Enzymatic profiling of tetanus and botulinum neurotoxins based on vesicle-associated-membrane protein derived fluorogenic substrates.
Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. In order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed. The assays were conducted with FRET peptides derived from SNAP-25, synaptobrevin and syntaxin. The substrates were cleaved by 2 ng/mL of toxin at different rates (K(cat)/K(M) from 0.028 to 75.9 microM.s(-)) at a single bond, as confirmed by Q-TOF mass spectrometry. Inhibition of the hydrolysis was obtained with EDTA or with specific antibodies directed to each neurotoxin. Different substrate selectivities, especially by BoNT- A and E, suggest that these substrates can be used as a putative method for clostridial toxin quantification and serotype differentiation and could be easily adapted to a high-throughput protocols.
Perpetuo
Elen A
EA
Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, Brazil.
Juliano
Luis
L
Juliano
Maria Aparecida
MA
Fratelli
Fernando
F
Prado
Sally M A
SM
Pimenta
Daniel C
DC
Lebrun
Ivo
I
D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D050683:R-SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D013744:Tetanus Toxin / Q000378:metabolism*
D010455:Peptides; D050683:R-SNARE Proteins; D013744:Tetanus Toxin; C035979:tetanospasmin; D008666:Metalloendopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E