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.
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.
Nuss
Jonathan E
JE
US Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 , USA.
Wanner
Laura M
LM
Tressler
Lyal E
LE
Bavari
Sina
S
D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D005504:Food Analysis / Q000379:methods; D057166:High-Throughput Screening Assays; D008744:Methylamines / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010447:Peptide Hydrolases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D017510:Protein Folding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012680:Sensitivity and Specificity; D015854:Up-Regulation / Q000187:drug effects
D008744:Methylamines; C096323:rimabotulinumtoxinB; D010447:Peptide Hydrolases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C005855:trimethyloxamine; C096326:botulinum toxin type E