Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin.

Abstract

Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.

• Authors: Sheng Chen, Cherisse Hall, Joseph T Barbieri
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 283 Issue 30 Pg. 21153-9 (Jul 25 2008) ISSN: 0021-9258 [Print] United States
• PMID: 18511417 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Tetanus Toxin
• Vesicle-Associated Membrane Protein 2
• rimabotulinumtoxinB
• tetanospasmin
• Glutathione Transferase
• Trypsin
• Metalloendopeptidases
• Botulinum Toxins
• Botulinum Toxins, Type A
• Alanine

Topics

• Alanine (chemistry)
• Botulinum Toxins (chemistry)
• Botulinum Toxins, Type A
• Cell Membrane (metabolism)
• Glutathione Transferase (metabolism)
• Humans
• Kinetics
• Metalloendopeptidases (chemistry)
• Models, Chemical
• Models, Molecular
• Point Mutation
• Protein Binding
• Protein Structure, Tertiary
• Tetanus Toxin (chemistry)
• Trypsin (chemistry)
• Vesicle-Associated Membrane Protein 2 (chemistry, metabolism)