RESUMO
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.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas/metabolismo , Metaloendopeptidases/metabolismo , Peptídeos/metabolismo , Proteínas R-SNARE/metabolismo , Toxina Tetânica/metabolismo , Sequência de Aminoácidos , Animais , Transferência Ressonante de Energia de Fluorescência , Hidrólise , Cinética , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Proteínas R-SNARE/químicaRESUMO
Proteases were identified and characterized from the culture supernatant of the C. diphtheriae and B. pertussis bacteria. The proteases were secreted in the media and detected at the end of the exponential growth phase. Activity was detected in some fluorescent substrates, based on selected protein sequences such as insuline beta-chain, bradykinin, and synaptobrevin. The proteases were purified by means of gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified proteins indicated, for the main secreted proteins, an estimated molecular mass of 30 kDa in C. diphtheriae and 69 kDa in B. pertussis culture media. The proteases were stable and presented enzymatic activity at 37 degrees C. These proteases were not related to the main toxic compounds described in these two bacteria, but could represent good markers for the fermentation process when the enzyme activity was measured with the fluorescent substrates.
Assuntos
Bordetella pertussis/enzimologia , Corynebacterium diphtheriae/enzimologia , Meios de Cultura/química , Peptídeo Hidrolases/análise , Peptídeos/metabolismo , Sequência de Aminoácidos , Toxinas Bacterianas/análise , Bordetella pertussis/crescimento & desenvolvimento , Soluções Tampão , Cromatografia em Gel , Corynebacterium diphtheriae/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Filtração , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Peptídeo Hidrolases/isolamento & purificação , Peptídeos/química , Toxina Pertussis/análise , Cloreto de Sódio/química , Especificidade por Substrato , Trometamina/químicaRESUMO
Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. The low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx. Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. The results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).