L2 - Toxins as Tools

Question 1

What are the Clostridium bacteria and what do they each cause?

Answer 1

bolulinum - botulism, food poisoning
difficile - fluid accumulation
perfringens - gangrene
tetanus - paralysis from infected wounds

Question 2

How many strains of toxin does C. botulinum and C. tetani produce?

Answer 2

botulinum - 7, A-G

tetani - 1

Question 3

What are the features of clostridial neurotoxins?

Answer 3

Domain structure homology
Secreted as 150kD single chain with single Di-S bond
Toxin cleaved into heavy and light chain

Question 4

What are the functions of the two chains of clostridial toxins?

Answer 4

Heavy - binding (Hc) / translocation (Hn)

Light - enzyme (LC)

Question 5

What do clostridial toxins bind to?

Answer 5

Gangliosides

Question 6

What are gangliosides and where are they located?

Answer 6

carbonhydrate modified sphingolipids on external leaflet of membrane (lots at synapse)

Question 7

What is the purpose of gangliosides for the toxin?

Answer 7

Binding to synapse

Binding to correct ganglioside will deliver toxin to correct neuronal type

Question 8

How do clostridial toxins enter the cell?

Answer 8

Co-docks with synaptotagmin and so is endocytosed with vesicle

Question 9

How does the clostridial toxin leave the vesicle?

Answer 9

Hn domain reacts to low pH and redox conditions of synaptic vesicle and forms a pore across membrane
Threads light chain through and reduced bond to release

Question 10

What did Mochida (1990) find?

Answer 10

Injection of light chain mRNA inhibits synaptic transmission

Question 11

Why do the clostridial toxins induce such different paralytic outcomes?

Answer 11

Site of action of BoTx in motorneurons

TeTx in inhibitory spinal interneurons

Question 12

What are the symptoms of the tetanus toxin?

Answer 12

Rigid paralysis

Question 13

What are the symptoms of clostridium toxin?

Answer 13

Floppy paralysis

Question 14

What happens internally with TeTx?

Answer 14

Binds motorneurons
Retrogradely transported along axon
Trans-synaptically transported into inhibitory interneuron
Blocks transmission

Question 15

What happens internally with BoTx?

Answer 15

Internalised by motorneurons

Blocks transmission

Question 16

What sequence homology does the light chain share?

Answer 16

metalloproteinase sequence

Question 17

What does the sequence homology in the light chain show?

Answer 17

Suggests clostridial toxins might cleave intracellular target

Question 18

What did Schaivo (1992) show about the clostridium toxins cleavage?

Answer 18

Block NT release by cleavage of synaptobrevin

Question 19

Why is the Leech Retzius neuron used for testing toxins?

Answer 19

Large with many vesicles
Intact pre and post synaptic neuron pair
Single cell can be used for western blot

Question 20

How was it shown there are similar structural requirement for toxin binding?

Answer 20

BotA inhibited cleavage of synaptobrevin by BotB,F,D,G or TetX

Question 21

What proteins are part of the SNARE complex?

Answer 21

Vesicel - synaptobrevin

membrane - syntaxin and SNAP-25

Question 22

What is a feature of the SNARE’S?

Answer 22

Make a complex resistant to chaotropic agent SDS

So must be thermodynamically stable

Question 23

What other 2 proteins bind to SNARE?

Answer 23

NSF (ATPase) and SNAP

Question 24

Which protein binds calcium?

Answer 24

Synaptotagmin

Question 25

What is the transglutaminase activity hypothesis?

Answer 25

TeTx activates transglutaminase causing covalent linkage of vesicle protein synapsin - blocking movement and release of vesicles

Question 26

What do synaptobrevin knockouts tell us?

Answer 26

Not essential for fusion | Essntial for rapid fusion and endocytosis

Question 27

What is an expression system used in flies and what are its advantages?

Answer 27

GAL4-UAS system | Allows expression in any tissue of choice