By : Zach Weiss, Tiffany Maestas & Amanda Lopez
Presented to: Dr. Toolson
Biology 445
April 21, 2010
The Malayan (Blue) Krait is a highly venomous
elapid snake, and is one of 12 species of kraits in
the Bungarus genus.
3rd deadliest snake in the world
We will discuss…
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Distribution
Habitat
Reproduction
Diet and Behavior
Venom
Where are they found?
Southeast Asia…
• Thailand
• Cambodia
• Vietnam
• Malaysia
• Singapore
• Indonesia
Habitat
• Flat and hilly country
• Found in areas at heights
of 2300 meters
• In habit open areas, fields,
grassy landscapes, and
forests
• Sometimes found in close
proximity to water
• Avoid the sun, so found
under fallen down trees or
rotting stumps
• Oviparous – lay eggs
• Mating season - March and April
• Female lays 4 to 10 eggs - 2 months after
mating season
• The females remain with their eggs until
they have hatched and guard them
• Incubation period of the eggs: 60-64 days
• The newborns are between 30 to 32 cm long
Venom!
Extremely potent neurotoxic venom
You do not want to mess with this snake!
B. candidus Venom
Neurotoxic
16 times more potent than cobra venom
LD50=3.5 μg (mice)
Venom is very powerful and quickly induces
muscle paralysis
• Venom contain mostly pre-synaptic
neurotoxins
• Effect the ability of neuron endings to
properly release the chemical that sends the
message to the next neuron
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B. candidus Venom Effects
• Frequently little or no pain at site of krait bite
• Leads to massive overexcitation, cramps,
tremors, spasms, eventually inducing paralysis
• Cause of death if often respiratory failure due
to paralysis of diaphragm (usually occurs 6-12
hours after bite
• Most bites occur at night since the snake is
nocturnal
Neurotoxins
• Target nerve cells (neurons)
• Snake neurotoxins especially
like to target the motor neurons
of the Peripheral Nervous System
• Inhibit proper neuron function
• Neuron inhibition causes paralysis.
• Death usually occurs by asphyxia
due to respiratory faiure.
Neurotoxins
• Snake neurotoxins target the
motor neurons of the
peripheral nervous system and
can act presynaptically,
postsynaptically, or both.
• Presynaptically acting
neurotoxins are called
β-neurotoxins
• Postsynaptically acting
neurotoxins are called
Alpha-neurotoxins
Neurotoxins
• Snake neurotoxins specifically target the
neuromuscular junctions in the
peripheral nervous system and block
neuromuscular transmission, either
presynaptically or postsynaptically,
leading to muscle paralysis
Neuromuscular Junction
The Neuromuscular
Junction
Three-Finger Toxin Family
Bungarus candidus venom contains several
toxins of the three finger toxin family
Candotoxin, a novel three-finger toxin from
the Bungarus candidus
Three-finger Toxin Family
• Three-finger toxins exhibit a broad range of
pharmacological activity:
Central and Peripheral neurotoxicty
Cytotoxicity
Cardiotoxicity
Inhibition of enzymes such as
acetylcholinesterase
• Platelet aggregation
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Three-Finger Toxin Family
• The three-finger toxin family is a very diverse group
of non-enzymatic polypeptides found only in the
venom of elapid snakes
• Family includes a large variety of toxins with different
functional activities that target different biological
sites.
General three finger toxin structure
Three-Finger Toxin Family
Common structure of several three finger toxins including
α-bungarotoxin (B) and candoxin (E) from B. candidus
venom, which will be discussed further.
Three-Finger Toxin Family
• All the proteins of this family have a similar structure, but
exhibit very different biological activity.
• Three finger toxin polypeptides contain 60-74 amino acid
residues
• Contain several disulfide bonds (all three finger toxins
have at least 4)
• All toxins share a common pattern of folding that has three
loops extending from a central core.
• They have the structural appearance of “three fingers”
Three-Finger Toxin Family
Structural comparison of three finger toxins bucadin (orange) and
bungarotoxin (purple) from B. candidus venom. Note differences in
loop regions, which ultimately lead to differences in action of toxin.
What’s in Snake Venom?
• It is highly modified saliva
• Mostly comprised of proteins, enzymes,
substances with cytotoxic effect,
neurotoxins, and coagulants
• Has about 20 different enzymes, of which, a
species usually has between 6 and 12
• The enzymes determine the toxicity of the
venom
• Venom’s primary importance is to capture or
kill its prey then to help it digest its prey
Components of B. candidus
Venom
3 types of neurotoxins in Bungarus candidus:
1.Candoxin: Novel three finger toxin from B.
candidus
2.Bucandin toxin: Novel presynaptic neurotoxin
isolated from B. candidus
3.Bungarotoxin: three finger toxin found in
Elapid snake venom
Candoxin
Is a novel toxin in B. candidus venom
•Classified as a three finger toxin
•Classified as a weak toxin
•Its affects are reversible
•Candoxin is a postsynaptic neurotoxin
•It shares similarities to α-neurotoxins,
but has some important differences
Amino Acid residues
in Candoxin
Candoxin protein
folded structure
Candoxin
• Produces a reversible postjunctional
neuromuscular blockade by binding with
muscle (αβγδ) receptors of the neuromuscular
junction and α7 neuronal receptors.
Nicotinic Acetylcholine receptor
Candoxin
• Candoxin binding prevents
acetylcholine from binding
to the postsynaptic
nicotinic acetylcholine
receptors
• The action potential from
the presynaptic neuron is
blocked, preventing
muscles from contracting
• This leads to muscle
paralysis.
Neuromuscular Junction
Candoxin
Candoxin:
•Binds reversibly to nicotinic acetylcholine receptors
in the neuromuscular junction
•Its fifth disulfide bridge is located at the tip of loop I
α-Neurotoxins:
•Bind irreversibly to nicotinic acetylcholine receptors
in the neuromuscular junction
•Its fifth disulfide bridge is located at the tip of loop II
Bucandin
Is a novel neurotoxin in B. candidus venom
• Classified as a three finger toxin
• A novel presynaptic neurotoxin
• 63 amino acid polypeptide
• 2 Antiparallel β-Sheets
• Has a fourth strand in second
antiparallel β-Sheet
• 5 dusulfide bonds
• Shares only 30-40% similarity with
most three finger toxins
Bucandin (A) protein structure
compared to α-neurotoxins
cobratoxin (B), erabutoxin
(C), and cytotoxin II (D)
Bucandin amino acid sequence and disulfide-bonding pattern
Bucandin
•Enhances presynaptic acetylcholine release from the synaptic
bulb.
•Bucandin causes excessive
acetylcholine to be released
into the synapse of the
neuromuscular junction
•Excess acetylcholine in the
synapse cannot be degraded
by acetylcholinesterase
***The mechanism of Bucandin
presynaptic action to cause excess
acetylcholine release is unknown
Neuromuscular Junction
Bucandin
•The large increase in acetylcholine in the neuromuscular
junction overwhelms the acetylcholine receptors and
cause over-excitation of the skeletal muscles.
•Over-excitation of skeletal muscles leads to muscle
spasms, tremors, seizures, etc.
•Ultimately, paralysis is induced.
Surface plot of Bucandin –
amino acid residues with potential
functional relevance are marked
Bungarotoxins
Three-dimensional structure of
α-bungarotoxin
Bungarotoxins
α-bungarotoxins
β-bungarotoxins
• Component of the venom
of the elapid krait snakes
• Acts postsynaptically
• Binds irreversibly and
competitively to the
acetylcholine receptor
found in neuromuscular
junction
• Causes paralysis,
respiratory failure, and
death
• A selective antagonist of
the nicotinic acetylcholine
receptor in the brain
• Fairly common in some
snake venoms
• Acts presynaptically
• Alters acetylcholine
release in presynaptic
terminal in both peripheral
and central nervous
systems
Bungarotoxins
B. Candidus venon contains
α-bungarotoxins
•A three fingered toxin
•Is a postsynaptic neurotoxin
•74 amino acid polypeptide
α-bungarotoxin
structure
Bungarotoxins
• α-bungarotoxin binds postsynaptically in the
neuromuscular junction to
nicotinic acetylcholine receptors
• α-bungarotoxin binds with very
high affinity and specificity for
the acetycholine receptor
nicotinic acetylcholine
receptors
Bungarotoxins
•α-bungarotoxin binding prevents
acetylcholine from binding to the
nicotinic acetylcholine receptors
on the postsynaptic side of the
neuromuscular junction.
•The inability of acetylcholine to
bind to its receptors prevents
sodium channels from opening in
the receptors.
•The action potential from the
presynaptic neuron is blocked and
muscle contraction is inhibited.
•This leads to muscle paralysis.
Neuromuscular Junction
B. candidus Attacks!
• B. candidus bites are very rare.
• They occur mostly in rural areas.
• Bites occur mostly at night, since the snake is
nocturnal.
• Death occurs quickly due to the extremely high
lethal toxicity and the neurotoxic mechanism of
its venom.
• Fatalities from bites are underreported because
most bites occur in rural areas and hospitals are
too far away to treat patients in time.
• Frequently little or no pain at site of bite
Venom Extraction (milking)
How to Make Anti-venom
• Milk the snake (make sure it has venom in it)
• Inoculate a horse (or other animal) and let it build
up antibodies to the venom
• Collect your horse serum with antibodies against the
toxins in the venom
• Make sure your batch of anti-venom is injected into
infected blood stream within an hour, if not, you
could be too late
• WARNING: Anti-venom only blocks further damage,
it can’t undo what has already been done
• SIDE EFFECTS: Could cause an allergic reaction
(i.e. serum sickness from the horse proteins)
Anti-venom
• Before anti-venom was developed there was
an 85% mortality rate
• Mortality rate of 50% even with anti-venom
• The polyvalent Elapid anti-venom usually
neutralizes effects
• If transport to medical care takes long a
permanent coma and brain death from
hypoxia can occur
Medical Value of Snake
Venom
• Some snake venoms may slow the growth of
cancerous tumors
• Snakes use venom to alter biological functions
which is similar to the action of medications
• ACE inhibitors, a class of drugs used to treat high
blood pressure and other cardiovascular disorders,
were developed from the venom of a Brazilian snake
• The variations between venom types and the number
of venomous snakes worldwide create a rich
molecular hunting ground for researchers, seeking to
find new drugs
• The advantage of these venom-derived toxins is that
they seem to act only on certain types of cells
Medical Value of Snake
Venom (cont.)
• Exanta, a blood thinner derived from the venom of
the cobra manages to thin the blood at a steady
level without major fluctuations
• Contortrostatin, a component found in copperhead
venom, is being used to attack breast cancer cells
and to prevent cancer from spreading
• A South American snake farm is harvesting venom to
make homeopathic medicine for AIDS patients.
• Amazingly, snake venoms may also hold cures to
many human diseases. Scientists have discovered
that natural poisons, toxins, and venoms contain
chemicals that can be used to create an array of
drugs for treating everything from chronic pain to
cancer
Medical Value of B. candidus
Venom
• The B. candidus snake is one of the medically
significant snakes in Southeast Asia
• The most obvious medical use of B. candidus
venom is to make anti-venom for snake bite
victims
• Neurotoxic proteins are isolated from the venom
and used as pharmacological tools to further
research the function of the nervous system
• In minimal amounts it could possibly be used to
bring about neuromuscular blockade like
botulinum toxin
Summary
The significance of our presentation:
The B. candidus species produces highly toxic venom
• Consists of three types of toxins
1. Candoxin
2. Bucandin
3. α-bungarotoxin
• Mechanism of Action of the three toxins
• Neurotoxic
• All three toxins belong to the three-finger toxin family
• Anti-venom is hard to obtain due to rare occurrence of
bites
• There are several medical uses of B. candidus venom
Thank You!
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