Recombinant forms of tetanus toxin engineered for examining and exploiting neuronal trafficking pathways.
Tetanus toxin is a fascinating, multifunctional protein that binds to peripheral neurons, undergoes retrograde transport and trans-synaptic transfer to central inhibitory neurons where it blocks transmitter release, thereby, causing spastic paralysis. As a pre-requisite for exploiting its unique trafficking properties, a novel recombinant single chain was expressed at a high level in Escherichia coli as a soluble, easily purifiable protein. It could be activated with enterokinase to produce a dichain that matched native toxin in terms of proteolytic and neuroinhibitory activities, as well as induction of spastic paralysis in mice. Importantly, nicking was not essential for protease activity. Substitution of Glu(234) by Ala created a protease-deficient atoxic form, which blocked the neuroparalytic action of tetanus toxin in vitro, with equal potency to its heavy chain; but, the mutant proved >30-fold more potent in preventing tetanus in mice. This observation unveils differences between the intoxication processes resulting from retrograde transport of toxin in vivo and its local uptake into peripheral or central nerves in vitro, dispelling a popularly held belief that the heavy chain is the sole determinant for efficient trafficking. Thus, this innocuous mutant may be a useful vehicle, superior to the heavy chain, for drug delivery to central neurons.
Li
Y
Y
Department of Biochemistry, Imperial College, London SW7 2AZ, United Kingdom.
Foran
P
P
Lawrence
G
G
Mohammed
N
N
Chan-Kwo-Chion
C K
CK
Lisk
G
G
Aoki
R
R
Dolly
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009469:Neuromuscular Junction / Q000187:drug effects; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013744:Tetanus Toxin / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity
D011994:Recombinant Proteins; D013744:Tetanus Toxin