemetson
2002). SV-LAAOs are widely distributed in the family of Viperidae, Crotalidae and
Elapidae (Du and Clemetson 2002; Pawelek et al. 2000). Sequence alignment of SV-
LAAOs is shown in Figure 1.3A.

Extensive studies have been carried out on SV-LAAOs only in the past ten years.

Studies have shown that SV-LAAOs elicit wide arrays of pharmacological actions. For
example, SV-LAAOs from Crotalus adamanteus and Crotalus atrox can associate

specifically with mammalian endothelial cells (Suhr and Kim 1996) and can either induce
(Ahn et al. 1997; Ali et al. 2000; Li et al. 1994) or inhibit platelet aggregation (Sakurai et
al. 2001; Suhr and Kim 1996; Takatsuka et al. 2001; Tan and Swaminathan 1992).

It was
found that SV-LAAOs can also induce apoptosis in

mammalian endothelial cells,
possibly through the production

of highly localized concentrations of hydrogen peroxide
(Abe et al. 1998; Ali et al. 2000; Souza et al. 1999; Suhr and Kim 1996, 1999; Torii et al.
1997). Two SV-LAAOs isolated from Pseudechis australis were identified to exhibit
potent anti-bacterial effect against gram-positive and gram-negative bacteria (Stiles et al.
1991). The exact mechanism of action(s) and physiological role(s) of SV-LAAOs in the
venom is still poorly understood.

X-ray crystallographic structure of SV-LAAO from Calloselasma rhodostoma
(PDB: 1F8R) was solved (Figure 1.3B) (Pawelek et al. 2000). SV-LAAO is a dimer and
each subunit consists of three domains, namely FAD-binding domain, substrate binding
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13
domain and helical domain. The structure of SV-LAAO resembles the reported structure
of porcine DAAO (D-amino acid oxidase) particularly within the FAD-binding domain
(Pawelek et al. 2000). Furthermore, the structure provides information about two

glycosylation sites at positions Asn172 and Asn361 which appear to be important for its
catalytic activity (Torii et al. 2000). The glycans identified from experimental results
were either of bis-sialylated, biantennary and/or core-fucosylated dodecasaccharide
(Geyer et al. 2001) which can be accommodated in the crystal structure obtained.

1.3.1.3 Three-finger toxins
This is a non-enzymatic protein family found only in the venoms of elapids (cobras,
kraits and mambas), hydrophids (sea snakes) and colubrids (mangrove snakes). The
common structural feature of all three-finger toxins is the three β-stranded loops that
emerge from the small, hydrophobic central core which is cross-linked by four conserved
disulfide bridges. Because of the 3-D structures appearance, this family of proteins is
called the three-finger toxin family (Endo and Tamiya 1991; Kini 2002; Menez 1998;
Servent and Menez 2001; Tsetlin 1999). The high cysteine content makes the toxins
stable and robust once isolated.

Generally, all three-finger toxins are basic proteins of 60 to 74 amino acid
residues with 4 or 5 disulfide bridges. They can be grouped according to their lengths.
Short-chain three-finger toxins, for example, erabutoxin-b (Figure 1.4A) and toxin-α
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14

* 20 * 40 * 60
C. rhodostoma : AHDRNPLEECFRETDYEEFLEIAKNGLTATSNPKRVVIVGAGMAGLSAAYVLAGAGHQVTVL
C. adamanteus : AHDRNPLEECFRETDYEEFLEIAKNGLTATSNPKRVVIVGAGMAGLSAAYVLAGAGHQVTVL
T. stejnegeri : ADDRNPLEECFRETDYEEFLEIARNGLKATSNPKRVVIVGAGMSGLSAAYVLAGTGHEVTVL
B. moojeni : ADDRNPLEECFRETDYEEFLEIAKNGLSTTSNPKRVVIVGAGMSGLSAAYVLANAGHQVTVL
B. jararacussu : ADDRNPLEECFRETDYEEFLEIAKNGLSTTSNPKRVVIVGAGMSGLSAAYVLANAGHQVTVL

* 80 * 100 * 120
C. rhodostoma : EASERVGGRVRTYR KKDWYANLGPMRLPTKHRIVREYIKKFDLKLNEFSQENENAWYFIK
C. adamanteus : EASERVGGRVRTYR KKDWYANLGPMRLPTKHRIVREYIKKFDLKLNEFSQENENAWYFIK
T. stejnegeri : EASERAGGRVRTYRNDEEGWYANLGPMRLPEKHRIVREYIRKFNLQLNEFSQENDNAWHFVK
B. moojeni : EASERAGGRVKTYRNEKEGWYANLGPMRLPEKHRIVREYIRKFDLQLNEFSQENENAWYFIK
B. jararacussu : EASERAGGQVKTYRNEKEGWYANLGPMRLPEKHRIVREYIRKFGLQLNEFSQENENAWYFIK


* 140 * 160 * 180
C. rhodostoma : NIRKRVREVKNNPGLLEYPVKPSEEGKSAAQLYVESLRKVVEELRSTNCKYILDKYDTYSTK
C. adamanteus : NIRKRVREVKNNPGLLEYPVKPSEEGKSAAQLYVESLRKVVEELRSTNCKYILDKYDTYSTK
T. stejnegeri : NIRKTVGEVKKDPGVLKYPVKPSEEGKSAEQLYEESLREVEKELKRTNCSYILNKYDTYSTK
B. moojeni : NIRKRVGEVNKDPGVLEYPVKPSEVGKSAGQLYEESLQKAVEELRRTNCSYMLNKYDTYSTK
B. jararacussu : NIRKRVGEVNKDPGVLDYPVKPSEVGKSAGQLYEESLQKAVEELRRTNCSYMLNKYDTYSTK


* 200 * 220 * 240
C. rhodostoma : EYLLKEGNLSPGAVDMIGDLLNEDSGYYVSFIESLKHDDIFGYEKRFDEIVGGMDQLPTSMY
C. adamanteus : EYLLKEGNLSPGAVDMIGDLLNEDSGYYVSFIESLKHDDIFGYEKRFDEIVGGMDQLPTSMY
T. stejnegeri : EYLIKEGNLSPGAVDMIGDLMNEDAGYYVSFIESMKHDDIFAYEKRFDEIVDGMDKLPTSMY
B. moojeni : EYLLKEGNLSPGAVDMIGDLLNEDSGYYVSFIESLKHDDIFAYEKRFDEIVGGMDKLPTSMY
B. jararacussu : EYLLKEGNLSPGAVDMIGDLLNEDSGYYVSFIESLKHDDIFAYEKRFDEIVGGMDKLPTSMY


* 260 * 280 * 300 *
C. rhodostoma : EAIKEKVQVHFNARVIEIQQNDREATVTYQTSANEMSSVTADYVIVCTTSRAARRIKFEPPL
C. adamanteus : EAIKEKVQVHFNARVIEIQQNDREATVTYQTSANEMSSVTADYVIVCTTSRAARRIKFEPPL
T. stejnegeri : RAIEEKVH FNAQVIKIQKNAEEVTVTYQTPEKDTSFVTADYVIVCTTSGAARRIKFEPPL
B. moojeni : QAIQEKVHL NARVIKIQQDVKEVTVTYQTSEKETLSVTADYVIVCTTSRAARRIKFEPPL

B. jararacussu : QAIQEKVHL NARVIKIQQDVKEVTVTYQTSEKETLSVTADYVIVCTTSRAARRIKFEPPL


320 * 340 * 360 *
C. rhodostoma : PPKKAHALRSVHYRSGTKIFLTCTKKFWEDDGIHGGKSTTDLPSRFIYYPNHNFTSGVGVII
C. adamanteus : PPKKAHALRSVHYRSGTKIFLTCTKKFWEDDGIHGGKSTTDLPSRFIYYPNHNFTSGVGVII
T. stejnegeri : PLKKAHALRSVHYRSGTKIFLTCTKKFWEDEGIHGGKSTTDLPSRFIYYPNHNFTSGVGVII
B. moojeni : PPKKAHALRSVHYRSGTKIFLTCTKKFWEDDGIHGGKSTTDLPSRFIYYPNHNFPNGVGVII
B. jararacussu : PPKKAHALRSVHYRSGTKIFLTCTKKFWEDDGIHGGKSTTDLPSRFIYYPNHNFPNGVGVII


380 * 400 * 420 *
C. rhodostoma : AYGIGDDANFFQALDFKDCADIVINDLSLIHELPKEDIQTFCHPSMIQRWSLDKYAMGGITT
C. adamanteus : AYGIGDDANFFQALDFKDCADIVINDLSLIHELPKEDIQTFCHPSMIQRWSLDKYAMGGITT
T. stejnegeri : AYGIGDDANFFQALDLKDCGDIVINDLSLIHQLPREEIQTFCYPSMIQKWSLDKYAMGGITT
B. moojeni : AYGIGDDANYFQALDFEDCGDIVINDLSLIHQLPKEEIQAICRPSMIQRWSLDKYAMGGITT
B. jararacussu : AYGIGDDANYFEALDFEDCGDIVINDLSLIHQLPKEEIQAICRPSMIQRWSLDKYAMGGITT


440 * 460 * 480 *
C. rhodostoma : FTPYQFQHFSEALTAPFKRIYFAGEYTAQFHGWIDSTIKSGLTAARDVNRASENPSGIHLSN
C. adamanteus : FTPYQFQHFSEALTAPFKRIYFAGEYTAQFHGWIDSTIKSGLTAARDVNRASENPSGIHLSN
T. stejnegeri : FTPYQFQHFSEALTSHVDRIYFAGEYTAHAHGWIDSSIKSGLTAARDVNRASENPSGIHLSN
B. moojeni : FTPYQFQHFSEALTAPVDRIYFAGEYTAQAHGWIDSTIKW
B. jararacussu : FTPYQFQHFSEALTAPVDRIYFAGEYTAQAHGWIASTIKSGPEGLDVNRASE


500
C. rhodostoma : DNEF
C. adamanteus : DNEF

T. stejnegeri : DNEL
B. moojeni :
B. jararacussu :



A






Chapter I Literature Review


15


B

Figure 1.3 Snake venom L-amino acid oxidases. (A) Sequence alignment of SV-
LAAOs. Sequences used are: Calloselasma rhodostoma (sp: O93364), Bothrops
moojeni (gb: 39841346), Bothrops jararacussu (gb: 39841344), Trimeresurus stejnegeri
(gb: 33355627) and Crotalus adamanteus (gb: 3426324). The conserved and identical
residues are highlighted. (B) Three-dimensional structure of apoxin I, an L-amino acid
oxidase functional dimer obtained from X-ray crystallography of the enzyme isolated
from Calloselasma rhodostoma (PDB: 1F8R) (Pawelek et al. 2000). FADs are labeled
pink and positions of the oligosaccharide residues are indicated.

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16
from Naja nigricollis, consist of 60 to 62 amino acid residues and are cross-linked by
four conserved disulfide bonds. They have cysteine connectivity of 1-3, 2-4, 5-6 and 7-8.
On the other hand, long-chain three-finger toxins, for example, α-bungarotoxin and κ-
bungarotoxin (Figure 1.4B and 1.4D), have 66 to 74 amino acids and possess five
disulfide bridges with the fifth disulfide bridge located in the second loop (loop II) (Endo
and Tamiya 1991; Mebs 1989). Long-chain neurotoxin and κ-bungarotoxin contain
cysteine connectivity of 1-3, 2-4, 5-6, 7-8 and 9-10 (Fry 1999).

Recently, another group of poorly characterized three-finger toxins was isolated
exclusively from the Elapidae and Colubridae families. They are called the non-
conventional three-finger toxins. The unique features that distinguish them from the
classical three-finger toxins are the length of the polypeptide chain and the location of
disulfide bridges. Non-conventional three-finger toxins contain 65 to 67 residues and five
disulfide bridges. The fifth disulfide bridge is located in the N-terminal loop (loop I) in

contrast to the classical long-chain three-finger toxins having their disulfide bonds at loop
II (Nirthanan et al. 2003). One such example is condoxin as shown in Figure 1.4E.

Although all ‘sibling’ three-finger toxins adopt the common three-finger fold in
their tertiary structures, they bind to different receptors/acceptors and hence exhibit a
wide variety of biological effects (Dufton and Harvey 1989; Kini 2002). Members of this
family include Erabutoxin-a, which antagonize muscle (α1) nicotinic acetylcholine
receptors (nAChR) (Figure 1.4A); cardiotoxins (cytotoxins), that exert their toxicity by
forming pores in cell membranes (Figure 1.4C); κ-bungarotoxins, which recognize
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17
neuronal (α3β4) nicotinic receptors (Figure 1.4D); fasciculins, that inhibit
acetylcholinesterase (Figure 1.4F); muscarinic toxins, with selectivity towards distinct
types of muscarinic receptors (Figure 1.4G); FS2 toxin, that block the L-type calcium
channels (Figure 1.4H); and dendroaspin, which are antagonists of various cell-adhesion
processes (Figure 1.4I) (Kini 2002). In many instances, the functional sites of interaction
between these pharmacologically diverse toxins and their molecular targets have been
successfully identified using a combination of theoretical and experimental approaches.
These functional motifs include the cytolytic, anti-platelet aggregation and anagelsic
sites (Kini 2002) (Figure 1.5).

1.3.1.4 Snake venom nerve growth factors
Nerve growth factor (NGF) was the first neurotrophic factor to be identified as a protein
that supports neuronal maintenance and survival (Cohen et al. 1954). Its activity was
described in two sarcoma tissues, 37 and 180 (Cohen et al. 1954); and later in snake
venom when an active ‘component’ promoting fibre outgrowth in spinal ganglia in vitro
was identified from the crude venom of Agkistrodon piscivorus (Cohen and Levi-
Montalcini 1956). Subsequent to that, reports on isolation and characterization of sNGFs

were mainly from the venom of Viperidae, Crotalidae and Elapidae families (Hogue-
Angeletti 1970; Hogue-Angeletti and Bradshaw 1979).
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A
B
D
E
F
G
H
I
C
α1 AChR
α1 & α7
AChR
Phospho-
lipids
α4 AChR
α7 & α1
AChR
Muscarinic AChR
L-type Ca
++
channel
α
IIb
β

3
AChE
α1 AChR
α1 & α7
AChR
Phospho-
lipids
α4 AChR
α7 & α1
AChR
Muscarinic AChR
L-type Ca
++
channel
α
IIb
β
3
AChE

Figure 1.4 Three-dimensional structural similarities among three-finger toxins from
snake venoms. (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); and (I)
Dendroaspin (1DRS). These β-sheeted loops are numbered right to left as loop I, II and
III, respectively. Although these toxins share similar three-finger fold, they are differ
from each other in their biological activities. Figure was adapted from Kini (2002).

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19

Figure 1.5 Functional sites of three-finger toxins. Various functional sites identified in
three-finger toxins are indicated as red segments. Figure is reprinted with permission
from Prof R. Manjunatha Kini.
Neurotoxic site Cytolytic site
Fasciculin site
Antiplatelet siteHypotensive site Analgesic site
Neurotoxic site Cytolytic site
Fasciculin site
Antiplatelet siteHypotensive site Analgesic site
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20
sNGFs can further be divided into four major groups based on their physical
properties (Kostiza and Meier 1996):
• Group I: sNGFs isolated from Agkistrodon piscivorus, Crotalus adamanteus,
Bothrops jararaca, Naja nigricollos, Naja melanoleuca and Vipera russelli. They
have a molecular mass of ~25,000 Da and are made up from two identical
subunits similar to the mouse β-NGF (Hogue-Angeletti and Bradshaw 1979).
• Group II: sNGFs isolated mainly from Bothrops atrox, Agkistrodon rhodostoma,
Vipera ammodytes and Vipera russelli. They differ from the first group only by
the presence of carbohydrate moiety. Thus the homodimer generally has a higher
molecular mass of ~35,000 Da (Hogue-Angeletti and Bradshaw 1979).
• Group III: sNGF isolated exclusively from Bitis arientans. It comprises of a

homodimer that is covalently linked by disulfide bonds (Smith et al. 1992).
• Group IV: sNGF is a heterodimer containing γ- and β-subunits. The γ-subunit was
found to be the serine proteinase with arginyl esterase activity (Perez-Polo et al.
1978).

Group I sNGFs shares ~72 % sequence similarity to mammalian NGF (mouse β-
nerve growth factor) as shown in Figure 1.6. Like mammalian NGF, sNGFs are known to
stimulate the growth of peripheral sympathetic neurons in vivo and nerve fibers from
embryonic sensory ganglia cultured in vitro (Hogue-Angeletti and Bradshaw 1979;
Kostiza and Meier 1996; Selby et al. 1987). Both the in vitro and in vivo experiments
have further shown that sNGFs are devoid of direct toxic effects (Hogue-Angeletti and
Bradshaw 1979). To date, PC-12 cells have been established as a standard bioassay that is
Chapter I Literature Review


21

* 20 * 40 * 60
Chinese cobra : EDHPVHNLGEHSVCDSVSAWV-TKTTATDIKGNTVTVMENVNLDNKVYKQYFFETKCKNP
Monocled cobra : EDHPVHNLGEHSVCDSVSAWV-TKTTATDIKGNTVTVMENVNLDNKVYKQYFFETKCKNP
Indian cobra : EDHPVHNLGEHSVCDSVSAWV-TKTTATDIKGNTVTVMENVNLDNKVYKQYFFETKCKNP
Mouse beta NGF : GEFSVCDSVSVWVGDKTTATDIKGKEVTVLAEVNINNSVFRQYFFETKCRAS


* 80 * 100 *
Chinese cobra : NPEPSGCRGIDSSHWNSYCTETDTFIKALTMEGNQASWRFIRIETACVCVITKKKGN
Monocled cobra : NPEPSGCRGIDSSHWNSYCTETDTFIKALTMEGNQASWRFIRIETACVCVITKKKGN
Indian cobra : NPEPSGCRGIDSSHWNSYCTETDTFIKALTMEGNQASWRFIRIDTACVCVITKKTGN
Mouse beta NGF : NPVESGCRGIDSKHWNSYCTTTHTFVKALTTDEKQAAWRFIRIDTACVCVLSRKA



Figure 1.6 Snake venom nerve growth factors. Sequence alignment of sNGFs with
mouse β-nerve growth factor. Sequences used are: chinese cobra (gb: 7438538),
monocled cobra (gb: 11275218), Indian cobra (gb: 7428567) and mouse β-nerve growth
factor (PDB: 1BET). The identical and conserved residues are highlighted. sNGFs shares
~72 % similarity with mouse β-NGF. The numbering of residues corresponds to that of
sNGFs.
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22
widely used to detect fractions with NGF activity after chromatographic procedure
(Greene and Rukenstein 1989).

The physiological role(s) of sNGFs in the venom is still not clear. It was
speculated that some neurotrophic-like molecules, such as the sNGFs, are utilized as a
carrier for neurotoxins to gain access into the central or peripheral nervous system and
subsequently exert their pharmacological effects (Levi-Montalcini 1987). It was also
hypothesized that prior contact of sNGFs to basophil cells may enhance the degranulation
process upon CVF or PLA
2
treatment (Kostiza and Meier 1996). There is no available
three-dimensional structure for sNGFs so far. Thus this may provide an opportunity for
structural biologists to solve its three-dimensional structure and compare with that from
the mouse β-NGF (PDB: 1BET).


1.3.1.5 Helveprins/ CRISPs proteins
Cysteine-rich secretory proteins (CRISPs) are single chain proteins with molecular
masses ranging from 20 to 30 kDa. CRISPs family possesses 16 strictly conserved
cysteines, all of them are engaged in disulfide bonds. Remarkably, 10 out of 16 cysteine
residues are clustered at the C-terminal end of the proteins (Osipov et al. 2005; Yamazaki
et al. 2003). CRISPs family was originally described in the male rodent reproductive tract
(Cameo and Blaquier 1976; Kasahara et al. 1989). Subsequently, proteins that belong to
this family have been identified in all mammals studied, such as horses and humans (Guo
et al. 2005). Most mammalian CRISPs members can be grouped into three classes,
namely CRISP-1, CRISP-2 and CRISP-3, which have functions that are related to sperm-

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23
egg fusion, binding of spermatocytes to Seritoli cells and innate host defence,
respectively (Guo et al. 2005). Interestingly, CRISPs are also found in the venom of
reptiles.

The first reported reptilian CRISPs was identified from the skin secretion of the
Mexican beaded lizard Heloderma horridum horridum (Mochca-Morales et al. 1990).
This protein was named helothermine. Later on, CRISPs were also identified from all
venomous snake families inhabiting in different continents, and thus they belong to a new
family of snake venom proteins (Yamazaki and Morita 2004). Based on the primary
structural similarity to helothermine, this new family was named helveprins
(Helothermine-related Venom Proteins). Some of the examples are trifflin from
Trimeresurus flavoviridis (Viperidae, Asia), ablomin from Agkistrodon blomhoffi
(Viperidae, Asia), pseudechetoxin from Pseudechis australis (Elapidae, Australia),
ophanin from Ophiophagus hannah (Elapidae, Asia) and tigrin from Rhabdophis tigrinus
tigrinus (Colubridae, Asia).

Helothermine from the lizard venom was shown to modulate the activity of a
variety of ion channels, including voltage-gated calcium channels, potassium channels
and ryanodine receptors (Mochca-Morales et al. 1990; Morrissette et al. 1995; Nobile et
al. 1996; Nobile et al. 1994). Helveprins, such as ablomin, latisemin, ophanin, triflin and
piscivorin, were found to inhibit the contraction of smooth muscle induced by high
concentration of K
+
but not caffeine. Thus these toxins are L-type Ca
2+
channel blockers
(Yamazaki et al. 2002b). Futhermore, PsTx and pseudecin were identified as CNG-

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24
channel blockers as both the toxins appear to act on the olfactory and retinal CNG
channel (Brown et al. 1999; Yamazaki et al. 2002a). Sequence alignment of helveprins is
shown in Figure 1.7A.

Crystal structures of stecrisp and natrin, isolated from Trimeresurus stejnegeri
and Naja atra, have been solved recently (Guo et al. 2005; Wang et al. 2005). Both these
structures contain two well-defined independent domains, namely pathogenesis-related
proteins of group 1 domain (PR-1) and cysteine-rich domain (CRD). Both the domains
are connected by a hinge. The 16 conserved cysteine residues form eight-paired disulfide
bridges, with three in PR-1 domain, two in the hinge region and three in CRD domain as
shown in Figure 1.7B. PR-1 domain was proposed to be the catalytic domain for Tex31,
another CRISPs protein isolated from cone snail (Milne et al. 2003). Hence the amino-
terminal moiety is suggested to be responsible for the proteolytic activity in some of the
CRISPs members. Another domain, CRD, shows no obvious sequence similarity, except
for its conservation in the cysteine pattern to Bgk and Shk. However, it is intriguing to
find that CRD shares similar fold with Bgk and Shk, which are the K
+
channel-blocking
toxins (Guo et al. 2005). This provides evidence for helveprins/ CRISPs proteins exhibit
various receptor-related activities. In this regard, CRD is likely to be the interacting
domain with different receptor(s) or acceptor(s) that governs diverse functions of
helveprins/ CRISPs members (Guo et al. 2005). The actual binding epitopes with the
target(s) are yet to be identified.


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25


* 20 * 40 * 60
Helothermine : EASPKLPGLMTSNPDQQTEITDKHNNLRRIVEPTASNMLKMTWSNKIAQNAQRSANQCTLEH
PsTx : SNKKNYQKEIVDKHNALRRSVKPTARNMLQMKWNSRAAQNAKRWANRCTFAH
Ablomin : NVDFDSESPRKPEIQNEIVDLHNSLRRSVNPTASNMLKMEWYPEAAANAERWAYRCIEDH
Stepcrisp : NVDFDSESPRKPEIQNEIVDLHNSLRRSVNPTASNMLRMEWYPEAADNAERWAYRCIESH
Natrin : NVDFNSESTRRKKKQKEIVDLHNSLRRRVSPTASNMLKMEWYPEAASNAERWANTCSLNH
Ophanin : NVDFNSESTRRQKKQKEIVDLHNSLRRSVSPTASNMLKMQWYPEAASNAERWASNCNLGH


* 80 * 100 * 120
Helothermine : TSKEERTIDGVECGENLFFSSAPYTWSYAIQNWFDERKYFRFNYGPTAQNVMIGHYTQVVWY
PsTx : SPPNKRTVGKLRCGENIFMSSQPFPWSGVVQAWYDEIKNFVYGIGAKPPGSVIGHYTQVVWY
Ablomin : SSPDSRVLEGIKCGENIYMSPIPMKWTDIIHIWHDEYKNFKYGIGADPPNAVSGHFTQIVWY
Stepcrisp : SSYESRVIEGIKCGENIYMSPYPMKWTDIIHAWHDEYKDFKYGVGADPPNAVTGHYTQIVWY
Natrin : SPDNLRVLEGIQCGESIYMSSNARTWTEIIHLWHDEYKNFVYGVGANPPGSVTGHYTQIVWY
Ophanin : SPDYSRVLEGIQCGENIYMSSNPRAWTEIIQLWHDEYKNFVYGVGANPPGSVTGHYTQIVWY


* 140 * 160 * 180
Helothermine : RSYELGCAIAYCPDQPTYKYYQVCQYCPGGNIRSRKYTPYSIGPPCGDCPDACDNGLCTNPC
PsTx : KSYLIGCASAKCSSS KYLY-VCQYCPAGNIRGSIATPYKSGPPCADCPSACVNKLCTNPC
Ablomin : KSYRAGCAAAYCPSSEYSYFY-VCQYCPAGNMRGKTATPYTSGPPCGDCPSACDNGLCTNPC
Stepcrisp : KSYRIGCAAAYCPSSPYSYFF-VCQYCPAGNFIGKTATPYTSGTPCGDCPSDCDNGLCTNPC
Natrin : QTYRAGCAVSYCPSSAWSYFY-VCQYCPSGNFQGKTATPYKLGPPCGDCPSACDNGLCTNPC
Ophanin : KTYRIGCAVNYCPSSEYNYFY-VCQYCPSGNMRGSTATPYKSGPTCGDCPSACDNGLCTNPC

* 200 * 220
Helothermine : KQNDVYNNCPDLKKQVGCGHPIMKD-CMATCKCLTEIK
PsTx : KRNNDFSNCKSLAKKSKCQTEWIKKKCPASCFCHNKII
Ablomin : TQEDVFTNCNSLVQQSNCQHNYIKTNCPASCFCHNEIK
Stepcrisp : TRENKFTNCNTMVQQSSCQDNYMKTNCPASCFCQNKII
Natrin : TIYNKLTNCDSLLKQSSCQDDWIKSNCPASCFCRNKII
Ophanin : TLYNEYTNCDSLVKQSSCQDEWIKSKCPASCFCHNKII

A













B

Figure 1.7 Helveprins/ CRISPs. (A) Sequence alignment of helveprins. Proteins
sequences used are: helothermine (gb: 2500711), PsTx (gb: 48428844), ablomin
(gb:48428846), stepcrisp (gb: 37694046), natrin (gb:32492059) and ophanin (gb:
48428838). The identical and conserved residues are highlighted in black. The numbering
of residues corresponds to that of helothermine. (B) Three-dimensional structure of
stepcrisp (PDB: 1RC9). The PR-1 domain, hinge region and CRD domain are labeled.