Inhibition of release of neurotransmitters from rat dorsal root ganglia by a novel conjugate of a Clostridium botulinum toxin A endopeptidase fragment and Erythrina cristagalli lectin.
Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH(N)/A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH(N)/A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved.
Duggan
Michael J
MJ
Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom.
Quinn
Conrad P
CP
Chaddock
John A
JA
Purkiss
John R
JR
Alexander
Frances C G
FC
Doward
Sarah
S
Fooks
Sarah J
SJ
Friis
Lorna M
LM
Hall
Yper H J
YH
Kirby
Elizabeth R
ER
Leeds
Nicola
N
Moulsdale
Hilary J
HJ
Dickenson
Anthony
A
Green
G Mark
GM
Rahman
Wahida
W
Suzuki
Rie
R
Shone
Clifford C
CC
Foster
Keith A
KA
D000818:Animals; D001483:Base Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000494:pharmacology*; D002478:Cells, Cultured; D017931:DNA Primers; D010450:Endopeptidases / Q000302:isolation & purification / Q000494:pharmacology*; D005727:Ganglia, Spinal / Q000187:drug effects* / Q000378:metabolism; D037102:Lectins / Q000494:pharmacology*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology*; D037121:Plant Lectins; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D017931:DNA Primers; D037102:Lectins; D018377:Neurotransmitter Agents; D010446:Peptide Fragments; D037121:Plant Lectins; D011994:Recombinant Proteins; C045629:erythrina lectin; D010450:Endopeptidases; D001905:Botulinum Toxins