By : Zach Weiss, Tiffany Maestas & Amanda LopezPresented to: Dr. ToolsonBiology 445 April 21, 2010

By : Zach Weiss, Tiffany Maestas & Amanda LopezPresented to: Dr. ToolsonBiology 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…We will discuss…

• Distribution• Habitat• Reproduction• Diet and

Behavior• Venom

• Distribution• Habitat• Reproduction• Diet and

Behavior• Venom

Where are they found?Where are they found?

Southeast Asia… • Thailand• Cambodia• Vietnam• Malaysia• Singapore• Indonesia

Southeast Asia… • Thailand• Cambodia• Vietnam• Malaysia• Singapore• Indonesia

HabitatHabitatHabitatHabitat

• Flat and hilly countryFlat and hilly country• Found in areas at heights Found in areas at heights

of 2300 metersof 2300 meters• In habit open areas, In habit open areas,

fields, grassy landscapes, fields, grassy landscapes, and forestsand forests

• Sometimes found in close Sometimes found in close proximity to waterproximity to water

• Avoid the sun, so found Avoid the sun, so found under fallen down trees or under fallen down trees or rotting stumps rotting stumps

• Flat and hilly countryFlat and hilly country• Found in areas at heights Found in areas at heights

of 2300 metersof 2300 meters• In habit open areas, In habit open areas,

fields, grassy landscapes, fields, grassy landscapes, and forestsand forests

• Sometimes found in close Sometimes found in close proximity to waterproximity to water

• Avoid the sun, so found Avoid the sun, so found under fallen down trees or under fallen down trees or rotting stumps rotting stumps

• OviparousOviparous – lay eggs– lay eggs

• Mating season - March and AprilMating season - March and April

• Female lays 4 to 10 eggs - 2 months after Female lays 4 to 10 eggs - 2 months after mating seasonmating season

• The females remain with their eggs until The females remain with their eggs until they have hatched and guard themthey have hatched and guard them

• Incubation period of the eggs: 60-64 daysIncubation period of the eggs: 60-64 days

• The newborns are between 30 to 32 cm The newborns are between 30 to 32 cm longlong

• OviparousOviparous – lay eggs– lay eggs

• Mating season - March and AprilMating season - March and April

• Female lays 4 to 10 eggs - 2 months after Female lays 4 to 10 eggs - 2 months after mating seasonmating season

• The females remain with their eggs until The females remain with their eggs until they have hatched and guard themthey have hatched and guard them

• Incubation period of the eggs: 60-64 daysIncubation period of the eggs: 60-64 days

• The newborns are between 30 to 32 cm The newborns are between 30 to 32 cm longlong

Venom!Venom!Venom!Venom!Extremely potent neurotoxic venom

You do not want to mess with this snake!

Extremely potent neurotoxic venom

You do not want to mess with this snake!

B. candidus VenomB. 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

• 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

B. candidus Venom Effects

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

• 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

• 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.

• 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.

NeurotoxinsNeurotoxins

• 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

• 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

NeurotoxinsNeurotoxins

• Snake neurotoxins specifically target the neuromuscular junctions in the peripheral nervous system and block neuromuscular transmission, either presynaptically or postsynaptically, leading to muscle paralysis

• 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 JunctionNeuromuscular JunctionNeuromuscular JunctionNeuromuscular Junction

The Neuromuscular Junction

Three-Finger Toxin Family

Three-Finger Toxin Family

Candotoxin, a novel three-finger toxin from the Bungarus candidus

Bungarus candidus venom contains several toxins of the three finger toxin family

Three-finger Toxin Family

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

• Three-finger toxins exhibit a broad range of pharmacological activity:• Central and Peripheral neurotoxicty • Cytotoxicity• Cardiotoxicity• Inhibition of enzymes such as

acetylcholinesterase • Platelet aggregation

Three-Finger Toxin Family

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.

• 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 FamilyThree-Finger Toxin Family

Common structure of several three finger toxins including α-bungarotoxin (B) and candoxin (E) from B. candidusvenom, which will be discussed further.

Three-Finger Toxin Family

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”

• 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”

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.

Three-Finger Toxin FamilyThree-Finger Toxin Family

What’s in Snake Venom?

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

• 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 Components of B. B. candidus candidus VenomVenom

Components of Components of B. B. candidus candidus VenomVenom

3 types of neurotoxins in Bungarus candidus:

• Candoxin: Novel three finger toxin from B. candidus

• Bucandin toxin: Novel presynaptic neurotoxin isolated from B. candidus

• Bungarotoxin: three finger toxin found in Elapid snake venom

3 types of neurotoxins in Bungarus candidus:

• Candoxin: Novel three finger toxin from B. candidus

• Bucandin toxin: Novel presynaptic neurotoxin isolated from B. candidus

• Bungarotoxin: three finger toxin found in Elapid snake venom

CandoxinCandoxin

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

Candoxin protein folded structure

Amino Acid residues in Candoxin

Candoxin Candoxin• Produces a reversible postjunctional

neuromuscular blockade by binding with muscle (αβγδ) receptors of the neuromuscular junction and α7 neuronal receptors.

• Produces a reversible postjunctional neuromuscular blockade by binding with muscle (αβγδ) receptors of the neuromuscular junction and α7 neuronal receptors.

Nicotinic Acetylcholine receptor

• 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.

CandoxinCandoxin

Neuromuscular Junction

CandoxinCandoxin

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

BucandinBucandinBucandinBucandin

• 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

• 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

Is a novel neurotoxin in B. candidus venom

Bucandin (A) protein structure compared to α-neurotoxins cobratoxin (B), erabutoxin (C), and cytotoxin II (D)

Bucandin amino acid sequence and disulfide-bonding pattern

BucandinBucandinBucandinBucandin•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

BucandinBucandin•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

BungarotoxinsBungarotoxinsBungarotoxinsBungarotoxins

Three-dimensional structure of α-bungarotoxin

BungarotoxinsBungarotoxins

α-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

α-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

β-bungarotoxins• Fairly common in some

snake venoms• Acts presynaptically• Alters acetylcholine

release in presynaptic terminal in both peripheral and central nervous systems

β-bungarotoxins• Fairly common in some

snake venoms• Acts presynaptically• Alters acetylcholine

release in presynaptic terminal in both peripheral and central nervous systems

BungarotoxinsBungarotoxins

B. Candidus venon contains α-bungarotoxins

•A three fingered toxin•Is a postsynaptic neurotoxin

•74 amino acid polypeptide

α-bungarotoxinstructure

BungarotoxinsBungarotoxinsBungarotoxinsBungarotoxins

• 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

• 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

BungarotoxinsBungarotoxins•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 B. candidus Attacks!Attacks! B. candidus B. candidus Attacks!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

• 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)

Venom Extraction (milking)

How to Make Anti-venom

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)

• 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 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

• 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

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

• 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.)

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

• 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

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

• 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

SummarySummary

The significance of our presentation:

The B. candidus species produces highly toxic venom• Consists of three types of toxins

1. Candoxin2. Bucandin3. 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

The significance of our presentation:

The B. candidus species produces highly toxic venom• Consists of three types of toxins

1. Candoxin2. Bucandin3. 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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Nirthanan, S., Charpantier, E., Gopalakrishnakone, P., Gwee, M.C., Khoo, H.E., Cheah, L.S., Bertrand, D., & Kini, R.M. 2002. Candoxin, a Novel Toxin from Bungarus candidus, Is a Reversible Antagonist of Muscle but a Poorly Reversible Antagonist of Neuronal Nicotinic Acetylcholine Receptors. The Journal of Biological Chemistry. 277: 17811-17820.

Nirthanan, S., Gopalakrishnakone, P., Gwee, M. C. E., Khoo, H. E. and Kini, R. M. 2002. Non-conventional toxins from elapid venoms.  Toxicon, 41: 397-407.

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Shivaji, P.G. 2004. Snake Venom Neurotoxins: Pharmacological Classification. Toxin Reviews.23:37-96.

Tanh, N.H., Poh, C.H., & Tan, C.S. 1989. The Lethal and Biochemical Properties of Bungarus candidus (Malayan Krait) Venom and Venom Fractions. Toxicon. 27:1065-1070

Torres, A.M., Kini, R. M., Selvanayagam, N., & Kuchel, P.W. 2001. NMR Structure of Bucandin, a Neurotoxin from the Venom of the Malayan Krait (Bungarus candidus). Biochemical Society. 360:539-548.

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