Analysis of the substrate recognition domain determinants of botulinum type B toxin using phage display.
The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.
Evans
E R
ER
Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, Wilts. SP4 0JG, UK. liz.evans@hpa.org.uk
Sutton
J M
JM
Gravett
A
A
Shone
C C
CC
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000378:metabolism; D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D003001:Cloning, Molecular; D017931:DNA Primers; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D019151:Peptide Library; D050988:Vesicle-Associated Membrane Protein 2; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A