The Osmolyte Trimethylamine N-Oxide (TMAO) Increases the Proteolytic Activity of Botulinum Neurotoxin Light Chains A, B, and E: Implications for Enhancing Analytical Assay Sensitivity

Abstract

Botulism, the disease caused by botulinum neurotoxins (BoNTs), secreted by the spore-forming, anaerobic bacteria Clostridium botulinum, has been associated with food poisoning for centuries. In addition, the potency of BoNTs coupled with the current political climate has produced a threat of intentional, malicious poisoning by these toxins. The ability to detect and measure BoNTs in complex matrixes is among the highest research priorities. However, the extreme potency of these toxins necessitates that assays be capable of detecting miniscule quantities of these proteins. Thus, signal-boosting strategies must be employed. A popular approach uses the proteolytic activity of the BoNT light chain (LC) to catalyze the cleavage of synthetic substrates; reaction products are then analyzed by the analytical platform of choice. However, BoNT LCs are poor catalysts. In this study, the authors used the osmolyte trimethylamine N-oxide (TMAO) to increase the proteolytic activities of BoNT LCs. Their data suggest that concentrated solutions of TMAO induce complete folding of the LCs, resulting in increased substrate affinity and enhanced enzyme turnover. The authors observed increases in catalysis for BoNT serotypes A, B, and E, and this increased proteolytic activity translated into substantial increases in analytical assay sensitivity for these medically relevant toxins. (Journal of Biomolecular Screening 2010:928-936)

Protein folding and stability in the presence of osmolytes

“…It appears that a number of osmolytes have stabilizing effects on proteins and can prevent their aggregation and increase the functional activities of proteins [1,[6][7][8][9][10][11][12][13][14][15][16][17][18][19]. High concentrations of osmolytes in the cytoplasm are observed not only during extreme pressure conditions, but also at extreme pH values, temperatures of the media, and concentrations of salts and denaturing agents [20].…”

The synthesis of 2,5-bis(4-amidinophenyl)thiophene derivatives providing submicromolar-range inhibition of the botulinum neurotoxin serotype A metalloprotease

“…Table 1 provides the K i values for inhibitors 1 and 4a – d when examined in vitro employing a well documented HPLC-based assay for BoNT/A LC inhibition [23–29]. The potencies of the derivatives, in support of our hypothesis, provide further evidence that tethering -(CH 2 ) n -4,7-ACQ components onto the termini of di-cationic lead BoNT/A LC inhibitors ( i.e.…”

Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

“…To determine if the SFK inhibitors were acting directly on BoNT/A-mediated cleavage of SNAP-25, a well-characterized HPLC-based in vitro assay for measuring BoNT/A LC proteolytic activity was used to evaluate the compounds (Nuss et al 2010) (Fig. 7a).…”

“…BoNT/A LC proteolytic activity was determined using a high-performance liquid chromatography (HPLC)-based assay, as described previously (Nuss et al 2010). Briefly, this assay utilizes an N-terminal acetylated, C-terminal aminated synthetic peptide that corresponds to the minimum substrate requirements of the BoNT/A LC.…”